JOURNAL OF BACTERIOLOGY, Dec. 1984, p. 958-9650021-9193/84/120958-08$02.00/0Copyright C) 1984, American Society for Microbiology

Vol. 160, No. 3

General Approach to Bacterial Nutrition: Growth FactorRequirements of Moraxella nonliquefaciens

ELLIOT JUNI,* GLORIA A. HEYM, AND REBECCA A. BRADLEY

Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-

0010

Received 9 July 1984/Accepted 14 September 1984

A general procedure was devised for the determination of growth factor requirements of heterotrophicbacteria based upon identification of individual nutrients as they are successively depleted from a limitedquantity of complex medium. By using this approach, it was possible to develop a defined medium for growth ofMoraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids,proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth.Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute forserine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was

antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodiumsulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartatefor this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23independently isolated strains of M. nonliquefaciens were essentially the same.

Many moraxellae have extensive nutritional requirements(1, 8). Henriksen (8) has characterized these organisms ashaving "moderate growth energy" since they generally formsmall colonies on the commonly used complex media. Intheir classic studies of the Moraxella group of bacteria,Baumann et al. (1) determined the complexity of the mediarequired to grow strains of several species of this genus.Strains of M. osloensis are able to grow on a defined mineralmedium containing NH4Cl as the nitrogen source with asingle carbon and energy source such as sodium lactate orsodium acetate (1). Strains of M. catarrhalis (Branhamellacatarrhalis) can grow in a casein hydrolysate-biotin mediumcontaining either lactate or succinate, but strains of othersimilar species (M. caviae [Branhamella caviae] and M. ovis[Branhamella ovis]) fail to grow in this medium even when itis supplemented with vitamins and purine and pyrimidinebases (1). Strains of M. bovis and M. nonliquefaciens growwell in a medium containing heart infusion and yeast extract,but strains of M. lacunata grow in this medium only when itis supplemented with either serum or oleic acid (1). In areport by Bivre (2) on growth of several Moraxella speciesin tryptone broth (Difco Laboratories), which is a pancreaticdigest of casein, it was shown that M. nonliquefaciens growsextremely slowly, failing to reach stationary phase after 15 hof growth, whereas other Moraxella species grow quitereadily in this medium.Because of our interest in the physiology and genetics of

M. nonliquefaciens, we undertook to determine the exactnutritional requirements of the same strain of this organismstudied by B$vre (2). During this investigation, a generalprocedure was developed that can be used to determine thenutritional requirements offastidious heterotrophic bacteria.Some nutritional features peculiar to strains of M. nonlique-faciens were discovered during the course of studies, leadingto development of a defined medium for this organism.

MATERIALS AND METHODSBacterial strains. Detailed growth studies were performed

with M. nonliquefaciens (ATCC 19975). Other strains of M.

* Corresponding author.

nonliquefaciens examined in this investigation are listed inTable 1.Media and chemicals. Cultures were grown routinely on

heart infusion agar (Difco) plates at 34°C. Medium Mn, adefined medium derived from the studies described in thisreport, was prepared by dissolving the following compo-nents in distilled water to a final volume of 1 liter: KH2PO4,1.5 g; Na2HPO4, 13.5 g; NH4Cl, 2 g; MgSO4, 0.1 g; CaCl2, 1ml of a 1% solution; FeSO4 * 7H20, 0.5 ml of a freshlyprepared 0.1% solution; L-glutamic acid, 1 g; monosodium L-glutamate, 11.5 g; L-proline, 10 g; L-serine, 10 g; L-phenylal-anine, 1 g; L-valine, 0.5 g; L-threonine, 0.5 g; L-methionine,0.5 g; L-tyrosine, 0.2 g; nicotinic acid, 5 mg; calciumpantothenate, 250 ,ug; d-biotin, 5 jxg; L-cysteine hydrochlo-ride, 1 g. The L-cysteine hydrochloride was incorporated inthe medium by dissolving 1 g in 10 ml of water, sterilized bymembrane filtration, and added to the other constituents ofmedium Mn, which were also sterilized by membrane filtra-tion, immediately before preparation of the semisolid medi-um. Semisolid medium Mn plates were prepared by mixingequal parts of liquid medium Mn and 3% melted agar. Thesalts used in medium Mn are the S2 salts solution of Monodand Wollman (14).

Spent GC medium was obtained by centrifugation of thecells of M. nonliquefaciens (ATCC 19975) after overnightgrowth in GC broth (Difco) at 34WC with shaking and wassterilized by membrane filtration.Amino acids and other growth factors were obtained from

Sigma Chemical Co. Beef extract and yeast extract wereproducts of Difco. Vitamin-free, salt-free casein hydrolysatewas obtained from ICN Nutritional Biochemicals.Growth factor pools. Pools of growth factors similar to

those of Holliday (9) were prepared by mixing 1 ml of each ofsix growth factor solutions. Pool 1 (milligrams per milliliter):uracil, 3.5; hypoxanthine, 0.5; cytosine, 5; guanine, saturat-ed; adenine, 0.5; thymine, 4. Pool 2 (micrograms per millili-ter): biotin, 1; folic acid, saturated; calcium pantothenate,50; pyridoxin hydrochloride, 50; thiamine hydrochloride, 1;riboflavin, saturated. Pool 3 (per milliliter): L-phenylalanine,10 mg; L-serine, 10 mg; L-tryptophan, 10 mg; L-tyrosine, 300,ug; potassium p-amino benzoic acid, 60 ,ug; nicotinic acid,

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GROWTH FACTOR REQUIREMENTS OF M. NONLIQUEFACIENS

50 pLg. Pool 4 (milligrams per milliliter): L-alanine, 10; L-cysteine hydrochloride, 14.5; L-threonine, 10; L-methionine,10; sodium thiosulfate, 5; choline chloride, 1. Pool 5 (milli-grams per milliliter): L-arginine hydrochloride, 12; L-aspara-gine, 10; monopotassium L-aspartate, 12.8; L-proline, 10;monosodium L-glutamate, 11.5; L-glutamine, 10. Pool 6(milligrams per milliliter): L-leucine, 10; glycine, 10; L-isoleucine, 10; L-histidine, 10; L-valine, 10; L-lysine hydro-chloride, 12.5.When testing for growth stimulation by pools, 1 ml of each

pool was added per 10 ml of growth medium.Procedure for measurement of growth. Growth in liquid

media took place in a total volume of 10 ml contained in 500-ml Erlenmeyer flasks with 14-mm-diameter side-arm tubes(Bellco Nephlo culture flasks). The cultures were aerated ina shaking 34°C water bath with an orbital motion of ca. 185rpm. At suitable intervals the culture in each flask waspoured into the attached side arm, and the absorbance of thesuspension was determined in a Klett-Summerson colorim-eter with a 660 (red) filter. The inoculum for each growthexperiment was prepared by seeding 10 ml of heart infusionbroth (Difco), contained in a 500-ml Erlenmeyer flask, withcell paste ofM. nonliquefaciens from growth on an overnightheart infusion plate and incubating with shaking at 34°C forca. 12 h. The cells in this liquid culture were centrifuged,washed with 10 ml of S2 salts solution, and suspended in 5 mlof S2 salts solution. In all growth experiments, 0.5 ml of thissuspension was added to each side-arm flask.

RESULTSUse of spent growth medium to determine required growth

factors. Initial studies of the growth of M. nonliquefaciens(ATCC 19975) were performed with a variety of complex

400

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FIG. 1. Growth of M. nonliquefaciens in complex media.Washed cells were inoculated into half-strength S2 salts solutioncontaining 0.8% vitamin-free casein hydrolysate and nicotinic acid(0.5 ,ug/ml (O), 0.25% monosodium glutamate and 0.1% yeastextract (Difco) (-), or 0.25% monosodium glutamate and 0.1% beefextract (Difco) (A); in other experiments, the medium used was GCbroth (Difco) (0) or GC broth (Difco) containing 0.3% sodiumlactate (0).

Z 10080J

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5 40 /

20>

2 -

TIME-HQURSFIG. 2. Growth of M. nonliquefaciens in spent GC medium.

Washed cells were inoculated into half-strength S2 sales solutioncontaining 15% spent GC medium (a), 12.5% spent GC medium and0.3% sodium lactate (0), or 50% spent GC medium and 0.3% sodiumlactate (0).

media. Growth in casein hydrolysate or in a glutamate-mineral medium containing either beef extract or yeastextract was extremely poor (Fig. 1). Good but limited growthwas obtained with GC broth (Difco) (Fig. 1). A higher finalcell yield was obtained when sodium lactate (0.3%) wasadded to GC broth (Fig. 1), a result indicating that growth inGC broth stopped because of carbon source limitation. Asimilar increase in final cell yield was also obtained when0.25% sodium glutamate was added to GC broth.

Since the addition of an excess of a suitable carbon source(lactate or glutamate) to GC broth resulted in an increase infinal cell yield, cessation of growth in such a supplementedcomplex medium .must then have resulted from depletion ofeither the nitrogen source or some required growth factor,assuming that no growth-inhibiting compound accumulatedin the medium. To explore these possibilities, spent GCmedium from an overnight culture of M. nonliquefacienswas prepared and shown to be incapable of supportinggrowth of the same strain (Fig. 2). Supplementation of spentGC medium with sodium lactate permitted growth of M.nonliquefaciens, the final cell yield being a function of theamount of spent GC medium used (Fig. 2).The addition of ammonium chloride to a medium contain-

ing spent GC broth and sodium lactate failed to increase thefinal cell yield (Fig. 3). Supplementation with aspartate as apossible nitrogen source resulted in a higher final cell yield,but aspartate was unable to serve as both a carbon sourceand a nitrogen source in the absence of lactate (Fig. 3).

Since glutamate could serve as a carbon source as well asa nitrogen source for growth ofM. nonliquefaciens (Fig. 3), aseries of growth media was prepared, each medium contain-ing glutamate, a small volume of spent GC medium (1.0 ml),and 1 ml of the pools of growth factors presented above todetermine which pool contained the nutrient that was growthlimiting for this medium. The addition of pool 4 resulted in asignificant increase in both the rate of growth and in the finalendpoint of growth (Fig. 4). There was also a positive butsmaller response to the addition of pool 1 (Fig. 4).

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FIG. 3. Nitrogen sources for growth of M. nonliquefaciens.Washed cells (washed and suspended in half-strength S2 saltssolution lacking NH4CI) were inoculated into half-strength S2 saltssolution lacking NH4CI but containing 10% spent GC medium and0.3% sodium lactate (0), 0.3% sodium lactate and 0.2% NH4Cl (0),0.3% sodium lactate and 0.25% monopotassium aspartate (O), 0.2%monosodium glutamate (a), or 0.25% monopotassium aspartate (A).

Because the stimulatory action of pool 4 resulted in amaximum cell yield (Klett reading = 200) (Fig. 4) that wasless than the final cell yield that can be obtained whengrowth takes place in a rich medium (Klett reading 400), itseemed evident that growth limitation in the presence of pool4 was probably the result of depletion from the medium ofsome other essential growth factor. When the mediumcontaining pool 4 (Fig. 4) was supplemented with each of theother pools of Table 1, however, no further increase in finalcell yield was obtained. To check the possibility that growthlitnitation in the presence of pool 4 (Fig. 4) was a conse-quence of simultaneous depletion of more than one essentialgrowth factor, the medium containing pool 4 was modifiedby adding two additional pools at one time. Considerable

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FIG. 4. Effect of nutrient pools on growth ofM. nonliquefaciens.Washed cells were inoculated into half-strength S2 salts solutioncontaining 0.2% monosodium glnutamate, 10% spent GC medium,and 1.0 ml of pool 1 (0), pool 2 (0), pool 3 or pool 5 (A), pool 4 (),pool 6 (A), or no pool (O).

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FIG. 5. Effects of combinations of three nutrient pools on growthof M. nonliquefaciens. Washed cells were inoculated into half-strength S2 salts solution containing 0.2% monosodium glutamate,1% spent GC medium, and 1.0 ml each of pools'3, 4, and 5 (0), pools1, 4, and 5 (0), pools 4, 5 and 6 (Li), pools 1, 3, and 4 (M), pools 1, 4,and 6 (A) or pools 3, 4, and 6 (A).

stimulation of growth was observed when the mediumcontaining pool 4 was supplemented with pools 3 and 5 (Fig.5). It shotild be noted that the growth curves in Fig. 5 wereobtained with a medium that contained 1/10 the amount ofspent GC broth used for the growth studies of Fig. 4 in whichonly pool 4 was included. Supplem, entation of the mediumcontaining pool 4 with pools 1 and 5 or pools 5 and 6 alsoresulted in significant stimulation of growth, but the final cellyields in these cases were lower than that obtained whenpools 3 and 5 were added together with pool 4 (Fig. 5).

Identification of growth-stimulatory components of pools 3,4, and 5. To deterrmine the factor(s) in pool 3 responsible forthe growth stimulation observed in Fig. 5 when pools 3, 4,

too

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FIG. 6. Effect of single components of pool 3 on the growth ofM. nonliquefaciens in the presence of pools 4 and 5. Washed cellswere inoculated into half-strength S2 salts solution containing 0.2%monosodium glutamate, 1% spent GC medium. 1.0 ml each of pools4 and 5, and (per milliliter) 200 ,ug of serine (0), 200 ,ug ofphenylalanine (0), 10 p.g of nicotinic acid (Li), or 200 ,ug oftryptophan or 200 ,ug of tyrosine or 12 F±g of potassium p-aminobenzoic acid (-).

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GROWTH FACTOR REQUIREMENTS OF M. NONLIQUEFACIENS

and 5 were present, the growth responses to single compo-nents of pool 3 were tested in the presence of pools 4 and 5and spent GC medium. It was found that the addition of onlyserine stimulated the rate of growth and also resulted in ahigher final cell yield (Fig. 6). The final cell yield obtainedupon the addition of serine (Fig. 6), however, was not asgreat as that observed when pool 3 was added (Fig. 5). Itseemed likely, therefore, that pool 3 contained more thanone required component. It was then shown that nicotinicacid, in addition to serine, seemed to further increase thefinal cell yield, although still not to the level obtained withpool 3 (Fig. 5). Phenylalanine was then demonstrated to be athird component of pool 3 required for growth of M. nonli-quefaciens.By using the same procedure, it was shown that proline

can substitute for pool 5 and that cysteine can substitute forpool 4 (data not shown).

Identification of other required growth factors. Goodgrowth of M. nonliquefaciens was demonstrated to takeplace in a medium containing glutamate as the carbon andnitrogen source, growth factor concentrations of cysteine,proline, serine, phenylalanine, and nicotinic acid, and asmall quantity of spent GC medium. Upon checking forfurther stimulation by any of the nutrient pools, it was shownthat the addition of pool 6 resulted in a significant stimulationof growth rate. The stimulatory component of pool 6 waseither valine or leucine. A mixture of these two amino acidsgave no better stimulation than was obtained with valine orleucine alone. At this point it was demonstrated that growthcould occur when spent GC mediurm was omitted.Upon inclusion of valine in a medium containing sodium

glutamate (carbon, nitrogen, and energy source), cysteine,proline, serine, phenylalanine, and nicotinic acid, the addi-tion of single pools was tested for possible further stimula-tion of growth. Pools 3 and 4 each showed a small stimula-tion of growth rate and final cell yield. Analysis of thecomponents of these pools responsible for these effects

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FIG. 7. Relation of glycine and hypoxanthine to the growthrequirement for serine by M. nonliquefaciens. Washed cells wereinoculated into half-strength S2 salts solution containing 0.2%monosodium glutamate, 200 ,ug each of cysteine, proline, valine,phenylalanine, tyrosine, threonine, and methionine per ml, and 10jig of nicotinic acid per ml. This medium was supplemented withglycine (200 ,ug/ml) (O), hypoxanthine (10 ,ug/ml) (-), serine (200 ,ug/ml) (0), or no addition (0).

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FIG. 8. Effect of lowering the concentrations of certain aminoacids on the final cell yield of M. nonliquefaciens growing in adefinied medium. Washed cells were inoculated into half-strength S2salts solution containing 0.2% monosodium glutamate and (permilliliter) 300 ,ug of cysteine, 1 mg of serine, 1 mg of proline, 300 ,ugof valine, 500 pLg of phenylalanine, and 2.5 pg of nicotinic acid (0).In separate growth flasks containing the above constituents, 100 ,ugof proline was substituted for 1 mg of proline (0), 100 ,ug of serinewas substituted for 1 mg of serine (O), or 100 ,ug of cysteine wassubstituted for 300 ,ug of cysteine (-).

showed them to be tyrosine, from pool 3, and threonine andmethionine, from pool 4 (data not shown). Although tyro-sine, threonine, and methionine were not absolutely requiredfor growth to occur in the defined medium containing theother components listed above, they were generally includedin further studies because of their stimulatory effects.

Relationships among requirements for serine, glycine, hypo-xanthine, and methionine. The studies shown in Fig. 5revealed that pools 1 or 6 could replace pool 3, although notas efficiently, when testing for essential growth factors in thepresence of pools 4 and 5 and a small volume of spent GCmedium. The factor in pool 1 responsible for this effect wasfound to be hypoxanthine, and the corresponding factor inpool 6 was found to be glycine (data not shown). Since serineis a known precursor of glycine and both serine and glycineare precursors of C-1 groups for purine biosynthesis (4, 15),it seemed likely that hypoxanthine (present in pool 1) andglycine (present in pool 6) must be capable of replacingserine (present in pool 3), at least in part. Glycine couldreplace serine, but hypoxanthine did so less effectively (Fig.7). Also, growth of M. nonliquefaciens in the defined medi-um used occurred at a slow rate in the absence of serine (Fig.7). Subsequent studies revealed that this slow rate of growth,in the absence of serine, required the presence of methio-nine.

Critical concentrations of some of the required growthfactors. Although growth of M. nonliquefaciens did occur inthe absence of spent GC medium when suitable growthfactors were present, the final cell yield obtained was alwayssignificantly lower than that observed when growth tookplace in a complex medium supplemented with a utilizablecarbon source. Preliminary experiments showed that thefinal cell yield was significantly dependent upon the concen-trations of cysteine, proline, and serine (Fig. 8). By using

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FIG. 9. Final growth yield of M. nonliquefaciens as a function ofconcentration of critical amino acids. Washed cells were inoculatedinto half-strength S2 salts solution containing 0.7% monosodiumglutamate and (per milliliter) 4 mg of serine, 300 ,ug of cysteine, 4 mgof proline, 300 p.g of valine, 500 ,ug of phenylalanine, and 2.5 ,ug ofnicotinic acid. When testing for the effect on growth of the concen-tration of a particular amino acid, that amino acid was omitted fromthe above medium and added to separate growth flasks in variousconcentrations. The amino acids tested for effect of concentrationon growth were cysteine (0), cysteine in the presence of 200 p.g ofmethionine per ml (0), serine (O), glycine (U), and proline (A).

high concentrations of two of these three amino acids, theconcentration of the third amino acid was varied to deter-mine the effect on final cell yield (Fig. 9). Since glycine couldreplace serine in a defined medium (Fig. 7), a curve showingfinal cell yield as a function of glycine concentration was alsoobtained (Fig. 9). Methionine was demonstrated to have asparing effect on the concentration of cysteine required foroptimal growth (Fig. 9). Optimum growth was obtained byusing growth factor concentrations (50 to 100 ,u/ml) of theremaining organic components of the medium.Because the optimum concentrations of the required fac-

tors cysteine, proline, and serine (or glycine) (Fig. 9) are ashigh as that required of the carbon source, a study was madeto determine whether any of these amino acids could substi-tute for glutamate as a carbon or nitrogen source. Slowgrowth, with a generation time of ca. 6 h as compared with a96-min generation time when glutamate was the carbonsource, occurred in the absence of glutamate or of lactateand aspartate (nitrogen source) (Fig. 10). The addition ofaspartate alone stimulated growth slightly, as did the addi-tion of lactate alone (Fig. 10). The addition of lactate andaspartate together, however, resulted in an optimal growthrate (Fig. 10). Glutamate was able to substitute for lactateplus aspartate (Fig. 10).

Studies of the requirement for valine. Since cysteine isknown to interfere with the synthesis of branched-chainamino acids in Escherichia coli (7, 10), the stimulatory effectof valine, described above, was tested when cystine orsodium sulfide was substituted for cysteine. Valine exerted a

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FIG. 10. Growth of M. nonliquefaciens in a defined medium inthe absence of the principal carbon and nitrogen sources. Washedcells were inoculated into defined medium that contained half-strength S2 salts solution and (per milliliter) 300 p.g of cysteine, 4 mgof proline, 4 mg of serine, 300 ,ug of valine, 500 ,ug of phenylalanine,and 2.5 ,ug of nicotinic acid. In addition to these constituents, theindividual growth flasks contained 0.7% monosodium glutamate(0), no additions (0), 0.5% sodium lactate (O), 0.5% sodium lactateand 0.5% monopotassium aspartate (U), or 0.5% monopotassiumaspartate (A).

very significant stimulatory action with cysteine as the sulfursource but exerted only a small stimulatory effect whencystine or sodium sulfide replaced cysteine (Fig. 11). A 4-hlag was observed with sodium sulfide as the source of sulfur.

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FIG. 11. Effect of valine on growth of M. nonliquefaciens withvarious sulfur sources. Washed cells were inoculated into half-strength S2 salts solution containing 0.7% monosodium glutamateand (per milliliter) 4 mg of proline, 3 mg of serine, 500 ,ug ofphenylalanine, 200 p.g of threonine, 200 p.g of methionine, 30 ,ug oftyrosine, and 2.5 ,ug of nicotinic acid. In addition to the aboveconstituents, individual growth flasks contained (per milliliter) 200,Jg of cysteine and 300 ,ug of valine (0), 200 ,ug of cysteine (0), 100,±g of cystine and 300 p.g of valine (O), 100 ,ug of cystine (U), 200 ,ugof sodium sulfide and 300 ,ug of valine (A), or 200 ,ug of sodiumsulfide (A).

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TABLE 1. Growth of strains of M. nonliquefaciens on medium Mn plates lacking one or two components of the mediumGrowth on medium missing the following component(s)':

Methio- Phenylal-Strain Proline Nicotinic Cysteine Methio- Senne nin and Pantothe- Threo- Phenylal- anineacid nine serine nate nine anine Tyrosine and tyro-serine sine

ATCC 19975 - - - _b _b _b + + + + _b +ATCC 17953 - - - _b _b _b _b + + + + +ATCC 19966 - - - _b _b _ + + + + + +Mn4 - - _ _b + _ -b + + + _b +Mn5 - - - + + _b -b + + + +Mn6 - - _ _b + _b -b + + + - +Mn7 - - _ b _b _b _ + + + + +Mn8 - - _ _b + _b + + + + +Mn9 - - _ _b + _b + + + + + +Mn25 - - - + + - + + + + + +Mn26 - - - - + + - + + + + +

ATCC 19968 - - - _b + _b + + + + + +Mn28 - - - - + - - + + + + +

ATCC 19969 - - - _b + _b + + + + + +Mn3O - - - + + _b _b + + + _b +Mn31 - - _ _b + _b + + + + +

ATCC 19967 - - - + _b _b _ + + + _b +ATCC 19990 - - - - _b _ + + + + + +Mn34 - - - - + _ + + + + _ +Mn35 - - - - + _ + + + + _b +Mn36 - - - -

Mn37 - - - - _b _ + + + + + +Mn58 - - _ _b + + + + + + +Mn78c - - _ _b + _b + + + + _b +a +, Good growth of isolated colonies; -, no growth.bGrowth in confluent region but no growth of isolated colonies.c Spontaneous mutant of strain Mn36 that grew on medium Mn.

The ability to obtain growth with pools 3, 4, and 5 in theabsence of valine or leucine, in the previously describedexperiments, was explained by the fact that the pools andsolutions of cysteine for these studies had been preparedseveral days before being used and that cysteine in thesesolutions had been largely oxidized spontaneously to cystinebefore the beginning of these experiments. Glutathione cansubstitute for cysteine for growth of M. nonliquefaciens.

Nutritional requirements of other strains of M. nonliquefa-ciens. A semisolid medium containing all required nutrientsfor growth of the strain of M. nonliquefaciens (ATCC 19975)used in these studies was found to give rise to good-sizedisolated colonies after 1 to 2 days of incubation at 34°C.When this medium was supplemented with small amounts ofthe nutrient pools described above, it was found that theinclusion of pool 2 resulted in slightly larger colonies thanappeared in its absence. The most significant component inpool 2 responsible for this growth stimulation proved to bepantothenate, which was then incorporated into the semisol-id medium. When tested in liquid culture, however, panto-thenate failed to show stimulation of growth rate or of finalcell yield. The addition of biotin also gave a small growthstimulation on the semisolid medium. The medium incorpo-rating pantothenate and biotin is called medium Mn (seeabove).Of 23 available strains of M. nonliquefaciens, 22 of them

grew and formed isolated colonies on medium Mn plates.One strain (Mn36) failed to grow on this medium but gaverise to spontaneous mutants (strain Mn78) able to grow onmedium Mn. The wild-type strain (Mn36) was able to grow ifmedium Mn was supplemented with growth factor quantitiesof isoleucine, valine, and leucine. In a study designed toassay all strains for specific growth factor requirements,

plates were prepared, each lacking either one or two ofcertain components of medium Mn, and all strains werestreaked on all the plates. The results of this assay are shownin Table 1. No strain was able to grow if nicotinic acid,proline, or cysteine was omitted from the medium. Most ofthe strains failed to show a requirement for serine, althoughmany strains showed only confluent growth on the mediumlacking serine and failed to form isolated colonies. A fewstrains grew in the absence of methionine, but most of thestrains showed either an absolute requirement for methio-nine or failed to form isolated colonies when plated on themedium lacking this amino acid. Although all strains grewwhen either phenylalanine or tyrosine was omitted from themedium, several strains failed either to grow or to formisolated colonies when both aromatic amino acids wereabsent. A similar growth response was observed uponomission of pantothenate (Table 1). No strain showed anabsolute requirement for threonine.Optimal pH and temperature for growth. The optimal pH

for growth of M. nonliquefaciens (strain ATCC 19975) wasbetween 7.0 and 7.5. No growth occurred at pH 6.0, andthere was very slow growth at pH 9.0. The optimal tempera-ture for growth of this strain was found to be 34°C.

DISCUSSIONA general approach to determination of the growth factor

requirements of nutritionally fastidious heterotrophic bacte-ria was developed during the process of divising a definedmedium for M. nonliquefaciens. The procedure involvedpreliminary determination of suitable carbon and nitrogensources, followed by identification of individual nutrients asthey were successively depleted from a growth-limitingamount of complex medium. An important advantage of this

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964 JUNI, HEYM, AND BRADLEY

technique over that of testing a variety of media for growthor lack of growth of a particular organism was the fact thateach nutrient solution used in the new procedure contained agrowth-limiting amount of complex medium and supportedgrowth until a single required component became exhausted.By contrast, when testing a series of defined media forgrowth of an organism under study, it was not clear whetherinability to grow in a particular medium resulted because ofthe absence of one or more growth factors, the presence of acompound that inhibited growth, or an extremely long lagperiod. Identification of the limiting growth factor at anytime in the present study was revealed upon its addition bycontinuation of growth further than the maximum growthobserved in the absence of the added component. Shouldany particular factor tested be growth inhibitory, this wouldbe evident from its effect on the limited growth that takesplace because of the presence of the small amount ofcomplex medium. At any point during the procedure, elimi-nation of the complex medium served to reveal whether allrequired components had already been identified, sincefailure to obtain growth was a clear indication that additionalrequired components must be identified. An increased lagperiod or a decreased growth rate after removal of thecomplex medium revealed that, although not absolutelyrequired for growth, other factors may be necessary toobtain optimal growth. It is then possible to identify thesefactors by the same procedure used to determine requirednutrients. By employing this methodology, defined mediahave already been devised for several other fastidious bacte-ria (Juni, unpublished data). Other methods for determiningnutritional requirements of bacteria have been previouslyreported (6).A successful modification of the method used in the

present study has been employed in identifying the nutrition-al requirements of other bacteria. In the alternate procedure,in addition to inclusion of growth-limiting amounts of com-plex medium, samples of all six nutrient pools were placed ina control flask, and five of the six pools were placed in eachof six other flasks, a different pool being deleted in each ofthese six flasks. Upon inoculation and incubation of allflasks, a decrease in final cell yield was observed only whena deleted pool contained a required nutrient. It was thuspossible to determine in one experiment which pools con-tained essential growth factors. In a similar manner, usinggrowth flasks containing either all components of a singlepool or all but one of the components of that pool, it waspossible to identify all required components in a single poolat one time (Juni, unpublished data).

For two of the other bacteria whose nutritional require-ments were investigated subsequent to the studies describedin this report, it was observed that compounds other thanthose contained in the six nutrient pools appeared to berequired for growth to occur. Despite this fact, it was stillpossible to determine other required nutrients (Juni, unpub-lished data). Further application of standard nutritionalmethodology should eventually result in identification of theunknown factors. The general approach described above canbe modified at any time by inclusion of other possible growthfactors, such as NAD, hematin, and fatty acids, for example,into some of the six nutrient pools or by creating yet anothernutrient pool.An unexpected finding of the current investigation was the

requirement for unusually high concentrations of cysteine,proline, and serine for optimal growth of M. nonliquefaciens.Although not yet examined, it seems possible that there maybe enzymes in M. nonliquefaciens that degrade these re-

quired amino acids so that they rapidly become unavailable.It cannot be the case that the high-concentration require-ments of these amino acids was the result of poor transportof these compounds, since concentrations of these growthfactors that resulted in half the maximum final cell yield, forexample (Fig. 9), were still very significantly higher thannormally required to give optimal growth of other bacteriarequiring these same factors. Utilization of the amount ofamino acid required for optimal growth would not be expect-ed to alter the high external concentration of that amino acidvery significantly.The requirement for a high concentration of serine, in

addition to the fact that slow growth of M. nonliquefacienscan take place in the absence of serine when methionine is acomponent of the medium (Fig. 7), implies that this bacteri-um must be capable of synthesizing serine but that most of itis probably destroyed as rapidly as it is synthesized. Bysupplying a product whose synthesis requires C-1 groups,normally derived from serine, methionine may serve to spareserine for synthesis of protein. Although the presence ofmethionine in the defined medium lowered the concentrationof cysteine required for optimal growth (Fig. 9), methioninewas unable to replace cysteine,completely.

In the experiments depicted in Fig. 5, in which variouscombinations of three nutrient pools were checked forstimulation of growth of N. nonliquefaciens, it was shownthat in the presence of pools 4 and 5 the addition of pool 1, 3,or 6 gave significant stimulation, but the effect of pool 3 wasconsiderably more than the effects observed upon the addi-tion of either pool 1 or pool 6. It was then demonstrated thatthe stimulatory components of pools 1 and 6 were hypoxan-thine and glycine, respectively, each of which could replaceserine (Fig. 7). The fact that, in addition to serine, pool 3 alsocontained nicotinic acid and phenylalanine, both requirednutrients for growth of M. nonliquefaciens, accounts for thegreater stimulation observed with pool 3 compared with thestimulation obtained with either pool 1 or 6 in the presence ofpools 4 and 5 (Fig. 5).The extremely poor growth of M. nonliquefaciens in

casein hydrolysate medium (2; Fig. 1) can be explained bythe growth-limiting concentrations of cysteine, proline, andserine in this medium. We have shown that excellent growthoccurred in casein hydrolysate medium when it was supple-mented with nicotinic acid and high concentrations of cyste-ine, proline, and serine. Failure to add any of these factorsresulted in a significantly lower final cell yield (data notshown). The requirement for unusually high concentrationsof these three amino acids may account for the fact that M.nonliquefaciens is only found to reside in animal tissues inwhich a ready source of amino acids is always available.The inability of NH4Cl to serve as a nitrogen source forM.

nonliquefaciens implies that this organism must lack NADP-glutamate dehydrogenase as well as glutamate synthase.Most microorganisms have been previously shown to con-tain at least one of these enzymes (3). Since glutamate canserve as carbon and nitrogen source for growth of M.nonliquefaciens, it appears that glutamate probably suppliesnitrogen via transamination to keto acid precursors of otheramino acids.The breakdown of glutamic acid required to supply ener-

gy, as well as carbon precursors for macromolecular biosyn-thesis, could occur via an NAD-glutamate dehydrogenase,as previously shown in other microorganisms (11, 12, 16), orthrough operation of the cyclic mechanism first described inE. coli by Marcus and Halpern (13) that makes use ofglutamate-oxalacetate amino transferase and aspartate am-

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GROWTH FACTOR REQUIREMENTS OF M. NONLIQUEFACIENS 965

monia lyase. If the requirement for a high concentration ofproline for optimal growth of M. nonliquefaciens occursbecause of degradation of proline, that process cannotinvolve conversion of proline to glutamate (5), since verypoor growth was observed in the absence of glutamate in amedium containing a high concentration of proline (Fig. 10).An interesting result of the present study is that all strains

of M. nonliquefaciens investigated have essentially the samenutritional requirements. All the strains have an absoluterequirement for cysteine, proline, and nicotinic acid (Table1). The stimulation of growth of the strain under study bymethionine, phenylalanine plus tyrosine, and pantothenate isalso evident for other strains (Table 1); in some cases, therequirement for some of these factors is absolute (Table 1).Only one strain (Mn36) (Table 1) of the entire group studiedappeared to be auxotrophic since unlike all the other strains,it showed a requirement for branched-chain amino acids.This strain was found to mutate spontaneously to givevariants able to grow on the defined Mn medium. Themutant strain Mn78 showed nutritional requirements similarto those of the other strains of M. nonliquefaciens (Table 1).

ACKNOWLEDGMENTSG. L. Gilardi, M. Maurer, and J. R. Lentino generously supplied

cultures of M. nonliquefaciens.This investigation was supported by Public Health Service grant

AI-10107 from the National Institute of Allergy and InfectiousDiseases.

LITERATURE CITED

1. Baumann, P., M. Doudoroff, and R. Y. Stanier. 1967. Study ofthe Moraxella group. I. Genus Moraxella and the Neisseriacatarrhalis group. J. Bacteriol. 95:58-73.

2. B$vre, K. 1970. Pulse RNA-DNA hybridization between rod-shaped and coccal species of the Moraxella-Neisseria groups.Acta Pathol. Microbiol. Scand. Sect. B 78:565-574.

3. Brown, C. M., D. S. Macdonald-Brown, and J. L. Meers. 1974.Physiological aspects of microbial inorganic nitrogen metabo-

lism. Adv. Microb. Physiol. 11:1-52.4. Dev, I. K., and R. J. Harvey. 1982. Sources of one-carbon units

in the folate pathway of Escherichia coli. J. Biol. Chem.257:1980-1986.

5. Frank, L., and G. Ranhand. 1964. Proline metabolism in Esche-richia coli. III. The proline catabolic pathway. Arch. Biochem.Biophys. 107:325-331.

6. Guirard, B. M., and E. E. Snell. 1981. Biochemical factors ingrowth, p. 79-111. In P. Gerhardt, R. G. E. Murray, R. N.Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B.Phillips (ed.), Manual of methods for general bacteriology.American Society for Microbiology, Washington D.C.

7. Harris, C. L. 1981. Cysteine and growth inhibition of Escherich-ia coli: threonine deaminase as the target enzyme. J. Bacteriol.145:1031-1035.

8. Henriksen, S. D. 1976. Moraxella, Neisseria, Branhamella, andAcinetobacter. Annu. Rev. Microbiol. 30:63-83.

9. Holliday, R. 1956. A new method for the identification ofbiochemical mutants of microorganisms. Nature (London)178:987.

10. Kari, C., Z. Nagy, P. Kovacs, and F. Hernadi. 1971. Mechanismof the growth inhibitory effect of cysteine on Escherichia coli. J.Gen. Microbiol. 68:349-356.

11. Kinghorn, J. R., and J. A. Pateman. 1973. NAD and NADP L-glutamate dehydrogenase activity and ammonia regulation inAspergillus nidulans. J. Gen. Microbiol. 78:39-46.

12. LeJohn, H. B., and B.-E. McCrea. 1968. Evidence for twospecies of glutamate dehydrogenase in Thiobacillus novellus. J.Bacteriol. 95:87-94.

13. Marcus, M., and Y. S. Halpern. 1969. The metabolic pathway ofglutamate in Escherichia coli K-12. Biochim. Biophys. Acta177:314-320.

14. Monod, J., and E. Wollman. 1947. L'inhibition de la croissancede l'adaption enzymatique chez les bacteries par le bacterio-phage. Ann. Inst. Pasteur (Paris) 73:937-956.

15. Newman, E. B., B. Miller, and V. Kapoor. 1974. Biosynthesis ofsingle-carbon units in Escherichia coli K12. Biochim. Biophys.Acta 338:529-539.

16. Strickland, W. N. 1969. Induction of NAD-specific glutamatedehydrogenase in Neurospora crassa by addition of glutamateto the media. Aust. J. Sci. 22:425-432.

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