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BACrERIOLOGICAL REVIEWSVol. 28, No. 2, pp. 97-125 June, 1964Copyright @ 1964 by the American Society for Microbiology

Printed in U.S.A.

COMPARATIVE PHYSIOLOGY OF PLEUROPNEUMONIA-LIKE ANDL-TYPE ORGANISMS

PAUL F. SMITHDepartment of Microbiology, University of South Dakota,

Vermillion, South Dakota

INTRODUCTION............................ 97CHEMICAL COMPOSITION............................ 98Composition of Intact Organisms ............................ 98Protein composition........................................................................ 98Nucleic acid composition.................................................................... 98Carbohydrate composition................................................................... 99Lipid composition............................ 100Elemental composition...................................................................... 103

Composition of Cell Envelopes............................ 103General composition........................................................................ 103Structure of cell envelopes................................................................... 104

NUTRITION............................ 105Chemical Requirements for Growth............................ 106Protein requirements........................................................................ 106Amino acid requirements.................................................................... 107Nucleic acid requirements................................................................... 107Carbohydrate requirements.................................................................. 108Lipid requirements............................ 108Vitamin requirements....................................................................... 110Inorganic requirements ............................... 110

Physical Requirements for Growth............................ 111Temperature..1..........................11Effects of pH..........................111Osmotic effects .............................................................................. 111

Gaseous Requirements for Growth............................ 112ENZYMATIC ACTIVITIES............................ 112Energy-Yielding Metabolism ............................ 112

Carbohydrate-utilizing organisms............................................................ 112Nonfermentative organisms............................ 115

Nitrogen Metabolism............................ 115Lipid Metabolism............................ 116Biosynthesis................................................................................. 117Enzymatic Activity Related to Pathogenesis ................................... 118

CONCLUSIONS.................................................................................. 119LITERATURE CITED...................................................... 119

INTRODUCTION (23) for the pleuropneumonia group, exists overCurrent knowledge concerning the pleuro- the relation or distinction of the two groups of

pneumonia group of organisms (PPLO, Myco- organisms. Much of this controversy stems fromplasma, Mycoplasmataceae) and L-type organ- the results of morphological studies hampered byisms has been reviewed in part (14, 18, 47) and the fragility and plasticity of these organismsin toto (2, 30, 48) during recent years. All of these and from serological data reflecting the lack ofreviews have treated either the Mycoplasma or good antigenic specificity due to the absence ofthe L organisms singly, or they have not covered typical bacterial cell-wall components. Nothe physiological aspects of the two types of critical review of the physiological similaritiesorganisms. Controversy, heightened by the re- and dissimilarities of these organisms has beencent classification scheme of Edward and Freundt made. Since the behavior and properties of bio-

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logical entities reflect physiological mechanisms,it is considered appropriate to examine thecomparative physiology of the Mycoplasma andthe L-type organisms.The physiological aspects covered by this

review include chemical composition, nutrition,metabolism, and biosynthesis. No morphologicalor immunological data are presented unless theybear directly upon some physiological aspect. Allorganisms currently accepted as belonging to thepleuropneumonia group are considered. Only L-type o0 ganisms which have been shown toproduce the typical L colony and which have notshown reversion to the bacterial form in theabsence of the agent inducing the L transforma-tion are included. Thus, both salt-requiring andsalt-nonrequiring L organisms from gram-nega-tive and gram-positive bacteria are included, butthe so-called transformation forms are excluded.

CHEMICAL COMPOSITIONComposition of Intact Organisms

Protein composition. The few reports pertainingto protein composition are directed mainlytoward determination of the presence or absenceof amino acids found in bacterial cell walls. M.hominis strain 07 contains 31 to 42% protein(60), and M. gallisepticum strain 5969 contains80% (70). Protein hydrolysates of the lattercontained 17 amino acids common to manyproteins. Proteus L 9 is composed of 50 to 75%protein and contains a, E-diaminopimelic acid,although in a smaller amount than does theparent bacterium (163, 165). This typical com-ponent of many bacterial cell walls has not beenfound in any other stable L organism or Myco-plasma (41, 79, 86, 141). Kandler and Zehender(41) found 17 common amino acids in threestrains of Mycoplasma (Findlay mouse, M.laidlawaii strain A, and Seiffert compost). Thesame amino acids were found in Proteus L 52(40). Panos et al. (79) demonstrated the absenceof muramic acid and ornithine, a relatively highcontent of serine and leucine, and a relatively lowcontent of aspartic acid, alanine, and lysine in thesalt-requiring L organism, AED-L. With theexception of one L organism from Proteus, bothtypes of organisms lack a, E-diaminopimelic acidand contain other amino acids common to mostproteins. This conclusion should be tentative,pending examination of other representativestrains of both types of organisms.

Nucleic acid composition. The total nucilec acidcontent of Mycoplasma exhibits a wide variation,depending upon the strain examined. Kandler etal. (43) reported that during the logarithmicphase of growth the total nucleic acid of theFindlay mouse strain amounted to 2.5 mg/mg ofprotein nitrogen and dropped to 1.7 in thestationary phase. Although accurate determina-tion of these values as percentage of dry weightfor comparative purposes is not possible, it canbe estimated that total nucleic acid in this strainrepresents 12 to 15% of the dry weight. M. homi-nis 07 contains 4 to 5% nucleic acid (59), whereasin 31. gallisepticum 5969 total nucleic acid repre-sents 12% of the dry weight of the organisms (70).Recently, Langenfeld and Smith (51) determinedthe nucleic acid phosphorus content of 13 strainsof Mycoplasma of varied origin (avian, human,caprine, bovine, tissue culture, sewage). Valuesranging from 3 to 13% of dry weight were found.Strains isolated as contaminants of tissue culturesconsistently possessed the higher values.

Salt-nonrequiring L organisms from only twogenera of bacteria (Proteus, Streptobacillus) havebeen analyzed for nucleic acid content. Kandleret al. (43) found 3 mg of nucleic acid per mg ofprotein nitrogen during the logarithmic phase forL 52, derived from P. vulgaris. As with theMycoplasma (43, 59), there was a reduction ofnucleic acid in the stationary phase, due toreduction of ribonucleic acid (RNA). Weibull(162), working with L 9, derived from P. mirab-ilis, found a total nucleic acid content of 14 to19% of the dry weight. The higher values foundby Weibull in comparison to those of Kandlerhave been attributed to loss of RNA by celldisruption in the latter case (163). Mandel et al.(63) reported values for nucleic acid in various-sized fractions of 18L, derived from P. morgani,but these values are not comparable because theyused defatted organisms. Langenfeld and Smith(51) found this strain to contain 8% and L 1,derived from S. moniliformis, 10% total nucleicacid.The total nucleic acid content of a salt-requir-

ing L organism, AED-L, derived from a group A0-hemolytic streptococcus, was reported byPanos et al. (79) to comprise 12% of its dryweight. The values of Langenfeld and Smith (51)for salt-requiring L organisms derived from groupA 0-hemolytic streptococci, diphtheroids, and

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stal)hylococci are considerably lower, i.e., 2 to3%.The variation in total nucleic acid content can

be accounted for not only by strain variation butalso by the age of the organisms used for analyses.Since RNA content increases during the earlylogarithmic phase and then declines rapidly (43,59), cells harvested in the stationary phase wouldbe low in RNA and, thus, total nucleic acid.Cellular disruption during handling of prepara-tions would liberate RNA into the nonsedi-mentable fraction as reported by Plackett (85),who found that freezing and thawing of cells of.31. mycoides liberated 80% of the RNA to thesupernatant fluid. The nature of the particles alsohas an effect on nucleic acid content. Mandel etal. (63) noted a great variation in the RNA anddeoxyribonucleic acid (DNA) of different-sizedparticles of 18 L. Which of these particlesrepresent the typical cell is not known. Theseauthors thought that the forms ranging from 1.9to 3.8 ,u in diameter represented the stage of activeproliferation, because this fraction possessed thehighest RNA-DNA ratio and the highest rate ofrespiration per unit of RNA (65). Weibull andBeckman (165) also found a difference in the verysmall elements ( <0.3 i in diameter) of L 9 ascontrasted to particles of >1.0 ,u in diameter.The smaller particles contained less RNA and es-sentially no DNA, and were considered to beorganized debris.DNA represents 1.5 to 4.0% of the dry weight

of both Mycoplasma and L organisms (43, 59,70, 79. 162) and, as in bacteria, remains relativelyconstant throughout the growth cycle (43, 163).The DNA of one human and one avian strain ofMycoplasma has been examined. Lynn andSmith (52) determined the nature of and themolar ratios of the bases found in Ml. hominis 07.Adenine, guanine, cytosine, and thymine, but nohydroxymethyl cytosine, were found. The ratioof adenine to thymine was 0.87; guanine tocytosine, 0.81; and purine to pyrimidine, 0.86.The ratio of guanine-cytosine (GC) to guanine-cytosine-adenine-thymine (GC-AT) was 0.46.Morowitz et al. (70), employing 31. gallisepticum5969, isolated a DNA preparation of a density of1.693 g/cm^3 and predicted a CG-to-CG-AT ratioof 0.33; by chemical procedures, they found thissame ratio. The ratio of adenine to thymine was0.96 and of guanine to cytosine, 1.360. Thislatter ratio was considered high and could not be

accounted for unless cytosine destructionoccurred. The low cytosine value could have aneffect on the overall base ratio obtained bychemical methods. Morowitz et al. presentedevidence of increased band width and densityupon heating in cesium chloride gradient,indicating that the DNA was double stranded.Thus, even with only two strains of Mlycoplasma,heterogeneity in DNA composition exists.RNA is found to the extent of 3 to 10% in the

two types of organisms, with larger amountsbeing found in the L organisms (43, 59, 70, 79,162). In both M1ycoplasma (43, 59) and L orga-nisms (43), RNA increases rapidly during theearly logarithmic phase and then declinesrapidly, reaching its lowest level during thestationary phase. This behavior is typical ofbacterial systems and reflects the requirementsfor the various species of RNA for proteinsynthesis (33). Starting at essentially the samelevel, RNA per unit of protein nitrogen ofProteus L 52 rose to a greater extent than did thecorresponding value of the Findlay mouse strainof Mlycoplasma (43). This difference may reflectdifferences between L organisms and Mycoplasmaor, more probably, the effect of the culturemedium which was nutritionally richer, com-paratively speaking, for the more enzymaticallycomplete Proteus L 52. Thus, the greater growthrate of the Proteus L organisms would result inmore rapid synthesis of RNA until it reached theRNA-protein ratio characteristic of that growthrate, whereas the slower growth rate of theMycoplasma would result in a slower rate of RNAsynthesis and a lower RNA-protein ratio. Suchhave been the findings with bacteria upontransfer from poor to rich culture media and viceversa (62).Only one strain, M. hominis 07, has been

examined for the base content of its RNA. Thisorganism contains adenine, guanine, cytosine,and uracil in molar proportions of 10.0, 17.5, 16.8,and 11.2, respectively. The purine-pyrimidineratio was 0.98 (59). Schlieren photographs of thesedimentation pattern of RNA from M. gal-lisepticum 5969 revealed 17S, 38S, 58S, 76S, 84S,and 117S ribosomal particles (70).

Carbohydrate composition. Australian workersare the only group who have investigated in anydetail the carbohydrate composition of eithertype of organism (7, 86, 88). Other investigationshave been limited to the qualitative detection of

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hexoses and hexosamines commonly found inbacterial cell walls. Kandler and Zehender (41)noted the almost complete absence of glucos-amine in L organisms from P. vulgaris. Sharpand Dienes (119) found a hexosamine-reducingsugar antigen in the L organism from P. morgani,normally associated with the cell wall of thisProteus strain. At the time of their report, this Lorganism was considered to be similar to the 3Btype described by Dienes (11), which is nowconsidered -to be an unstable transformationform. Repeated subculture has resulted in astable L organism. It is probable that thisorganism better fits the classification given byKandler and Zehender (41) as a C type, i.e.,initially revertible to the bacterium in theabsence of the inducing agent but stabilizing inthe L phase after many passages in the presenceof the inducer. Several investigators have notedthe presence of the glycolipid-containing 0antigen in L organisms of gram-negative bacteria[Proteus (69, 158) and Vibrio (155)]. None or onlytrace amounts of glucosamine and rhamnose andno group-specific polysaccharide are found insalt-requiring L organisms of group A streptococci(79, 120).The carbohydrate content of Mycoplasma is

variable according to the findings of Morowitz etal. (70) for M. gallisepticum 5969, in which itconstituted less than 0.1% of the dry weight, andthe findings of Plackett, Buttery, and Cottew (88)for several strains, in particular M. mycoides inwhich as much as 10% of the dry weight couldbe accounted for as a galactose-containing poly-saccharide. Glucose, sometimes galactose, andprobably mannose, but no uronic acid and onlytraces of hexosamine, were found in bovine,caprine, ovine, saprophytic, and avian strainsThe purified galactan of M. mycoides appeared tobe composed mainly of galactofuranose unitscontaining 6-0-f-D-galactofuranosyl-1 linkages.The galactan contained 4% bound triglyceride,containing predominately palmitic and somestearic acid. This polysaccharide was morealkaline-labile than the lipopolysaccharides ofgram-negative bacteria, although it was antigenicand reacted in serological reactions. A glucanisolated by this group from a bovine arthritisorganism contained primarily 2-O-3-i)-gluco-pyranosyl-1 linkages. Polysaccharide haptenshave been detected in various body fluids of sheepand cattle infected with M. mycoides (35) and un-

doubtedly arose from the infecting organism.Their relation to the galactan and glucan ofPlackett et al. is not known.Mycoplasma and L organisms are similar with

respect to their lack of cell-wall carbohydrateconstituents of bacteria. The other types ofcarbohydrate probably depend upon the specificstrain of organism. There is no reason to believethat any significant distinction or similarity be-tween the two types of organisms can be antici-pated, other than lack of cell-wall carbohydrate.

Lipid composition. The first indication thatlipids might be of significance in L-type organismsand Mycoplasma was the observation of Partridgeand Klieneberger (81) that L 1, the L organismderived from S. moniliformis, appeared toconcentrate oily droplets containing cholesterol.Subsequent nutritional studies by several in-vestigators indicated that lipids were among theessential growth requirements for several strainsof both types of organisms. Poetschke (89) notedthat L organisms derived from Corynebacterium,but not the parent bacteria, reduced the choles-terol content of the culture medium duringgrowth. Cholesterol could be isolated from drycolonial growth, and microscopy indicated cho-lesterol ester formation based on changes incrystalline form. Lynn and Smith (60) dem-onstrated that Lieberman-Burchard positivesterol comprised a significant portion of the dryweight of intact cells of Mycoplasma. Sterol, freeand esterified, was detected in two strains of M.hominis, 07 and Campo, and in one avian strain,J, but none was found in two sterol-nonrequiringstrains, M. laidlawii strains B and B 15. Theamount of total sterol varied with the strain, andthe amount of esterified sterol varied unpredic-tably with the age of the cells. Smith and Roth-blat (142), studying the incorporation of choles-terol-4-C'4 by Mycoplasma, L-type organisms,and their parent bacteria, found that both grow-ing and resting cells of Mycoplasma and certainL-type organisms removed cholesterol from thesurrounding medium and incorporated it solelyinto the nonsaponifiable lipid fraction of theorganism. The one salt-requiring L-type organismdid not incorporate cholesterol significantly,whereas two salt-nonrequiring strains behaved asdid Mycoplasma (142, 143).A comparative study of the lipid composition

of representative strains of Mycoplasma and L-type organisms (143) demonstrated that sterol-

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requiring Mycoplasma strains contain 10 to 20%Vototal lipid, of which 50 to 65% is nonsaponifiable;sterol-nonrequiring Mycoplasma strains contain8 to 9% total lipid, of which 50 to 65% is non-

saponifiable; and salt-requiring L organisms con-

tain 3 to 5% total lipid of which 30 to 50% isnonsaponifiable. Salt-nonrequiring L organismswere similar to the two types of Mycoplasma. Al-though the culture media varied for differentorganisms used in this study, it was shown thatstrains not requiring serum supplementation con-

tained equivalent quantities of lipid when grown

with or without serum. The presence of sterol inthe culture medium did, however, alter the quali-tative composition, for every strain of Myco-plasma or L organism thus grown contained slow-acting Lieberman-Burchard positive sterol. Sterol-nonrequiring strains of both types grown in theabsence of sterol contained nonsaponifiable lipid,which gave a positive reaction with the non-

specific ferric chloride-sulfuric acid reagent but a

negative Lieberman-Burchard reaction and was

not digitonin-precipitable. Rebel and co-workers(98, 99) found Proteus 18 L to contain 14 to 16%total lipid of which 6.5% was cholesterol. This or-

ganism was grown on a serum-containing culturemedium from which the sterol was derived. Thenature of the nonsaponifiable lipids of L orga-

nisms when grown in a medium necessitatingtheir own synthesis of lipid is not known. Recentstudies with Proteus 18 L grown in a definedmedium indicate the presence of a long-chainsaturated molecule possessing a carbonyl group(Smith, unpublished data).The nature of the nonsaponifiable lipids of

several strains of sterol-requiring and sterol-non-requiring Mycoplasma has been thoroughlyexamined. The organisms used for analysis were

grown on culture media from which all neutrallipids were removed and, in the case of sterol-requiring strains, to which was added purifiedsterol. One sterol-requiring strain of humanorigin incapable of carbohydrate utilization (M.hominis 07) contained nonsaponifiable lipidcomposed entirely of the sterol in which it was

grown, e.g., cholesterol (110), cholestanol, or

ergosterol (132). One sterol-requiring avianstrain capable of glucose utilization (J) containednonsaponifiable lipid comprised entirely ofcholesterol, and cholesteryl-3-D-glucoside whengrown in the presence of cholesterol. The relativeproportions of these two compounds depended

upon the amount of glucose supplied in theculture medium (110). Identity of the abovecompounds was proven by extensive physical,chemical, and enzymatic analyses. Any alterationof the basic molecular structure of a sterol, otherthan addition to the 3-hydroxyl group, could beaccounted for by air oxidation. The correlation ofthe presence of cholesteryl glucoside with abilityto utilize glucose has been extended to otherstrains. A tissue culture strain, T5, isolated fromHeLa cells, utilized glucose and contains theglucoside, whereas another strain of humanorigin, Campo, unable to degrade glucose,contained only cholesterol (Smith, unpublisheddata). A portion of the sterol is found esterified(60). The ratio of volatile to nonvolatile fattyacids in ester linkage with the sterol was 0.86 forthe glucose-fermenting avian strain J and 49.5for the glucose-nonfermenting human strain 07.The volatile fraction of the former strain wasprincipally acetic with a trace of propionic, andof the latter strain was principally butyric, lessacetic, and a trace of propionic acid. Rodwell(104), working with M. mycoides var. mycoides,found no reduction in the specific activity ofC'4-labeled cholesterol upon its isolation from theorganisms were such that no cholesterol ester orexisted in the form of free cholesterol. It ispossible that the age and metabolic state of theorganisms was such that no cholesterol ester orglycoside was present. That the ester andglycosidic forms of sterol are in a dynamic statehas been shown (60, 110).The first evidence that sterol-nonrequiring

strains of Mycoplasma contain lipid of a naturedistinct from sterol was the reported finding ofalcohol-ether soluble pigments in M. laidlau'istrains B and B-15 (130). Rothblat and Smith(110) subsequently isolated a hydrocarbon pig-ment from the former strain and identified it asneurosporene. Two other pigments were detectedin this strain and, based on behavior on silicic acidcolumns, were suspected to be a hydroxylatedcarotenoid and a carotenyl glucoside. A morethorough analysis (133) of four strains of sterol-nonrequiring Mycoplasma revealed the presenceof three pigments in each. The most polar pig-ment was identified as neurosporene, based uponcomparison of physical and chemical propertieswith those of authentic neurosporene. Thesecond pigment was concluded to be a hydroxyl-ated carotenoid, probably a C 40 compound

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containing two hydroxyl groups. This carotenolwas found to exist in the esterified form withacetate as the sole volatile fatty acid in esterlinkage. A carotenyl glucoside was isolated fromall four strains. The carotenyl moiety of thecompound was identical to the dihydroxycarotenol found free and esterified, based on itsphysical properties after removal of the glucoseby #3-glucosidase. The naturally occurringglucoside was found to contain 1 mole of glucoseper mole of carotenol. In two strains, 31. laid-lawii B and M. inocuum, the glucoside linkagewas beta; in the other two, M11. laidlawii A andcaprine strain KHS, probably alpha. Cellsharvested at the peak of the logarithmic phasecontained relative amounts of the differentpigments as follows: glucoside > carotenol >neurosporene.

Little is known about the content and com-position of other neutral lipids, phospholipids,and glycolipids in Mycoplasma and L organisms.The few reported studies have employed cellsgrown in crude culture media containing a massof ill-defined lipids. The lipid fractions of theorganisms merely mimic the lipid composition ofthe media (148). One probable criterion as to thecontamination of microbial lipids with lipids ofthe culture medium is the presence of sphin-gomyelin, not known to occur in any micro-organism grown on a defined medium (4). Thefindings of O'Leary (75) that the total fattyacids of M. hominis 07 contained only 13% un-saturated fatty acid and a preponderance of C 16saturated acid may reflect the fatty acid com-position of the crude medium. However, hisdemonstration of a C 17 acid identical with cis-9,10 methylene hexadecanoic acid, a C 15 acidwith properties resembling a cyclopropane acid,and another acid which may be a C 15 un-saturated precursor of the C 15 cyclopropane acidis indicative of fatty acids synthesized by theorganisms rather than incorporated from theculture medium. Tourtellotte et al. (152) ex-amined the total lipids of M1. gallisepticum 5969grown in chloroform-methanol extracted mediumand found free fatty acids, cholesterol, cho-lesterol esters, di- and triglycerides and cephalins,inositides, phosphatidyl choline, and sphingo-myelin. Phosphorus-32 incorporation was de-tected in the four phospholipid fractions. Ap-proximately 90% of the phosphatides wereinositides and lecithin. Although some radio-

activity was found in a fraction considered to besphingomyelin, it is doubtful that this representsa phosphatide synthesized by this organism forreasons stated above. Phosphatidic acids, a majorlipid component of bacteria (4) and consideredby these workers as degradation products in thestrain 5969, are eluted with several fractionsupon silicic acid chromatography. These com-pounds could account for some of the radio-activity found, especially in the "sphingo-myelin" fraction. Fatty acids of the variousfractions were shown to vary from C 6 throughC 18 with palmitic, stearic, and oleic predominat-ing. Saturated acids predominated, as previouslyshown by O'Leary (75), except in the cholesterolester fraction in which unsaturated acids were ingreater abundance.

Plackett (87) isolated a polyglycerophosphatecompound bearing some similarity to cardio-lipin from 31. mycoides. Carbon-14 labeledglycerol was incorporated into this compoundwhich yielded the hydrolysis products, glycerol,glyceryl phosphoryl glycerol, a- and 3-glycero-phosphate, glyceryl phosphorylglycerylphos-phoryl glycerol, and glyceryl phosphoryl glycero-phosphate. This component undoubtedlyrepresents a polymer synthesized by thisorganism.Rebel et al. (98), employing Proteus 18 L grown

in a crude medium, found 4.5, 77, and 8.5% of thetotal lipid as neutral lipid, phosphatides, and gly-colipids, respectively. Free and esterified choles-terol, mono-, di-, and triglycerides, phosphatidylethanolamine, and small amounts of phosphatidylserine were detected.

Lipid phosphorus determinations have beenperformed on several strains of both Mycoplasmaand L-type organisms. Kandler et al. (43) found120 ,ug of lipid phosphorus per mg of proteinnitrogen in Proteus 52 L and 40,ug in the Findlaymouse strain of Mycoplasma. MI. hominis 07contained lipid phosphorus as 0.13 to 0.29% ofdry weight (59). Proteus L 9 contains lipidphosphorus as 0.57% of dry weight (165).Recent studies of a representative collection ofMycoplasma and L-type organisms (51) showedthe lipid phosphorus content of the Mycoplasmato vary from 0.12 to 0.44 mg of phosphorus per100 mg of dry weight, of salt-nonrequiring Lorganisms from 0.24 to 0.65, and of salt-requiringL organisms from 0.01 to 0.09.The lipid content and composition of both

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types of organisms is, in many instances, cloudedwith the probability that lipids from the culturemedia contaminate the organisms and mask thetrue nature of their lipids. Nevertheless, it can beconcluded that the lipid content of Mycoplasmaand salt-nonrequiring L organisms is similar,but the salt-requiring L organisms are low in lipid,in particular phospholipid. This conclusionprompted the hypothesis that high salt require-ment for some L organisms reflected stabilizationof monolayers of ionized long-chain lipids bycondensation and solidification by metal ions(143). In spite of the fact that L organisms andMycoplasma not requiring sterol for growthcontain sterol if supplied exogenously, not enoughis known to make an accurate comparison of the"true" lipids of the various organisms. Super-fically, it appears that a structural relationshipexists with the nonsaponifiable lipids, i.e., 3-hydroxy sterols, 3-hydroxy carotenoids, andpossibly long-chain aldehydes or ketones.

Elemental composition. No reports of studies onthe elemental or ash composition of eitherMycoplasma or L organisms has ever been made.In this connection, the finding of an extremelyhigh content of acid-soluble phosphorus in salt-requiring L organisms is of interest (51). Noexplanation can be given for this result, unless itrepresents an effort by the organisms to attemptcell-wall synthesis resulting in the accumulationof intermediate nucleotides. Serum preventedaccumulation of large amounts of acid-solublephosphate in the streptococcal, but not thediphtheroid, L organisms.

Composition of Cell EnvelopesDefinition of cell envelopes or membranes is

made complex, particularly with L organisms,because of the inconclusive morphologicalfindings and the controversial modes of re-production. Generally, when grown in liquidmedium, Ml ycoplasma cells contain protoplasmsurrounded by a limiting membrane whether theorganism be spherical or filamentous in form(13, 31, 55). Rupture of these elements releasesthe protoplasmic constituents from the mem-brane. Centrifugation results in the sedimenta-tion of at least the maj or portion of the mem-branes. Depending upon the method and degree ofrupture, membranous fragments may be retainedin the supernatant fraction. An exception appearsto be a strain of MI. gallisepticum, the mem-

branes of which are not sedimentable (P. J.VanDemark, personal communications). Mem-brane fractions employed in all studies of Myco-plasma thus far have constituted washed sedi-mentable fragments after sonic lysis (144) oralternate freezing and thawing (93). In no casewere preparations treated with various enzymes,such as ribonuclease and deoxyribonuclease, tofree them from nonmembranous material. Thus,data relating to membranes of Mycoplasma canbe considered only relative and not quantitative.That such treatments give rise to membranousfragments was shown by Razin (93).L organisms, on the other hand, appear to

exist as the so-called large bodies within whichlie smaller viable granules (48). Dienes (12) doesnot consider that any significant differenceexists between the two types of organisms exceptfor the larger size and greater pleomorphism ofthe L organisms. With Proteus L organisms,various sizes are apparent (65), and the smallestgranules are considered to be organized debris(65, 163). Sonic treatment of the salt-requiring Lorganisms results in reduction of optical densitybut not of viability, allowing Panos et al. (80) toconclude that small granular elements dem-onstrated by electron microscopy were viableelements. Suspension in a nonhypertonic milieuresulted in lysis of even these granules (Panos,personal communication). Membrane fractions ofsalt-nonrequiring L organisms used in studies ofmembrane composition have been obtained bysubjecting the organisms to sonic lysis in distilledwater, thereby insuring complete lysis. As withthe Mycoplasma, no effort was made to rid themembranes of nonmembranous material. Mem-branes of salt-requiring L organisms have beenobtained by lysis in a hypotonic environment orby this method plus sonic treatment. Thus, themembrane fractions, although impure, do consistof the cell envelopes, whether they are from largebodies or elementary granules. As with theMycoplasma, data regarding the membranes areof a relative nature.

General composition. Razin (93) reported theonly analysis of the total composition of.the cellmembranes of either of the two types of orga-nisms. With cell membranes of M. laidlawii B,obtained by freezing and thawing, he found 55to 67% protein, 25 to 30I% total lipid, 0 to 3.5%sterol (depending upon whether sterol was presentin the growth medium), 7 to 8%7, carbohydrate,

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2.1 to 4.7% RNA, and 0.15 to 0.5% DNA. Thepresence of RNA, and especially DNA, is sug-gestive of impure cell-membrane preparations.

Essentially all of the data reported concerningcomposition of the cell membranes of these orga-nisms relate to lipid composition. Lynn andSmith (60) reported slightly greater amounts oftotal sterol in the sedimentable residue aftersonic lysis of Mycoplasma strains 07, Campo, andJ. Esterified sterol was found in greater amount inthis residue than in the supernatant fraction.Phospholipid was likewise unequally distributedbetween the two fractions, the greater quantitybeing associated with the residue. Smith andRothblat (142) in their study of sterol incorpora-tion by Mycoplasma found the major portion ofcholesterol, as measured chemically or radio-metrically, in the residue of sonic-lysed strain 07.Extension of this study to other strains of Myco-plasma and to L-type organisms (143) revealedthat 69 to 86% of nonsaponifiable lipid (steroland carotenoids) was associated with the cellmembrane fraction of six strains of Mycoplasmaand L 1. One salt-nonrequiring L organism,Proteus 18 L, and three salt-requiring L orga-nisms contained equivalent amounts in bothfractions or greater amounts in the supernatantfraction. These results could be accounted for bythe greater lability of the membranes of L orga-nisms, particularly the salt-requiring types.Extended sonic treatment of the cell membranesof Mycoplasma resulted in liberation of moresterol to the supernatant fraction but never tothe extent of complete solubilization.The galactan and the polyglyceropbosphate

compounds of M. mycoides, treated earlier in thisreview, have been suggested to comprise part ofthe surface structure of this organism (7, 87).

Structure of cell envelopes. Thin sections ofMycoplasma and L-type organisms when viewedby electron microscopy reveal the presence of adouble-layered enveloping membrane (24, 118,157). Such a double-layered membrane, consist-ing of two dense outer layers each 20 A in thick-ness and presumably composed of protein, and aninner transparent layer 35 A in thickness, pre-sumably composed of lipid, making total mem-brane thickness of 75 A (157), is typical of cellmembranes in general (131). Lacking the rigidcell wall typical of bacteria, both Mycoplasmaand L organisms can be considered similar to oneanother and to animal cells. The pliability of the

cells of these organisms is attested to by the verynumerous reports on their pleomorphism and insome respects the resistance of Mycoplasma toosmotic shock (94, 144). The absence of thetypical cell-wall components, a, E-diaminopimelicacid and hexosamines, was already referred to ina previous section of this review. The well-established ineffectiveness of penicillin againstboth Mycoplasma and L-type organisms (159),indeed the use of penicillin to induce formation ofthe latter (14), and the resistance of the organismsto other antibiotics exerting their action againstbacterial cell walls, e.g., erythromycin andcycloserine (100, 160), substantiate the lack of atypical bacterial cell wall. Taubeneck (150)showed further the absence of specific phagereceptors in stable L forms of P. mirabilis.Lysozyme, known to attack the ,3(1-4) glyco-sidic bonds between acetyl muramic acid andacetyl glucosamine of the mucocomplex compos-ing bacterial cell walls (113), is without effect oneither Mycoplasma or L organisms (94, 86).

Evidence of the lipoidal nature of the surface ofMycoplasma was first presented by Tang et al.(149) for M. mycoides and by Smith (145) forthis organism and strains of M. laidlawii. Smithand Rothblat (142) demonstrated the need forsterol in maintenance of the membrane structureby showing the lytic action of digitonin onMycoplasma grown in the presence of sterol. M.laidlawii B when grown without sterol wasunaffected by digitonin, a result confirmed byothers (94). Proteus 18 L was unaffected bydigitonin (142), and L 1 (143) was agglutinatedbut not lysed by digitonin. Other relatively non-specific surface-active agents effective in dis-solution of lipoprotein membranes have beenshown to lyse Mycoplasma. Thus, lecithin,bile salts, long-chain fatty acids, soaps, alcohols,and anionic and cationic detergents have beenshown to disrupt Mycoplasma strains of humanorigin (45, 136, 144), sterol-nonrequiring strains(94), and M. mycoides (101). L organisms arealso susceptible to the action of these non-specific lytic agents (90). Osmotic shock haslittle effect on the viability of Mycoplasma (94,144), although loss of intracellular constituentsundoubtedly occurs (101) from which theorganisms are capable of recovery. Salt-non-requiring L organisms, such as L 1, are notadversely affected by osmotic shock (94); otherL organisms, such as those from Proteus, are

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lysed to a significant degree, presumably due totheir larger size (12). Salt-requiring L organismsare very susceptible to lysis outside a hypertonicenvironment (78). This effect is not strictly anosmotic one; suspension in equimolar con-centrations of glycerol, phosphate, and sucrosewas attendant witb considerable differences inresultant viability. It is possible that the highcation requirement is needed for stabilization ofthe lipid surface as mentioned previously.

Phospholipids also appear to play a part in themaintenance of the membrane structure. Uranylions which attach to the phosphate groups ofphospholipids inhibit the incorporation of sterolinto the membrane, indicating that a portion ofthe membrane, probably the inner transparentlayer described by van Iterson and Ruys (157),may be composed of phospholipid interspersedwith sterol (135). Uranyl ions also have beenshown to protect the organisms against theaction of anionic and cationic detergents (94).Rodwell and Abbot (105) and Rodwell (103)found that glycerol deficiency and fatty aciddeficiency resulted in lysis of M. mycoides after abrief period of growth. This effect of glyceroldeficiency could be negated by instituting auracil deficiency or by the presence of chlor-amphenicol. It was concluded that glycerol andprobably fatty acids were required for synthesisof this structure which was more sensitive toglycerol deficiency than was synthesis of thecytoplasm. The glycerol deficiency was alsoattendant with the appearance of enlarged cellswith shrunken appendages (103). This con-clusion was further verified by the finding thatthe requirement for glycerol was greater underaerobic conditions where a greater rate ofoxidation and hence greater loss of glycerolphosphate for synthesis occurred. Whether theglycerol-containing structure required was thepolyglycerophosphate of Plackett (87) orphospholipid, or both, is not known.Mycoplasma in the presence of sterol possesses

the capacity to bind polyene antibiotics (J. 0.Lampen, personal communication; P. F. Smith,unpublished data). The polyene antibiotics areknown to complex with sterols (50). Growthinhibition is noted with organisms which syn-thesize their own sterol, whereas with Myco-plasma no inhibition occurs unless the antibioticlevel is high enough to cause a deficiency ofexogenous sterol. No effect is noted with sterol-

nonrequiring strains of Mycoplasma, nor is itknown whether these strains bind the polyenes.Nothing is known with respect to polyene bindingby L organisms. Protoplasts of sterol-containingfungi will bind the polyenes (46), but it is un-likely that any sterol-noncontaining L organismsor Mycoplasma will do so.

Pancreatic lipase induces lysis of both Myco-plasma and L forms (94). Clostridium welchii,lecithinase C, and 0 hemolysin do not effect lysisof M. mycoides (101).Only superficial examination of the effect of

proteolytic enzymes on Mycoplasma and Lorganisms has been made. Morowitz et al. (70)stated that protein of M. gallisepticum 5969,lysed by the action of trimethylamine forpreparation of DNA, was degraded to aminoacids by trypsin. Razin and Argaman (94) foundpapain to be ineffective against both Mycoplasmaand L organisms and trypsin to be effectiveagainst Mycoplasma only after subjection toheating at 70 C. Trypsin proved to be ineffectiveagainst M. hominis 07 when either intact cells orcell membranes were exposed to the action ofthis enzyme (Smith and Rothblat, unpublisheddata).

It can be tentatively concluded that the cellenvelopes of both Mycoplasma and L-typeorganisms consist of a lipoprotein, the morpho-logical and molecular structures of which aresimilar. Differences exist as to the nature andamount of lipid found in these structures andprobably account in part for the relative sta-bility of the various types and strains.

NUTRITIONMore effort has been extended to elucidate the

nutritional requirements, particularly of theMycoplasma, than in any other area of thephysiology of Mycoplasma and L-type organisms.There has yet to be devised a completely definedmedium which will support adequate growth toenable the study of the physiology without doubtas to interference from unrequired constituents.Innumerable reports have been publisheddescribing the requirements for supplementationof basal culture media, comprised mainly of aheart infusion, with different mammalian bloodsera, egg yolk, yeast extracts or autolysates,detoxifying agents such as erythrocytes oralbumin, etc. Likewise, many reports of theability of cultured tissue cells, chick embryos, the

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egg chorioallantoic membrane, etc., to sustain thegrowth of Mycoplasma are available in theliterature. It is not the intent of this review todocument these reports, which have beenvaluable, to say the least, in providing a basisupon which to study in a more refined way thenutrition of these organisms. This review willexamine in a comparative fashion the definedchemical and physical factors essential forgrowth.

Chemical Requirements for GrowthDefined media which support good growth of

Proteus L forms have been devised. Medill andO'Kane (68) reported better growth than incrude media in a defined basal medium consistingof glucose, lactate, nicotinamide, potassiumphosphates, Mg+, Na+, Fe++, and Mn+ salts,and 15 amino acids or vitamin-free acid-hy-drolyzed casein to which serum was added.Abrams (1) further refined this medium tocontain the Vitamin Free Casamino Acids,citrate, potassium phosphates, magnesium sul-fate, and nicotinamide. No effort has beenspent to devise a defined medium for morefastidious L organisms such as L 1. The onlysemidefined requirements which have been foundare bovine serum albumin and the acetone-insoluble lipid fraction of egg yolk (20). Un-published findings (P. F. Smith) have shown thata lipoprotein fraction of bovine serum replacesthe serum requirement only when added intact.Panos formulated a partially defined lipid-freemedium for the cultivation of a salt-requiring Lorganism, AED-L, derived from a group A 3-hemolytic streptococcus (personal communica-tion). Not enough different strains have beenexamined to make any conclusion as to thenutritional requirements of L organisms ingeneral. From the meager data available, therequirements appear to vary and, as can bededuced from the following information, areprobably less exacting than Mycoplasma.

Protein requirements. Smith and Morton (139)first reported the separation and characterizationof a protein component of mammalian serumcapable of supporting growth of serum-requiringMycoplasma. This protein, presumed to be oflow molecular weight, was rendered lipid-freewithout significant alteration of its activity, andwas subsequently shown to contain eight dif-ferent species of amino acids, with lysine pre-

dominating and with considerable quantities ofleucine, arginine, and glycine (140). Additionalstudy of this protein factor by Smith et al. (137)resulted in refinement of the fractionationprocedures with the isolation of an electro-phoretically and ultracentrifugally pure proteinwith properties similar to the alpha-1 lipoproteinof serum. It contained esterified cholesterol andphospholipid, possessed an electrophoretic mo-bility of 7.98 X 10lO per see per v per cm, anisoelectric point of 5.2, a sedimentation constantof 3.76 Svedberg units, and a molecular weightof 1.35 x 105. The lipid-extracted protein was in-active in lipid-free media, as described later. Thisprotein could be replaced by larger quantities ofbovine albumin (21, 130, 136) or f-lactoglobulin(136). These proteins may contain traces oflipoprotein which would account for theiractivity. Rodwell (101) independently, by aprocedure similar to that of Smith and Morton(139), isolated a protein from horse serum which,although not so well characterized, appears to besimilar to that of Smith and Morton and satisfiespart of the protein requirement for M. mycoides.This protein, too, contains phospholipid andcholesterol esters. The additional protein require-ment was met with fraction V bovine albuminrendered lipid-free (105).The function of the lipid-free fraction V

albumin for M. mycoides in a semidefined mediumwas considered to be as a carrier for requiredfatty acids, acting as a detoxifier by allowing onlyrestricted release of fatty acids as the organismsutilized them (102, 105). Smith and Boughton(136) studied the possible role of the proteinmoiety of the lipoprotein growth factor. Theability of a protein to support growth of Myco-plasma correlated with its ability to regulate theincorporation of sterol into the cells. All proteinswhich were capable of supporting growth werealso capable of neutralizing the lytic effect ofsurface-active agents. However, this detoxifica-tion property was not the sole explanation for theprotein requirement, since proteins inactive insupporting growth also neutralized the surface-active agents. Although the protein increased theaqueous solubility of sterol, it was not significant.No uptake of protein by the organisms could bedemonstrated based on growth of the Mycoplasmain 2,4-dinitrobenzene-labeled protein. Alterationof the terminal groups of the lipoprotein in aneffort to determine the functional groups on the

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protein required for regulation of sterol in-corporation revealed that destruction of sulf-hydryl groups and periodate oxidation of hy-droxyl groups resulted in diminution or loss ofgrowth-promoting capacity and diminution ofsterol uptake. It was concluded that the functionof the protein growth factor was to regulatesterol uptake. Such a function is considered byRodwell and Abbot (105) to apply to theirprotein isolated from horse serum. Razin andKnight (96) reported the requirement of crystal-line serum albumin for growth of the sterol-nonrequiring 11. laidlawii strains A and B on apartially defined medium. Its function is thoughtto be one of detoxification.Amino acid requirements. 31. hominis 07 was

shown to be capable of suboptimal growth in thepresence of nine amino acids (arginine, asparticacid, cysteine, glutamic acid, glutamine, iso-leucine, methionine, phenylalanine, and tryp-tophan). Isoleucine was not an absolute require-ment, but stimulated growth (125). Rodwell andAbbot (105) found that the amino acid require-ments for Al. mycoides were satisfied withvitamin-free acid-hydrolyzed casein plus atryptic digest of casein together with additionalcystine and tryptophan. 31. laidlawii strains Aand B were found to grow in a medium containingVitamin Free Casamino Acids supplemented withcystine and tryptophan (96). Further analysis ofthe growth requirements of these strains (95)revealed that in the presence of ammoniumsulfate only cystine, isoleucine, glutamine, andasparagine were absolute requirements. Sevenother amino acids were added because they werestimulatory. In the absence of serum, there wasan additional requirement for methionine andthreonine. Apparently, the amino acid require-ments of L 1 are satisfied at least partially byCasamino Acids supplemented with cystine andtryptophan, since the serum-supplemented basalmedium for the Laidlaw strains supported growthof this L organism (96).

Nucleic acid requirements. Smith (125) dem-onstrated a requirement for RNA, DNA,guanine, and hypoxanthine for 31. hominis 07.Lynn (56) further defined the requirements ofthis strain for nucleic acid precursors, findingthat guanine, uracil, cytosine, ribose, anddeoxyribose would replace the nucleic acids andpurines when a solidified medium was employed.In liquid defined medium, a mixture of deoxy-

adenosine, deoxyguanosine, deoxyinosine, deoxy-cytidine, and thymidine provided all the require-ments for nucleic acid precursors including thepentose sugars. In the presence of the ribosides,adenosine, guanosine, inosine, xanthosine, cyt-idine, and uridine, only deoxycytidine andthymidine of the deoxyribosides were required.That these requirements are compatible with thesynthetic capabilities of this organism is dis-cussed in another section of this review. Edwardand Fitzgerald (22) noted that certain strains ofMycoplasma found in the genital tract of cattlerequired mucin for growth and that mucin couldbe replaced with calf thymus DNA. These organ-isms lost this requirement upon repeated sub-culture. M. laidlawii strains A and B wereinitially shown to require RNA and DNA whencultivated on a partially defined medium (96).Partial hydrolysis of RNA to its oligonucleotidesdid not destroy its activity, whereas ribonucleasedid (97). Deoxyribonuclease did not effect thegrowth-promoting ability of DNA, nor didremoval of the purine bases to yield apurinicacid. Thymidine was the only deoxyribosiderequired and could effectively replace DNA. 31.laidlawii B was shown not to have a requirementfor RNA. Interference between deoxyribosidesand thymidine and noninterference of RNAwhen thymidine substituted for DNA indicatedthat DNA must be degraded to be of use to theorganisms. M. mycoides was shown to requireboth RNA and DNA, with the latter being onlypartially replaceable with thymidine. Rodwelland Abbot (105), on the other hand, found allnucleic acid precursor requirements for 31.mycoides to be satisfied by the purine andpyrimidine bases, adenine, guanine, uracil, andthymine. Folinic acid (leucovorin) was found toproduce better growth in the defined medium.Upon re-examination of the requirements fornucleic acid precursors, Razin (92) found DNAto be replaceable with thymidine and folinic acid(leucovorin) for M. laidlawii A. RNA could bereplaced by adenine, hypoxanthine, guanosine,and cytidine. His medium was less well definedthan that of Rodwell and Abbot (105), because itcontained 10% dialyzed human serum. The 2' and3' mononucleotides of guanine and cytosine wereinactive, as were the pyrimidine precursors,ureidosuccinic and orotic acids. RINA was notrequired by L 1, and thymidine could replaceDNA for this organism (96).

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Carbohydrate requirements. The pentose sugarsfound in the nucleic acids are required either inthe form of free sugar or as the nucleosides forM. hominis 07 (56). Although M. laidawii cells donot appear to require free pentose, it is probablethey derive all their pentose requirements fromguanosine and cytidine and their deoxypentoserequirements from the required thymidine (95).That nucleoside interconversions occur wasdemonstrated by Lynn (56). M. mycoidesapparently synthesizes its own pentose sugars,since none are required in a defined medium (105).No other carbohydrate requirement is essential

for the growth of carbohydrate-nonutilizingstrains (125). Avian strains (34) M. mycoides(105) and M. laidlawii (95, 110, 133) possess arequirement for glucose, which serves as thecarbon and energy source. Glucose is replaceablefor M. mycoides by maltose, less effectively bymannose or fructose, but not by galactose (102).M. laidlawii A will utilize maltose in place ofglucose, but mannose, fructose, galactose,sucrose, and lactose are ineffective substitutes(95). Rodwell (102) noted the requirement byM. mycoides for high concentrations of lactate,the L isomer being the more effective. More wasrequired under aerobic than under anaerobicconditions. Various crude supplements wereeffective substitutes for lactate at concentrationslower than lactate. Rodwell thought that lactatefunctioned as more than an energy source, sincelittle lactate is metabolized. Although its realfunction is unknown, it is possible that lactateserves to maintain the osmotic requirements ofthe medium or serves as a precursor for glycerol,which is also a requirement as is discussed below.Razin and Knight (96) reported that lactateinhibited growth of the M. laidlawii. Glucoseappears to satisfy the energy requirements forthe L organisms (90, 1, 96).

Lipid requirements. Edward and Fitzgerald(21), in a study of the serum requirement forgrowth of certain Mycoplasma, found that thelipid extract of serum, as well as the protein, wasnecessary. This lipid extract could be replacedcompletely by an ether extract of egg yolk, inpart by cholesterol, and completely by cholesterolplus the acetone-insoluble lipids of egg yolk. Thecephalin fraction of these acetone-insoluble lipidswas as effective as the total acetone-insolublelipid, but purified cephalins, lecithins, andsphingomyelin were ineffective replacements.

Cholestanol and stigmasterol could substitute forcholesterol, but ergosterol, coprostanol, andcholesterol esters could not. Cholesteryl hydrogenphthalate was a poor substitute for cholesterol.A definite balance between sterol and acetone-insoluble lipid was essential. Smith et al. (137)found that M. hominis 07 when grown in a lipid-free medium required, in addition to the proteinmoiety of the lipoprotein, cholesterol andlecithin. Cholesteryl laurate could substitute forfree cholesterol, probably by virtue of thepresence of a cholesterol esterase (129) in theorganisms making available a supply of freecholesterol. Other cholesterol esters were poorsubstitutes for cholesterol. It is also possible thatlauric acid supplied a part of the fatty acidrequirement of the organism. Cephalin was lessactive than lecithin when added with choles-teryl laurate but more active when added withcholesterol. In this case, too, fatty acid require-ments may explain the results. There was aninterrelationship between the levels of sterol andphospholipid giving optimal growth. Twice asmuch cholesterol on a molar basis was requiredas phospholipid (130). Rodwell (101) demon-strated a requirement for cholesterol by M.mycoides. Cholesterol could be replaced only withcholestanol or lathosterol (104). In addition tocholesterol, the lipid requirements derived fromserum could be replaced with Tween 80 or sodiumoleate. Like the previous two groups (21, 137),a balance between sterol and other lipid wasnecessary or growth inhibition occurred. Rodwellalso noted a requirement for acetate, whichappeared to have only an osmotic effect forunivalent salts; sucrose could replace it. Cho-lesterol and the protein fraction isolated byRodwell from horse serum prevented lysis of theorganisms by oleate.

Further analysis of the sterol requirement(130, 134, 138) of human strains of Mycopasrmashowed that several sterols were capable ofcompletely or partially effective substitution ofcholesterol (,B-sitosterol, stigmasterol, ergosterol,cholestanol). Cholesterol esters of short-chainfatty acids were less effective. Cholesterolprecursors (acetate, squalene, and mevalonicacid), 7-dehydrocholesterol, sitosterol, and stig-masterol acetates, estrone, estradiol, testosterone,cholic acid, cholestan-3-one, o-tocopherol, vita-min K1 (138), zA4 cholesten-3-one (142), epicho-lesterol, epicholestanol, coprostanol, and epi-

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coprostanol (134) were ineffective in support-ing growth. Cholestane, bicholesteryl ether,As cholesten-3-one, epicholesterol, epicholestanol,coprostanol, and epicoprostanol not only failedto substitute for cholesterol but actually in-hibited growth by competing with cholesterol forincorporation into the organisms (134, 142).Critical examination of the configurationalrequirements of the sterol molecule necessary tosupport growth of Mycoplasma (134, 138, 142)lead to the conclusion that the cyclopentanophen-anthrene ring (the saturated ring being moreeffective than unsaturation at C5), an equatorial3-hydroxyl group at C3, a planar ring structure(i.e., A/B rings in the trans configuration), and ahydrocarbon side chain (preferably an eight-carbon saturated chain) were essential for growth.Further elucidation of the necessity for theseconfigurational requirements demonstrated that:the hydrocarbon side chain was essential forincorporation of sterol into the cell membrane,being held there presumably bound to phos-pholipid by means of van der Waal forces (135);the equatorial 3-hydroxyl group was necessary forenzymatic action by sterol esterase (129) andglucosidase (38); and the planar ring structurewas necessary for proper fit into the lipid matrixof the cell membrane and to facilitate a proposedrotational movement in the membrane necessaryfor substrate and end product membrane permea-tion (135). The configurational requirements forsterol are dictated by these probable functions,since sterols capable of supporting growth areincorporated intact (132, 142) and the organismsare incapable of alteration of the sterol moleculeexcept for esterification and glycosidation at the3-hydroxyl group (129, 135). The inactivity ofergosterol noted by Edward and Fitzgerald (21)was probably due to the inhibitory activity ofoxidative degradation products shown to occur inergosterol preparations and to inhibit growth ofMycoplasma (132). Rodwell and Abbot (105)concluded from their studies, which demonstratedinitial growth followed by lysis of M. mycoidesin the absence of cholesterol and the proteinfraction from horse serum, that cholesterol wasneeded for the synthesis of an undetermined cellcomponent necessary for the structural integrityof the cell. Smith and co-workers concluded thatthe sterol is required not only for cell membranestructure, in which it is interspersed with otherlipid in the central lipid layer, but also for a

dynamic function of transport of metabolizablesubstrate and end product across the cell mem-brane (135).

Certain strains of Mycoplasma do not possess arequirement for serum under optimal growthconditions (17, 133). Butler and Knight (6)noted a requirement for serum by M. laidlawiistrains A and B, which was partially replaceableby cholesterol. This effect of cholesterol isexplainable by the fact that these sterol-non-requiring strains are capable of synthesis of theirown nonsaponiflable lipid which consists ofcarotenoids, this synthesis being spared by cho-lesterol (133) which is incorporated in place ofcarotenoids (143) and probably serves the samefunction (135). Certain 3-keto derivatives ofcholesterol were found to replace this cholesterolrequirement noted by Butler and Knight (6),whereas increasingly unsaturated analogues ofthese 3-keto steroids and corticosterone-typecompounds inhibited growth, showing a com-petition with cholesterol. These investigators alsodemonstrated inhibition with these types ofcompounds of sterol-requiring Mycoplasma andL 1. Shifrine et al. (121) demonstrated growthinhibition of M. laidlawii strains A and B by thesteroid inhibitors, benzmalecene and triparanoland Shoenhard and Padgett (116) noted growthinhibition by deoxycorticosterone of an avianMycoptasma. Inhibition by the steroids contain-ing a hydrocarbon side chain can be explained bytheir incorporation into the sites required for thecarotenol or sterol, with their subsequent inter-ference with growth as a result of not being ableto carry out the function of the carotenol orsterol. Inhibition by the inhibitors of sterol syn-thesis is probably the result of their interferencewith synthesis of the triterpenoid precursors ofcarotenoids, the synthetic pathway of whichbears a great similarity to that of cholesterol (90).Inhibition by the corticosteroids is explainablefrom the work of Lester et al. (54), who found thedeoxycorticosterone inhibited uptake of sugars,amino acids, and rubidium ions by Neurosporaand that this inhibition could be relieved bycholesterol. The effect of corticosteroids on Myco-plasma adds to the evidence that sterol and caro-tenol may be involved in permeation mechanisms.The function of the phospholipid requirements

for growth noted earlier (21, 137, 138) was furtherexamined by Smith (130) and Smith and Bough-ton (136). It was postulated that phospholipid

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served as a solubilizing agent for water-insolublelipids or was incorporated into the cell as partof the phospholipid of the organism where itmight serve in the many postulated functions ofthese compounds (130). Examination of thespecificity of this requirement (136) demon-strated that any surface-active agent, be it phos-pholipid, bile salt, or salt of long-chain fattyacid, promoted satisfactory growth. Althoughsome incorporation of oleate-1 -C'4 occurred,lecithin was not hydrolyzed by the organismsand so could not serve as a source of fatty acid.Some compounds which replaced the phospho-lipid contained no fatty acid (e.g., bile salts). Theonly correlation of some property of the activecompounds with ability to support growth wasobtained with the soubilization of cholesterol inaqueous medium. It was concluded that the so-called phospholipid requirements fulfilled thefunction of aqueous solubilization of sterol, mak-ing it more available for incorporation into thecell membrane. An additional function may be toalter the surface structure of the cells to allowentrance of the cholesterol into the inner lipidlayer of the cell membrane (135).The studies of Smith and collaborators on

lipid requirements for growth of Mycoplasma didnot employ culture media completely devoid offatty acids. The incorporation of oleate (136) in-dicated that exogenous fatty acids are utilized.Acetate was shown to stimulate growth of M.hominis 07, and was considered to function as asparker for fatty acid oxidation by this organism(138). Working with a defined medium, Rodwelland Abbot (105) demonstrated a definite re-quirement by Ml. mycoides for palmitate orstearate and oleate. Laurate was a poor substi-tute, but myristate almost completely satisfiedthe requirement for a saturated fatty acid. Lino-leate and linolenate were less effective as thesource of unsaturated fatty acid than was oleate,by virtue of their greater toxicity. A C 17 fattyacid, margaric, effectively substituted for pal-mitic or stearic. Fatty acid insufficiency led topleomorphism and branching of the organismsand eventually to lysis, indicating that the fattyacids were required for synthesis of some surfacestructure, probably phospholipids and glycerides.

Additional requirements for precursors oflipids in Mycoplasma have been shown to includecholine and m-inositol (96, 125) and glycerol(102, 103). Glycerol, as mentioned previously, is

the precursor of the polyglycerophosphate de-scribed by Plackett (87) and also of phosphatidesand glycerides (102). Glycerol deficiency leadsto unbalanced growth and lysis, indicating itspresence in the cell membrane.

Mlycoplasma strains requiring serum for growthpossess a requirement for nonsaponifiable lipidand for saturated and unsaturated fatty acidswhich they are unable to synthesize. The othercomponents of the serum, phospholipid and pro-tein, are necessary only to permit incorporationof the required lipids into the cell: the proteinfor regulation of sterol and possibly fatty aciduptake and the phospholipid for solubilization oflipid in aqueous media. Protein also acts second-arily in reducing the toxicity of some lipids.Those M31ycoplasma strains having no serum re-quirement, and also L organisms, both of whichappear capable of de novo lipid synthesis, possessa requirement for protein under certain culturalconditions for purposes of detoxification only.These latter two groups of organisms are similarto the lipid-requiring Mycoplasma in that exoge-nous lipid is incorporated, thereby sparing de novolipid synthesis.

Vitamin requirements. L organisms derivedfrom Proteus appear to possess a requirement fornicotinamide (1, 68). 11. laidlawii strains A andB require nicotinic acid, riboflavine, folinic acid,pyridoxine, and thiamine. Nicotinamide, nico-tinamide adenine dinucleotide (NAD), and nico-tinamide adenine dinucleotide phosphate (NADP)are less effective than nicotinic acid, but flavinadenine dinucleotide (FAD), thiamine pyrophos-phate, and pyridoxal or pyridoxal phosphate couldsubstitute for the parent vitamins. There was norequirement for cobalamin, biotin, thioctic acid,or putrescine (95, 96). AM. mycoides requirespantothenate, riboflavine, pyridoxal, nicotin-amide, thiamine, and probably biotin and a-lipoic acid (105). Both the Laidlaw strains andM11. mycoides require inositol and choline and,under conditions requiring extensive synthesis ofnucleic acid precursors, also leucovorin. Humanstrains of Mycoplasma appear to require choline,inositol, biotin, folic acid, pantothenate, pyri-doxine, and thiamine (125). Too little is knownto compare accurately the vitamin requirementsof the different types of organisms, but it wouldappear that the requirements are similar.

Inorganic requirements. Only one critical exam-ination of the inorganic requirements of either

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type of organism has been made. Razin andCohen (95), working with M. laidlawii A, foundrequirements for sodium and potassium chlorides,magnesium sulfate and phosphate, and ferrous,manganous, cupric, and molybdate ions. Rodwelland Abbot (105) obtained growth of M. mycoidesin the presence of added sodium and potassiumphosphates and magnesium, manganese, andferrous sulfates; Smith (125) obtained growth ofa human strain of Mllycoplasma with sodium,potassium, and calcium chlorides, sodium phos-phate, magnesium sulfate, ferric nitrate, and theash of peptone (Difco). Cadmium, cobalt, andzinc ions were poor substitutes for this peptoneash, but stannous ions almost completely replacedit. Abrams (1) found that addition of potassiumchloride and magnesium sulfate to his CasaminoAcids medium supplied the necessary inorganicrequirements for a Proteus L form. In general,the inorganic requirements do not differ fromthose of any other free-living biological entity.

Physical Requirements for Growth

Temperature. Numerous reports are scatteredthroughout the literature relating to the optimaltemperature for growth of Mycoplasma in particu-lar. Since cataloging the information regardingthese two types of organisms is not the purpose ofthe review, only a few selective references arecovered. Early workers first noted that the patho-genic strains of Mycoplasma grew best at tempera-tures near 37 C (5, 74) and that M. laidlawiistrains (49) grew at 22 C but best at 30 C. Ed-ward (17), in his analysis of the various biologicalproperties of various strains of Mycoplasma,confirmed the generalizations made from theseearlier findings. Gill (34) found the optimaltemperature for growth of an avian strain to be38 C. This slightly higher optimal temperaturefor avian strains could be accounted for by thehigher body temperature of fowl, the naturalhabitat of these strains. Ford (28) noted growthof the so-called T strains of Mycoplasma from thehuman genital tract at 30 to 36 C, 36 C being op-timal. Lower temperatures, although not permit-ting growth, are not deleterious to the organisms(144), but loss of viability occurs rapidly andcompletely between 45 and 50 C. Thus, the half-life of Mlycoplasma at 50 C has been determinedto be less than 2 min (144). No studies on theoptimal temperature for growth of various L or-ganisms has been reported. It would appear that

the optimal temperature of an L organism is ithesame as for its parent bacterium. L 1 undergoeslysis at 50 C, as does Mycoplasma (94).

Effects of pH. As with temperature, numerousreports exist regarding the optimal pH requiredfor growth. Early investigators noted thatMycoplasma required a pH alkaline to 7, prefer-ably between 7.5 and 8.0, to multiply (5, 49, 74).L-type organisms also require an alkaline pH(14, 117). Edward, in his study of the compara-tive biological properties of Mycoplasma and Lorganisms, noted that some strains of both typesof organisms can grow over a relatively widerange of pH, i.e., 6.8 to 9.2. Of interest is thefinding that Ml. mycoides and L 1 grew at thepH acid to 7. Ford (28) found that certain humangenital strains also grew at pH 6.8, and Shepard(personal communication) has found some of thesestrains to grow optimally at a pH around 6. Thus,there appears to be some variation as to optimalpH for growth by both Mycoplasma and L organ-isms, but the majority of strains studied requirea pH between 7.5 and 8.0. In the case of definedmedia, the optimal pH appears to be about 7.8(1, 95, 105, 125). Suspension of Mycoplasma andL forms in media with pH less than 7.0 andgreater than 9.0 generally results in loss of viabil-ity and lysis (94, 144).

Osmotic effects. The requirement for a mediumwith a high concentration of salt by the salt-requiring L organisms was mentioned above.These organisms will grow in a range of sodiumchloride concentrations of 0.25 to 1.1 M (117).Other salts can substitute for sodium chloride,e.g., the chlorides of potassium, ammonium, cal-cium, and magnesium, and dibasic sodium phos-phate. Although concentrations of univalentsalts lower than 0.25 M are ineffective, divalentcations are effective at 0.18 M and trivalent at0.14 M. Certain salts are ineffective by virtue oftheir toxicity, e.g., lithium chloride (13). Theseresults would imply, as stated by Panos andBarkulis (78), that the effect is not merely a strictosmotic one but may be a combination of osmoticeffects and stabilization of the membrane bycation binding onto phospholipid residues (143),as previously described. Panos and Barkulis (78)noted that 0.25 to 0.88 M sucrose allowed con-sistent survival of the salt-requiring L organisms.Sucrose in this instance could fulfill the osmoticrequirement, allowing the salts in the culturemedium to bind onto phospholipid and thereby

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reducing the total salt requirement. Salt-requir-ing L forms can be adapted to grow in the pres-ence of lower salt concentrations if serum ispresent. Salt-nonrequiring L organisms appearto be able to multiply in an environment withan osmotic pressure found in usual bacteriologi-cal culture media, i.e., 5 to 10 atm. They appearto be able to adapt to variable osmotic conditions,since the Proteus L organisms multiply in bothisotonic (1) and hypertonic media (65). Rodwell(102) reported that M. mycoides requires a me-dium with an osmotic pressure of 12 atm, withacetate, various inorganic salts, or sucrose serv-ing equally well as the solutes. Leach (126), inthe only detailed study of the osmotic require-ments of either type of organism, found theoptimal osmotic pressure of various strains ofMycoplasma to lie between 6.8 and 14.0 atm.Some strains such as M. gallisepticum grew onlyin a narrow range, i.e., 6.8 to 14, whereas strainsof M. laidlawii grew in a wide range, i.e., 2.7 to27 atm. An osmotic pressure of 41 atm greatlyretarded the growth of M. mycoides. Sodiumchloride, sodium sulfate, potassium chloride,sucrose, and a balanced salt solution servedequally well as solutes. There was little adapta-tion by Mycoplasma. for hypo- and hypertonicenvironments.Mycoplasma cells are in general relatively re-

sistant to osmotic shock, as shown by Smith andSasaki (144) and confirmed by Razin and Arga-man (94), presumably due to their small size andplasticity. The greater susceptibility of some Lorganisms can be explained by their larger sizeand, in the case of salt-requiring organisms, bythe different nature of their enveloping mem-branes. L 1 is as resistant to osmotic shock as isMycoplasma (94).

Gaseous Requirements for GrowthVirtually every report on the isolation of a

Mycoplasma or the production of an L-typeorganism relates whether the particular organismgrows aerobically, anaerobically, or requiresadded carbon dioxide. Only a few selected reportsare mentioned. Bridr6 and Donatien (5) foundaerobic and anaerobic conditions equally favor-able for the organism causing agalactia. Laidlawand Elford (49) noted the same effect with thesterol-nonrequiring strains from sewage. Warren(161) found that several rodent strains and theorganisms of agalactia and bovine pleuropneu-

monia grew anaerobically only in the presence ofadded carbon dioxide. L 1 grew better aerobicallythan anaerobically (37). Mfycoplasma strains iso-lated from the human genital tract grow aerobi-cally, anaerobically, or in the presence of addedcarbon dioxide (16, 17, 71, 76). Newing and Mac-Leod (73), as well as Rodwell (107), obtained agreater yield of Ml. mycoides by aeration of cul-tures. Human oral strains appear to be anaerobicin nature (122). Pathogenic strains of avian andhuman origin apparently prefer added carbon di-oxide, at least for initial isolation (27, 28). Growthof established cultures of avian strains is not stim-ulated by carbon dioxide (26). Gaseous require-ments for the L organisms appear to mimic thegaseous requirements of the parent bacteria (14).The true nature of the gaseous requirements is nodoubt clouded by the use of many different crudeculture media. Evidence that the medium con-stituents exert an effect on the gaseous require-ments is the report by Morton et al. (71) thatadded carbon dioxide improved growth only insuboptimal media, the finding by Rodwell (102)that glucose is not attacked anaerobically byM. mycoides, and the finding of Gill (34) thatanaerobic growth of M. gallisepticum occurredonly if glucose and pyruvate were added.

ENZYMATIC ACTIVITIESEnergy-Yielding Metabolism

Mycoplasma strains can be divided into twogroups based upon their capacity to ferment car-bohydrates. Those which ferment carbohydratesinclude M. mycoides (4, 107, 149, 161), most ofthe avian strains (3, 36, 66, 151), goat strains(5, 9, 19), rat strains (161), sterol-nonrequiringstrains (30, 49, 57, 82, 151), and one type of hu-man strain (165). Strains which fail to utilizecarbohydrates include those of human origin(23, 30, 53, 57, 131, 151), some of the mousestrains (15), and the agent of ornithosis (66). Thefew L organisms studied are capable of carbo-hydrate utilization, e.g., salt-requiring strepto-coccal L forms (77), L 1 (161), and Proteus Lforms (40, 42, 65).

Carbohydrate-utilizing organisms. The Myco-plasma and L organisms examined usually fer-ment glucose, fructose, mannose, maltose, dex-trin, starch, and glycogen with the production ofacid, except for Proteus L forms which in manyinstances also produce gas (3, 9, 19, 36, 42, 66,77, 107, 151, 161, 168). Sucrose is rarely fer-

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mented as is galactose, and is fermented princi-pally by avian strains (3, 36, 149, 151, 168). Noneof the strains active against hexoses has beenshown to ferment lactose, pentoses, or polyols.

Glucose degradation by M. mycoides was ex-amined by Rodwell and Rodwell (102, 106, 107,109) following the early work of Holmes andPirie (39). Direct demonstration of hexokinaseand aldolase was made, and glucose-6-phosphate,fructose-6-phosphate, and fructose-1, 6-diphos-phate were oxidized. No activity was notedagainst 6-phosphogluconate. Maltose apparentlyis cleaved to glucose prior to phosphorylation.Whether this reaction is accomplished by a hydro-lytic reaction or a transglycosidation is notknown. Mannose and fructose enter the pathwayby way of their phosphorylated intermediates.Glycerol enters the scheme by initial phospho-rylation to L-a-glycerophosphate followed byoxidation to L-glyceraldehyde-3-phosphate. Thislatter reaction is irreversible, accounting for thefact that glycerol, which also is used to syn-thesize the polyglycerophosphate component ofthe membrane, is a required nutritional factor.lodoacetate inhibits glucose utilization, suggest-ing that an NAD triosephosphate dehydrogenaseis present. However, iodoacetate would inhibitany sulfhydryl-containing enzyme. Fluoride isalso inhibitory, indicating the presence of anenolase. This inhibition may also be explainedby the possibility that fluoride inhibits the bio-synthesis of (108) some factor required for theterminal respiratory system like flavin nucleotidein the pyruvic oxidase system. The organisms arecapable of reduction of pyruvate to lactate, thedehydrogenase being NAD-requiring. It wouldappear that glucose degradation is accomplishedby the Embden-Meyerhof pathway. Anaerobi-cally, pyruvate undergoes the dismutation reac-tion and aerobically is oxidized by the pyruvicoxidase system which requires inorganic phos-phate, coenzyme A, a-lipoic acid, NAD, andcocarboxylase. The inability of intact or brokencells of M. mycoides to oxidize di- and tricar-boxylic acid cycle intermediates led to the con-clusion that these cycles are nonexistent in thisorganism. An interesting phenomenon noted bythe Rodwells was the inability of intact cells ofM. mycoides to degrade glucose anaerobically.They explained their results by postulating thatpyruvate is removed preferentially by the dis-mutation reaction, and the organism possesses

no other anaerobic mechanism for the reoxida-tion of reduced NAD (NADH) formed by oxida-tion of triose phosphate. Depletion of pyruvatetherefore results in accumulation of NADH. Thishypothesis received support by their finding thatadded pyruvate resulted in a synergistic effectand that exogenously added yeast alcohol dehy-drogenase and acetaldehyde increased anaerobicbreakdown of glucose by disrupted cells. Thepossibility of a permeation mechanism requiringenergy derived from some oxidation, or of lossof some glucose activation system, should not beoverlooked.

Other strains capable of carbohydrate utiliza-tion appear to possess the same degradative path-way as M. mycoides. Tourtellotte and Jacobs(151) found that the end products of glucosemetabolism in growing organisms of severalanimal, avian, and sterol-nonrequiring strains tobe predominately lactate, together with pyru-vate, acetate, and acetylmethylcarbinol. Theminor end products could have been derivedfrom the crude growth medium. Resting cellswere shown to oxidize glucose and pyruvatequantitatively to acetate and CO2. Lactate wasalso rapidly oxidized but acetate, a-ketogluta-rate, succinate, malate, and oxalacetate were veryslowly oxidized. Citrate, acetaldehyde, andethanol were not oxidized. Anaerobically, pyru-vate was dismutated to lactate, acetate, andcarbon dioxide. Neimark and Pickett (72) wereable to class seven strains of Mycoplasma ascarrying out either homolactic or heterolacticfermentations during growth. The work of Gill(34) with an avian strain supports the existenceof the Embden-Meyerhof cycle in the carbo-hydrate-fermenting strains.Rodwell and Rodwell (108) concluded that

pyruvate breakdown by 31. mycoides proceededthrough the intermediate formation of acetyl co-enzyme A and acetyl phosphate, although theycould not demonstrate acetyl phosphate forma-tion. Castrejon-Diez et al. (8) demonstrated bythe hydroxamic acid reaction a Mg -i- or Mn §-activated acetokinase in several fermentativestrains of Mycoplasma. This enzyme was specificfor acetate in M. laidlawii, 31. gallisepticum, M.agalactiae, and M. spumans. i1. gallinarium wascapable of phosphorylating several short-chainfatty acids. The hydroxamic acid method wouldnot differentiate between acyl phosphate andacyl coenzyme. Thus, the reaction described by

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this group (8) could just as well be an acetocoen-zyme A kinase. Conclusive evidence for an aceto-kinase could be obtained if phosphotransacetylasewere shown to be present. Acetokinase appearsto be limited to organisms containing phospho-transacetylase (32).Early workers (39, 82, 83, 84, 161) noted that

methylene blue could serve as a hydrogen ac-ceptor in the oxidation of glucose, lactate, andother substrates by various animal strains ofMycoplasma and the L 1 organism. These oxida-tions were stimulated by the addition of catalase.Peroxides were inhibitory in low concentrations.Although Pirie and Holmes (84) found reductionof methylene blue by the organism of agalactiato be cyanide-sensitive, further work by Pirie(82, 83) indicated the absence of a cytochromesystem. Kandler and co-workers (40, 42) andWeibull and Hammarberg (166) conclusivelydemonstrated the absence of catalase or peroxi-dases in many fermentative and nonfermentativestrains of Mycoplasma. The presence of catalasein ll. mycoides (107) may represent a straindifference. Rodwell and Rodwell (106, 107, 108),working with M. mycoides, found that methyleneblue, ferricyanide, or NAD would replace oxygenas electron acceptor in oxidation reactions, andin a study of pyruvate oxidation they noted that2,4-dinitrophenol and sodium azide were non-inhibitory. They concluded that terminal oxida-tions by this organism were fiavoprotein-cata-lyzed and that no cytochrome system existed.In further support of this conclusion was theirfinding that catalase was required for glyceroloxidation, suggesting the same initial steps asoccur in Streptococcus faecalis. VanDemark et al.(156) made a thorough examination of an avianstrain of Mycoplasma to determine the presenceof enzymes involved in terminal respiration.These workers noted the absence of catalase andcytochromes and the presence of NAD-linkedlactic dehydrogenase, NADH-menadione reduc-tase, NADH-ferricyanide reductase, and aNADH oxidase, but no cytochrome oxidase orNADH peroxidase. NADH oxidase activity re-sulted in the accumulation of hydrogen peroxide.Oxidation of lactate was sensitive to atabrine,but not to cyanide, azide, or carbon monoxide.The fact that no cytochromes were found andthat rate of oxygen uptake increased with in-crease of oxygen level to a maximal rate at 100%

oxygen led to the conclusion that the respiratorysystem of this avian strain was flavin-terminated.The pathways for carbohydrate breakdown by

L organisms are less well defined. Superficialexamination in many instances has led to theconclusion that the L organisms possess the samepathways as the parent. Proteus L forms carryout glycolysis (42, 65). The L organism has lessoxidative activity than does the parent bac-terium. This greater degree of glycolysis by the Lorganisms than by the parent may be a reflectionof the degree of disruption of the L organisms(164). Kandler and co-workers (40, 42) found noqualitative difference in the oxidative activitiesof the Proteus L forms and their parent bacteria.Respiration was inhibited by cyanide, 2,4-dini-trophenol, azide, arsenate, arsenite, iodoacetate,and fluoride. These inhibitions would suggestnot only the Embden-Meyerhof pathway but alsoa cytochrome-dependent terminal respiratorypathway. Catalase activity of various Proteus Lforms was shown to be equivalent to the activityof parent organisms (166, 167).Only one detailed study of salt-requiring L

organisms has been carried out. Panos (77) foundthat a group A fl-hemolytic streptococcus andits L form carry out a homolactic fermentationof glucose, glucosamine, and N-acetylglucosa-mine. Hexoses are utilized at a greater rate thanare pentoses, phosphorylated hexoses, and twocarbon compounds. No activity was detectedagainst glyoxylate and only with the L formagainst acetate. The L form displayed greateractivity against galactose, rhamnose, ribose, glu-cose-6-phosphate, fructose-1 ,6-diphosphate, glyc-eraldehyde, and dihydroxycetone, but loweractivity against glucose, glucosamine, N-acetyl-glucosamine, mannose, and fructose than did theparent bacterium. The L form exhibited an adap-tive response to glucose, whereas the parent didnot. The lower efficiency in utilization of acetylhexosamines probably was due to accumulationof acetate which the L form possibly could nottransport out of the cell. The lack of selectivityshown by the increased activity in media con-taining high levels of glucose suggested loss of arepression mechanism found in the parent bac-terium which acquired greater degradativeactivity against the hexosamines than againstglucose in media containing low levels of glucose.These findings led to the conclusion that the Lorganisms possess inherent metabolic activities

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different from the parent. Assessment of enzy-

matic activity of salt-requiring L organisms musttake into account the effects of high concentra-tion of sodium chloride (57), which may explainsome of the above differences.Although catalase and a cytochrome-containing

terminal respiratory pathway are definitely pres-

ent in some L forms but absent in all fermenta-tive strains of .Mlycoplasma thus far examined,the variety of organisms studied is too limited toconclude that a well-defined difference exists.The oxidative pathway for a streptococcal Lform appears closely related to the Mycoplasma.

Nonfermentative organisms. This group includesprimarily only strains of Mycoplasma, althoughsome diphtheroid L forms appear to be limitedin their capacity to oxidize glucose (141). Somer-son and Morton (146) noted that nonfermentativeMycoplasma strains possess the capacity to reducevarious tetrazolium compounds when grown

under anaerobic conditions. Tetrazolium reduc-tion was made use of by Lecce and Morton (53)in a study of the oxidative activity of theseorganisms. Activity was noted against lactate,fructose, and ribose, and slight activity withglutamate, a-ketoglutarate, and formate, butnone with Embden-Meyerhof intermediates,tricarboxylic acid cycle intermediates, glycerol,amino acids, and pentoses. By far the greatestactivity was displayed when monohydric short-chain alcohols served as substrates. Lactate oxi-dation was not NAD-dependent, and ethanoloxidation was cyanide-sensitive in contrast tothe fermentative strains. Although 2,4-dinitro-phenol is not growth inhibitory even at saturationconcentrations (Smith, unpublished data), a cyto-chrome pathway could exist since bacterial sys-

tems are relatively insensitive to this inhibitor(123). Lynn (57) found that these strains were

capable of oxidizing short-chain fatty acids, suchas butyrate, caprylate, and valerate. Whetherthese represent the major energy source for thenonfermentative Mycoplasma is not known. Ifsterol and carotenol serve as carrier molecules inpermeability processes as described below, withacetate (the metabolic end product of glucosedegradation in the fermentative strains) beingthe only volatile fatty acid found in sterol andcarotenol esters, the finding of butyric as themajor fatty acid in the cholesterol ester of non-

fermentative strains suggests a butyric acid typefermentation in these organisms. Unpublished

data (Smith) demonstrating some oxygen uptakein the presence of glyoxylate, malate, and succi-nate by iMl. hominis 07, together with the knownoxygen uptake with short-chain fatty acids,could suggest the presence of a glyoxylate path-way or a portion of the tricarboxylic acid cycle.

Nitrogen Mletabolism

Proteolysis as determined by liquefaction ofinspissated serum or gelatin has been reportedfor M. mycoides (30, 102), certain goat strains(19), and sterol-nonrequiring strains (30). Slightammonia production appears characteristic ofmany strains (30). Nitrate reduction and indoleformation have never been reported. Hydrogensulfide production has been noted only in certainanimal and sterol-nonrequiring strains (30, 40).A Proteus L form was shown to form hydrogensulfide, reduce nitrate, and form indole like theparent bacterium but to differ from the latter bybeing unable to decompose urea or liquify gelatin(40). Aside from these general biological activi-ties, the metabolism of nitrogen-containing com-pounds has been examined only for some humanstrains of Mycoplasma and, to some extent, fora few other strains of diverse origin.Smith (124, 131) demonstrated, by means of

measurement of loss of substrate or ammoniaproduction, that human strains of M7Iycoplasmautilize arginine, glutamine, glutamic acid, andaspartic acid very readily and exhibit slowaerobic utilization of histidine, leucine, and threo-nine and slow anaerobic utilization of tyrosineand tryptophan among the 18 amino acidsstudied. Glutamine (126) was shown to undergohydrolytic deamidation at alkaline pH but toundergo phosphorolysis at acid pH, formingglutamic acid and adenosine triphosphate (ATP)and ammonia. The latter reaction is reversible,but the equilibrium favors ATP formation, indi-cating that the glutamine phosphorolysis may bea source of energy for the nonfermentativeorganisms. Glutamic acid is not further degraded,but undergoes cyclization to form Al-pyrroline-5-COOH which is further reduced by the actionof reduced NADP to form proline (127, 131).Arginine undergoes quantitative hydrolytie des-imidation to citrulline (124, 131). The citrullineformed then undergoes phosphorolysis to yieldornithine and carbamyl phosphate, which in thepresence of adenosine diphosphate (ADP) iscleaved to yield ammonia, C02, and ATP. The

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overall reaction of citrulline degradation is re-versible, the equilibrium favoring synthesis ofcitrulline. Although ornithine appears to be a pre-cursor of proline similar to glutamic acid, the rateof ornithine removal is too slow to negate itsinhibitory activity on citrulline degradation.Thus, this pathway probably represents a syn-thetic one in the organisms. Schimke and Barile(115), on the other hand, concluded that argininebreakdown to ornithine represents a majorenergy-yielding mechanism based on stimulationof growth by arginine and the rapid conversion ofarginine to ornithine by growing organisms. How-ever, they did not present any experimental dataon the effect of varying levels of ornithine onornithine transcarbamylase and carbamyl phos-phokinase. Aspartic acid appears to be deam-inated, and may possibly be utilized for thesynthesis of homoserine and threonine (131). Notransamination reactions were detectable in hu-man strains of Mycoplasma except for some ala-nine formation from pyruvate with glutamineserving as the amino donor (124, 131).Rodwell (102) also failed to find transaminase

activity in M. mycoides. Only serine and threo-nine of several amino acids were degraded, andthese by dehydrases to yield the correspondinga-ketoacids which were oxidized by the pyruvicoxidase system. Razin and Cohen (95) failed todemonstrate any activity toward amino acids byM. laidlawii A. Gill (34) found a glutamic-aspar-tic transaminase system in an avian strain whichfavored the formation of aspartic acid. Powelson(91) noted a reduction of several amino acids intissue cultures contaminated with sheep strainsof Mycoplasma. Arginine and glutamine wereamong those amino acids principally depleted.Lynn (56), using Al. hominis 07, was the first

to demonstrate enzymatic activity by Myco-plasma toward nucleic acid constituents, al-though preliminary studies (N. L. Somerson,unpublished data) indicated a conversion of ade-nine to hypoxanthine and cytidine to uridine bya human strain. Lynn demonstrated the presenceof a nucleoside phosphorylase, which was capableof phosphorolytic cleavage or arsenolysis ofthymidine, deoxyinosine, deoxyadenosine, anddeoxyguanosine, but not deoxycytidine, with theliberation of the free base and phosphorylateddeoxypentose. The nucleoside phosphorylase wascapable of transfer of the deoxypentose moietyfrom thymidine to hypoxanthine and xanthinebut not to adenine to form the corresponding

purine deoxyribosides. Thymidine could not actas a deoxypentose acceptor with purine deoxy-ribosides as deoxypentose donors.

Plackett (85) demonstrated the presence of anenzyme similar to phosphodiesterase in M. my-coides. This enzyme liberated by freeze-thawdisruption of the cells depolymerized RNA withthe liberation of adenosine-5'-phosphate. Rod-well and Rodwell (109) found a fluoride-resistantadenosine triphosphatase in this same organism.The results of Razin and Knight (97) on theeffects of RNA and DNA on growth of M. laid-lawii A indicate that this organism is capable ofdegrading both types of nucleic acids.

Salt-requiring L organisms from group A strep-tococci contain acid and alkaline phosphatasesassociated with the intact cells (58). Alkalinephosphatase activity was lower, and acid phos-phatase possessed a different pH optimum in theL organism than in the parent bacterium. Thedifference in pH optimum may reflect loss of cellwall, since acid phosphatase of protoplasts wassimilar to that of the L organisms.

Lipid MetabolismIt has already been mentioned that certain

nonfermentative strains of Mycoplasma possessthe capacity to oxidize short-chain fatty acids.On the other hand, they appear unable to degradephospholipids, e.g., lecithin (130), or to oxidizecholesterol. The fact that the sterol supplied inthe growth medium is identical to the sterolfound in the cells (110, 132) indicates that theorganisms cannot carry out sterol transforma-tions.Mycoplasma (129) and some L organisms (141)

have been shown to contain a sterol esterase (129)located primarily in the cell membrane. Thisesterase is capable of hydrolytic or thiolyticcleavage of sterol esters and, in the presence ofcoenzyme A and ATP, of synthesis of the estersfrom free cholesterol and fatty acids. In thecourse of thiolytic cleavage, there appears to beproduced an acyl coenzyme A reaction product.The enzyme possesses little specificity with regardto the fatty acid moiety, but does possess speci-ficity toward the sterol moiety of the ester.Esters of cholestanols are more readily hydro-lyzed than are those of the A5 cholestanols, indi-cating that unsaturation in the B ring impedesthe esterase (134). With sterols containing a cisfused A/B ring, i.e., the coprostanols, esters of

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the axial 3-hydroxyl radical are more easilyhydrolyzed than are those of the equatorial3-hydroxyl, whereas no difference is noted inesterase action due to the configuration of the3-hydroxyl group in the sterols with a trans fusedA/B ring, i.e., the cholestanols. Activity againstesters of sterols with longer side chains thanthose of the cholestane and coprostane series isless, e.g., sitosterols and stigmasterol.A lipase capable of hydrolyzing triglycerides

and natural fats has been detected in Mycoplasma(129). It appears to be distinct from the sterolesterase.A membrane-associated glucosidase has been

detected in Mfycoplasma strains which are capableof carbohydrate utilization and which containsterol or carotenol glucosides. This enzyme is notfound in nonfermentative strains and appearsto possess a specificity, i.e., a or f3, correlativewith the configuration of the sterol or carotenolglucoside found in a given strain (38).Smith (135) postulated that the sterol esterase

and the glucosidase function in the organisms asmediators of substrate and end-product trans -port across the cell membrane. The sterol orcarotenol would act as carriers. Thus, externalglucose could be coupled with cholesterol orcarotenol, the hydroxyl groups of which face theoutside of the cell, by the action of the glucosi-dase. The increase in polarity of the sterol orcarotenol molecule as a result of glucosidationwould give reason for rotational or translationalmovements in the membrane, causing rearrange-ment of the molecule and making the glucosylgroup available to the inside of the cell. In thisorientation, the glucosidase could cleave theglucosyl group from the glucoside. Such a reac-tion would be favored, for the enzymatic mech-anism for glucose degradation is present in thecell and would force the glucosidase reaction inthe direction of cleavage. The free alcohol wouldremain in this orientation, being prepared toaccept the end product of glucose metabolism,acetate. After degradation of glucose to acetateinside the cell, the acetate would be coupled tothe sterol or carotenol by the action of theesterase in the membrane. Esterification resultsin a decrease in polarity of the molecule, givingreason for movement in the membrane, this timemaking the acetyl group available to the outsideof the cell. Again, the esterase could catalyzecleavage of the ester with the production of the

free alcohol and the liberation of acetate. Thefree sterol or carotenol would be in the properorientation to participate in another cycle oftransport. The dihydroxy carotenol by virtue ofits having a hydroxyl group at either end of themolecule could achieve transport in and out ofthe cell in one movement. Although this mech-anism is outlined for fermentative strains, thereis no reason to believe that fatty acids, the pre-sumed substrate for the nonfermentative strains,could not be transported in a similar fashion.The analogy could be extended to any compoundcapable of reaction with a hydroxyl group. Thispermeability mechanism necessitates presump-tion of like enzymes in both inner and outer pro-tein layers of the cell membrane. This functionfor sterol and carotenol does not detract fromthe structural function of these compounds inthe cell membrane. Although theoretical, thismechanism of function explains the necessity fora sterol of highly specific molecular configura-tion, the presence in the membranes of free,esterified, and glucosidyl derivatives of steroland carotenol, and the existence, specificity, andcellular location of the esterase and glucosidase.No data are available on the lipid metabolism

of L organisms. Although sterol and carotenolare not integral parts of the membranes of Lorganisms thus far studied, other lipids contain-ing functional groups with properties similar tohydroxyl groups could exist. There is no reasonto believe that other lipids could not replace thesterol in those Mycoplasma strains requiring it,provided that these compounds possess the appro-priate molecular configuration to make a properfit into the membrane, that they can be incor-porated from an exogenous source, and that theycan function in the required fashion.

BiosynthesisThe biosynthetic capabilities of Mycoplasma

and L organisms, as assessed from results ofstudies on nutritional requirements, are variabledepending upon the strain examined. Thus,Proteus L organisms are capable of synthesizingmost of their cellular constituents from a supplyof amino acids and glucose, whereas most otherL organisms studied and the Mycoplasma arelimited in their ability to synthesize the funda-mental chemical units comprising the complexmolecules but can synthesize the complex mole-cules themselves.

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Gill (34) isolated two peptides from culturesupernatant fluids of an avian strain. The-e pep-tides contained alanine, glutamic acid, glycine,and lysine, and may represent precursors of cell-wall peptide which the organisms are attemptingbut are' unable to synthesize. Tourtellotte andMorowitz (153) demonstrated amino acid acti-vation and incorporation of C'4-labeled phenyla-lanine, valine, leucine, and isoleucine into proteinof M. laidlawii A. Their results were suggestiveof separate enzymes for each amino acid.The ability to synthesize certain amino acids

has been demonstrated. The inability to syn-thesize certain other amino acids can be corre-lated with the growth requirement for these com-pounds. Glutamic acid can be produced bydeamidation of glutamine, and citrulline is pro-duced by desimidation of arginine (124); orni-thine can arise by decarbamylation of citrulline(128); proline can be synthesized from glutamicacid (127) and probably also from ornithine(131); homoserine and threonine can undoubt-edly be synthesized from aspartic acid (131). Theability to transaminate glutamine with alanineexplains why alanine is not a required aminoacid; conversely, the inability to aminate theketo analogues of glutamic and aspartic acidscorrelates with the requirements of these twoamino acids for growth (131).The results of Lynn's study (56) on the nucleic

acid metabolism of a human strain of Mlycoplasmaindicated that pyrimidines cannot be formedfrom purines. The growth requirement for thymi-dine and deoxycytidine indicated that thesepyrimidine deoxyribosides cannot be synthesizedfrom other ribosides and deoxyribosides. Theinability of thymine to participate in base inter-conversion by the nucleoside phosphorylase con-firmed the growth requirement for thymidine.Conversely, the ability of thymidine to serve asdonor of deoxypentose correlated with the find-ing that purine deoxyribosides were not requiredfor growth.

Little is known, too, about carbohydratesynthesis in these types of organisms. 31. my-coides can synthesize 3-D-galactoside bonds asshown by the presence of a galactan (7). AMyco-plasma strains which utilize carbohydrates arecapable of synthesizing a or f glucosides. The Lforms of group A f-hemolytic streptococci are un-able to synthesize the group-specific polysaccha-ride of the parent organism (120).

Lipid synthesis in most instances is not amen-able to assessment, since the lipids of crudemedia in which the organisms were grown con-taminate the lipids of the organisms. Cyclo-propane fatty acids (C 15 and C 17) can cer-tainly be synthesized, since these fatty acidswould not have been present in the crude culturemedium used for growing M. hominis 07 (75).Glycerol-C'4 has been shown to be incorporatedinto the lipids of M. mycoides (102) and also intothe cardiolipinlike polyglycerophosphate de-scribed by Plackett (87). Lecithin is not formedfrom its component parts by resting cells ofhuman strains of Mycoplasma (130), althoughthe organisms must synthesize their own phos-phatides. Oleic acid incorporation occurs in bothhuman (136) and in avian strains (152). Phos-phatides are synthesized by an avian strain, asshown by incorporation of P32 (152). The possi-bility exists that some of this incorporationrepresents phosphate exchange reactions. Sterol-nonrequiring Mllycoplasma and L organisms arecapable of synthesis of their nonsaponifiablelipid from C'4-labeled acetate or mevalonate(143). In the case of the Mllycoplasma, this non-saponifiable lipid is carotenoid (133). Synthesispresumably occurs through intermediate tri-terpenoid compounds. Cholesterol can spare thissynthesis (133).

Enzymatic Activity Related to PathogenesisSabin (112) recovered a thermolabile exotoxin

from culture filtrates of a murine strain of Myco-plasma. No information is available concerningthe nature or mode of action of this toxin. Somer-son et al. (147) demonstrated /3-hemolysis byM. pneumoniae, the agent considered responsiblefor atypical pneumonia in humans. This hemoly-sin appeared to be soluble, in that diffusion fromthe colonies occurred. Whether this hemolysin isan enzyme or a component such as a lipidderived from disrupted cells is not known. Itsoccurrence only in fully developed cultures andits requirement for oxygen (N. L. Somerson,personal communication) would tend to indicatethe latter. Other strains of Mycoplasma producea-hemolysis with erythrocytes of various sources.Avian strains of Mycoplasma are known tohemagglutinate avian erythrocytes (10). Whetherthis hemagglutination has any analogy to virushemagglutination is not known.One feature used to distinguish between L

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organisms and Mycoplasma is the lack of patho-genicity of the former. There are, however, re-ports in the literature attesting to the pathogenicproperties of L organisms. These reports shouldbe rare, since the pathogenic properties of mostmicroorganisms are associated with surfacestructures. In the case of bacteria whose patho-genicity is the result of internal factors, L formsderived from them possess pathogenic properties.Mattman et al. (67) showed the presence ofcoagulase in staphylococcal L forms. L forms ofVibrio have been shown to be pathogenic in mice(154), and Madoff et al. (61) demonstratedneuraminidase production by L forms of V.cholerae. Although L forms of Clostridium per-fringens produce no pathological response inguinea pigs (44), L forms of C. tetani produce thetypical tetanus toxin (111, 114).

CONCLUSIONSA definitive comparison of the physiology of

Mlycoplasma and L organisms is complicated bythe variable culture media and cultural condi-tions employed by various investigators and thevery few select strains, particularly of L or-ganisms, studied. Although much effort has beenexpended to devise defined culture media forboth types of organisms, little effort has beenmade to examine diverse strains of L organisms.Essentially all the information regarding the Lorganisms has been collected for Proteus L or-ganisms. These data have been used as thoughtypical of all L forms and to substantiate adifference between L organisms and Mycoplasma.Such a comparison is not valid, for the few dataconcerning other L organisms does not indicatethat all physiological aspects of diverse L or-ganisms are similar. When these variables aretaken into account, certain similarities anddissimilarities remain.

Protein and carbohydrate composition is simi-lar in both types, the differences reflecting straindifferences only. The lack of cell-wall proteinand carbohydrate constituents of bacteria isnotable in both. The possibility exists that bothattempt synthesis of cell-wall constituents, sincepeptides and carbohydrates possessing similari-ties to wall components appear under certainconditions. Total lipid content, although similarin Mycoplasma and salt-nonrequiring L organ-isms, is low in salt-requiring L organisms. Mostof the lipid is membrane-associated. The non-

saponifiable lipids appear to possess structuralsimilarities in all organisms examined and couldfunction in a similar fashion.A major difference between the two types of

organisms is their reaction to osmotic changesand lytic agents. In general, the greater labilityto osmotic changes of salt-requiring L organismsappears to be due to the low lipid content in andprobably the structure of the membrane; of salt-nonrequiring L organisms, to their larger size ascompared with the Mycoplasma. Small L or-ganisms like L 1 behave as do llycoplasma in thisrespect. Lytic agents which act by dissolution oflipoproteins disrupt both types of organismsequally well, whereas specific agents, such asdigitonin, lyse only organisms which containdigitonin-precipitable sterol. This difference isnot fundamental, since other lipid possessingthe same functional capacity of sterol is found insome of the organisms and is nonreactive withdigitonin.

There is wide variation in the nutritionalrequirements among different strains of Myco-plasma and L organisms, as well as between thetwo types. These variations represent straindifferences rather than differences between typesof organisms, and reflect the variations in enzy-matic activity.The key to establishment of the similarity or

distinction of L organisms and Mycoplasmaappears to lie in the fundamental nature of theenveloping membranes. Studies of this physi-ological aspect necessitate the examination ofmore strains of both types of organisms, par-ticularly L organisms, by physical, chemical, andimmunochemical techniques.

ACKNOWLEDGMENTThis work was supported in part by research

grant AI-04410-02 from the National Instituteof Allergy and Infectious Diseases, U.S. PublicHealth Service.

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111. RuIio-HUERTOS, M., AND C. GONZALEZ-VAZQUEZ. 1960. Morphology and patho-genicity of L-forms of Clostridium tetani in-duced by glycine. Ann. N.Y. Acad. Sci. 79:626-631.

112. SABIN, A. B. 1938. Identification of the fil-trable, transmissible neurolytic agent iso-lated from toxoplasma-infected tissue as anew pleuropneumonia-like microbe. Sci-ence 88:575-576.

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121. SHIFRINE, M., H. E. ADLER, S. AARONSON,AND S. H. HUTNER. 1961. Susceptibility ofMycoplasma to steroid inhibitors. Bac-teriol. Proc., p. 125.

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129. SMITH, P. F. 1959. Cholesterol esterase ac-tivity of pleuropneumonia-like organisms.J. Bacteriol. 77:682-689.

130. SMITH, P. F. 1960. Nutritional requirementsof PPLO and their relation to metabolicfunction. Ann. N.Y. Acad. Sci. 79:508-520.

131. SMITH, P. F. 1960. Amino acid metabolism ofPPLO. Ann. N.Y. Acad. Sci. 79:543-550.

132. SMITH, P. F. 1962. Fate of ergosterol and cho-lestanol in pleuropneumonia-like organ-isms. J. Bacteriol. 84:534-538.

133. SMITH, P. F. 1963. The carotenoid pigments ofMycoplasma. J. Gen. Microbiol. 32:307-319.

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137. SMITH, P. F., J. G. LECCE, AND R. J. LYNN.1954. A lipoprotein as a growth factor forcertain pleuropneumonia-like organisms.J. Bacteriol. 68:627-633.

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141. SMITH, P. F., AND G. H. ROTHBLAT. 1960. Re-lation of PPLO to bacteria. Ann. N.Y.Acad. Sci. 79:461-464.

142. SMITH, P. F., AND G. H. ROTHBLAT. 1960. In-corporation of cholesterol by pleuropneu-monia-like organisms. J. Bacteriol. 80:842-850.

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144. SMITH, P. F., AND S. SASAKI. 1958. Stabilityof pleuropneumonia-like organisms to somephysical factors. Appl. Microbiol. 6:184-189.

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156. VANDEMARK, P. J., S. L. SMITH, AND J.FABRICANT. 1962. Respiratory pathways ofan avian strain of PPLO. Bacteriol. Proc.,p. 125.

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