Vol. 61, No. 8INFECTION AND IMMUNITY, Aug. 1993, p. 3164-31740019-9567/93/083164-11$02.00/0Copyright C 1993, American Society for Microbiology

Enhancement of Extracapsular Polysaccharide Synthesis inKlebsiella pneumoniae by RmpA2, Which Shows

Homology to NtrC and FixJROCHAPORN WACHAROTAYANKUN,lt YOSHICHIKA ARAKAWA,1* MICHIO OHTA,l

KENJI TANAKA,2 TOMOHIRO AKASHI,2 MASASHI MORI,3 AND NOBUO KATO'Department ofBacteriology' and Laboratory ofMedical Mycology, Research Institute for DiseaseMechanism and Control, Nagoya University School ofMedicine, 65 Tsurumai-cho, Showa-ku,Nagoya 466, and Nagoya City Health Research Institute, 1-11 Hagiyama-cho, Mizuho-ku,

Nagoya 467, JapanReceived 24 November 1992/Accepted 5 May 1993

We determined the complete nucleotide sequence of a 2.1-kb HindlII-EcoRI fragment that was cloned from aresident large plasmid of Klebsiella pneumoniae Chedid, a highly virulent and mucoviscous strain of the 01:K2serotype. This fragment encoded an ability to enhance K2 capsular polysaccharide synthesis in K. pneumoniae,and a 636-bp open reading frame (rmpA2) was found. The 411-bp rmpA reported to be involved in the virulenceand mucoid phenotypes of K. pneumoniae by Nassif et al. (Mol. Microbiol. 3:1349-1359, 1989) was a part ofrmpA2. Eighty percent homology in nucleotide sequence was found between rmpA2 and rmpA in the correspond-ing regions. The central domain of the deduced amino acid sequence of RmpA2 showed considerable homologyto the central domains of NtrC ofK. pneumoniae and Escherichia coli, to which the cf factor of RNA polymerasebinds. The C-terminal domain ofRmpA2 also demonstrated considerable homology with the putative helix-turn-helix motifs of LuxR of Vibrio fischeri and FixJ of Rhizobium meliloti. Moreover, RmpA2 also showed somehomology in its N- and C-terminal regions to those of RcsA, a transcriptional activator for colanic acid synthesisin E. coli. On the other hand, a sequence upstream of rinpA2 was found to be highly homologous to insertionsequence 3 of members of the family Enterobacteriaceae. Southern hybridization analysis suggested that rmpA2exists on the large plasmids of all mucoviscous virulent K2 strains but not on those of the slightly mucoviscousavirulent strains. Freeze substitution electron microscopy and fluorescent-antibody staining with anti-K2 serumrevealed that K. pneumoniae Chedid has a dense and thick capsule (180 nm) with dense extracapsular substance,whereas K. pneumoniae K2-215, one of the slightly mucoviscous and avirulent strains, has a capsule which islooser and thinner (120 nm) than that of strain Chedid and no extracapsular substance. Introduction of rmpA2into K2-215 as well as reference strains K. pneumoniae K9 and K72 resulted in a change of the colony phenotypeto highly mucoviscous through abundant production ofextracapsular substance which reacted with anti-K2, -K9,or -K72, respectively, as did their parental strains. From these results, it is suggested that RmpA2 belongs to thefamily of transcriptional regulators and confers a highly mucoviscous phenotype on cells of various serotypes ofK. pneumoniae by enhancing extracapsular polysaccharide synthesis.

Klebsiella pneumoniae is an important opportunisticpathogen that usually causes serious disease in immunocom-promised hosts. Among at least 77 K serotypes distinguished(17, 19),K pneumoniae strains belonging to serotype 2 (K2)were frequently isolated from patients with bacteremia (3, 7,12). We found that seven of nine K pneumoniae O1:K2strains tested, including five clinical isolates, one laboratorystrain, and one reference strain, showed strong virulence inmice, with 50% lethal dose (LD50) values of as low as 2 to 10CFU after intraperitoneal injection (16). However, two ofthe nine K pneumoniae K2 strains were found to beavirulent in mice (LD50, >108 CFU). These two avirulentstrains grow as slightly mucoviscous colonies, while sevenvirulent strains are highly mucoviscous but with some vari-ations in the mucoviscosity.When we inserted transposon TnS into the structural

genes for capsule synthesis ofK pneumoniae Chedid, thevirulence disappeared concurrently with loss of the muco-viscous phenotype through abolition of K2 capsule produc-

* Corresponding author.t Present address: Department of Biopharmacy, Faculty of Phar-

macy, Silpakorn University, Bangkok, Thailand.

tion. We speculated that production of K2 capsular polysac-charide might be indispensable for bacterial virulence. Wealso speculated that in addition to production of K2 capsularpolysaccharide, the factor responsible for the mucoviscousphenotype inK pneumoniae might play an important part inthe virulence of this organism. The substances surroundingthe cell surface, including capsular polysaccharide and lipo-polysaccharide, were reported to be relevant, at least in part,to the pathogenicity of K pneumoniae (8, 23). The K- and0-antigens of K pneumoniae were reported to protect theorganism from complement-mediated serum killing andphagocytosis (31). It was also reported that the extracellularlipopolysaccharide of K pneumoniae serotypes 1 and 2enhanced the virulence of these bacteria when coinjectedintraperitoneally with the bacteria into mice (8).

Nassif et al. (18) reported that the mucoid phenotype ofKpneumoniae is a virulence factor encoded by a 180-kbplasmid, and two genes, nnpA and rmpB, were identified onthis plasmid. When we cloned CpSK* (2), encoding a part ofthe structural genes for K2 capsule synthesis, from thechromosome of strain Chedid and tried to express this K2capsule in Escherichia coli HB101, we found that HB101could produce thick K2 capsule through the functions of

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant characteristicsa Reference

K pneumoniaeChedid (O1:K2) Laboratory strain; highly virulent, highly mucoviscous 16K2-112 (O1:K2) Clinical isolate; virulent, mucoviscous 16K2-215 (O1:K2), K2-277 (O1:K2) Clinical isolates; avirulent, slightly mucoviscous 16K2-324 (O1:K2), K2-350 (O1:K2) Clinical isolates; virulent, highly mucoviscous 16K2-410 (01:K2), K2-694 (O1:K2) Clinical isolates; virulent, highly mucoviscous 16B5055 (O1:K2) Reference K2 strain; virulent, highly mucoviscous 19K9, K72 Reference strains 19

E. coliHB101 F- hsdS20 (rK- MK-) recA13 ara-14proA2 lacYl galK2 rpsL20 5, 6

xyl-5 mtl-l supE44 X-1JM109 recAl A(lac-proAB) endAl gyrA96 thi-I hsdR17 reLAI supE44 32

(F- traD36proA+B+ lacP'ZAM15)PlasmidspROJ3 Cosmid clone carrying rmpA2 isolated from a resident large 2

plasmid of Chedid; Apr; vector is pHC79pROJ31 Recombinant plasmid carrying rnpA2; Cm'; vector is pHSG398 26M13 mpl8, M13 mpl9 Bacteriophage vectors for DNA sequencing 32a Abbreviations: Ap, ampicillin; Cm, chloramphenicol.

both CPSK* and a gene locus encoding nnpA cloned from theresident large plasmid of Chedid (2).We report here the involvement of RmpA2, a putative

transcriptional regulator, in serotype-specific extracapsularpolysaccharide production that may be important for themucoviscous phenotype of K pneumoniae.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are listed in Table 1. Bacterialcultures were grown in Luria broth (LB) (21). Cells harbor-ing recombinant plasmids were grown on LB agar plates orin medium supplemented with appropriate antibiotics.Media and reagents. Tryptone, yeast extract, and agar as

medium constituents were purchased from Difco Laborato-ries, Detroit, Mich. Agarose for electrophoresis and immu-nodiffusion was from Wako Pure Chemical Industries, Ltd.,Osaka, Japan. Low-melting-temperature agarose for prepar-ing the DNA probes was purchased from InternationalBiotechnologies, Inc., New Haven, Conn. Restriction endo-nucleases, DNA polymerases, and T4 DNA ligase were fromNippon Gene Co., Ltd., Tokyo, Japan. ATP and chloram-phenicol were from Sigma Chemical Co., St. Louis, Mo.DNA sequencing. The 2.5-kb SalI-EcoRI DNA fragment of

pROJ31 was cloned into phages M13mpl8 and M13mpl9,and deletion mutants were generated by using exonucleaseIII, mung bean nuclease, Klenow fragment, and T4 ligasewith the Deletion kit supplied by Nippon Gene Co., Ltd.DNA sequencing was done by the method of dideoxy-mediated chain termination (22) on both strands.

Preparation of antisera. Polyclonal antisera against Kieb-siella K2, K9, and K72 capsular polysaccharides wereprepared as described previously (9, 17).

Preparation of DNAs. Plasmid DNA was prepared by thealkaline lysis method and purified by NA agarose (Pharma-cia, Uppsala, Sweden) gel electrophoresis followed by elec-troelution (21). Large plasmids were prepared by the methodof Kado and Liu (11).

Southern hybridization. The large plasmids prepared fromK pneumoniae K2 strains to be tested were electrophoresedand blotted onto a nitrocellulose membrane (Cellulosenitrate;Schleicher & Schuell). The 422-bp DraI fragment carrying a

part of nnpA2 on pROJ31 was used as the DNA probe.Hybridization was performed under conditions in which 60%homology can be detected, as described before (1).

Double-immunodiffusion analysis. Cell surface polysaccha-rides were extracted by the method of Sugiyama et al. (26).Double immunodiffusion was performed as described previ-ously (17).

Fluorescent-antibody staining. The bacterial cells weretreated for fluorescein isothiocyanate (FITC) staining essen-tially as described previously (27) except that the fixed cellswere incubated with rabbit anti-K2 serum diluted to 1:20with 10% nonimmune goat serum overnight at 4°C afterblocking with nonimmune goat serum.

Phase-contrast microscopy of antibody-treated cells. Bacte-ria to be tested were picked directly from agar plates andsuspended in phosphate-buffered saline (PBS) to 105 cells perml. A drop of each bacterial suspension was placed on a glassslide, and rabbit anti-K2 antiserum was added to give a finaldilution of anti-K2 serum of approximately 1:40. The slidewas incubated at room temperature for 24 h, covered with acoverslip, and observed under a phase-contrast microscope.

Freeze substitution electron microscopy. A bacterial colonygrown on an LB agar plate supplemented with appropriateantibiotics was picked up on a copper grid and rapidly frozen(KF80; Reichert-Jung) with liquid propane as a cryogen.Substitution fixation was performed in acetone containing2% osmium tetroxide cooled with dry ice-acetone (-80°C).Samples were embedded in Epon 812, cut into thin sections(Ultracut; Reichert-Jung) with a diamond knife before be-ing stained with uranyl acetate and lead citrate, and observedwith a transmission electron microscope (JEM-100SX elec-tron microscope; JEOL Ltd., Tokyo, Japan) at 80 kV.

Neutral-sugar component analysis of bacterial surfacepolysaccharides by HPLC. The quantitative analysis of neu-tral-sugar components of the surface polysaccharides ofeach bacteria by high-pressure liquid chromatography(HPLC) was done as described previously (27).

RESULTS

DNA sequencing and homology search. A 636-bp openreading frame, rnpA2, coding for 212 amino acids was found

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3166 WACHAROTAYANKUN ET AL.

AHindIII

10 20 30 40 50 60AAGCTTAAACGATACTGAAATTCAGGCGCTTCGCGAAGTATGTCGCGGCCTTTTGGAGGA

* ** ***** * ************** *************************

TAAAGACATACTTCATTTCAGGCGCTTCGCAAAGTATGTCGCGGCCTtTGGAGGA

70 80 90 100 110 120TGGCCAGTTCCTCAGCCTGCTCCGCCAGTTGTCGTTTAAGGCGGACATTTTCAGCGGCCA* *********** ***************** ** ** ****** ***************

TAGCCAGTTCCTCTGCCTGCTCCGCCAGTTGCCGCTTCAGGCGGGCATTTTCAGCGGCCA

130 140 150 160 170 180GTTCGCTTTCGCGCTCTGACGAACTCATTTGTTGCTGCTGTTTACTGCGCCAGGCATAAA**** ***** ***** ***** **** ********* **** ******** ***** *

GTTCACTTTCACGCTCAGACGAGGTCATCTGTTGCTGCAGTTTGCTGCGCCACGCATAGA

190 200 210 220 230 24GCTGAGATTCGTACAGGTTGAGTTCACGGGCTGCGGCGGCCACACCGATGCGTTCAGCGA********** ****** * ***************** ** ***************** *

GCTGAGATTCATACAGGCTAAGTTCACGGGCTGCGGCAGCAACACCGATGCGTTCAGCAA

250 260 270 280 290 300GTTTCAGGGCTTCGTTACGAAATTCAGGCGTATGTTGTTTACGGGGCTTCTTGCTGATTG* *********** * ************* *** ******** * ** ****** ***

GCTTCAGGGCTTCACTGCGAAATTCAGGCGAATGCTGTTTACGTGTTTTTTTGCTGGTTG

310 320 330 340 350 360ATACTGGTTTTGTCATGAGTCACCTCTGGTTGAGAGTTTACTCACTTAGTCCTGTGTCCA****** ********************* ************

ATACTGTTTTTGTCATGAGTCACCTCTGACTGAGAGTTTACT - S D I S3

370 380 390 400 410 420CTATTGGTGGGTAAGATTAATCTAATCCCCAAGGGTATTGTTGTTTTTAATAAAATTATC

-35 -10430 440 450 460 470 480

T TATTTCAAAAGAGG3 ATTA AAAATAATTCCTAAATAAACAAATATCCAA

490 500 510 520 530 540TTCTTTTTTATGAATTCTTAATCACACAGGGAAATGCTTTATTTATTTTTTCAAAGTAAA

EcgRI

550 560 570 580 590 600ATCAATTTGTTACTACCATGCAAACACAAACACAAACCTTATTAAGCACATAAAATGAAT

610 620 630 640 650 660AATAATTCATCATGAAACTGTAAACATTATGTTTCATGTAAAATGGTATTATGTTGCTTT

DraI670 680 690 700 710 720

730 740 750 760 770 780ACTGGTGTTTC TGGAAAAATATATTTACTTTATGTGCAATAAGGATGTTACATTAGTG

M E K Y I Y F M C N K D V T L V

790 800 810 820 830 840TTAACTGATGATTATTATTTTTATTTCGGCCTAAAGCAGTTAACTGGACTACCTCTGGTTL T D D Y Y F Y F G L K Q L T G L P L V

850 860 870 880 890 900

Y I T Y E G S M D K P I V I K Q K R N I

B Hindu! BcaRI

500

1&000

BamHI EccRIIl

IS3 rpA21000 200

%s s $' *s***vv^%s s%~~C

INFECT. IMMUN.

910 920 930 940 950 960AGAGTATTGGTTGATAGCCGGATTTTTTATTCAGGGAAATGGGATGGTTATAAAATGTTAR V L V D S R I F Y S G K W D G Y K M L

970 980 990 1000 1010 1020AGGAAAACATTAAATATGATAAGTCAATGGATGTGGCTTGACATTTCGGGGGGGGGGGAGR K T L N M I S Q W M W L D I S G G G E

1030 1040 1050 1060 1070 1080AAGTTTTATCCTAAAGGGTGTGATTATGACATCTATGTCAACATGCAAGGAAATTTAAAAK F Y P K G C D Y D I Y V N M Q G N L K

DraI1090 1100 1110 1120 1130 1140

AAAAACATTGAAGAGCTATATTATGCATACTrTAAAGAAAAATGTTAGCCGGATTGGAAATK N I E E L Y Y A Y L K K N V S R I G N

1150 1160 1170 1180 1190 1200CATTACCCACAACTAACAAAAAAAGAACAAATCATTCTACAATGCTTACTCTCCAGGAGGH Y P Q L T K K E Q I I L Q C L L S R R

1210 1220 1230 1240 1250 1260GAGGGCATCCATGAATTAAAAAGCCGTCTAAAAATTGAAGAGAAAACACTATCGTGTCACE G I H E L K S R L K I E E K T L S C H

1270 1280 1290 1300 1310 1320AGATGTAAAATAACAAGAAAATTTGGTTGTAAAAGATTCATAAGATTTATGTATCTTTACR C K I T R K F G C K R F I R F M Y L Y

1330 1340 1350 1360-a

70 1380AACTTAAATAAAGAAATAACTGATGAAAAATGGTGCACATCAAATACC rAGATAAAAAGGN L N K E I T D E K W C T S N T

ApaLI1390 1400 1410 1420 1430 1440

GAGGAGGAGGTGAACTATCCATCCATTATATTTATAATTCTTTTTCTATTTTATTTGTCA

1450 1460 1470 1480 1490 1500ATTTATAATTTTTATTCTTACATAGCTCAAAGAAGGAGGTTGAGAAACATATTCCTATCA

1510 1520 1530 1540 1550 1560AGAAGAAGGAGGCGCAAAAAAAAATAATCACCATAAAACGTCTCACACTCAATAAATAAT

1570 1580 1590 1600 1610 1620ATAATAATCATATATAATTAATGACAGTTTTAGTTGATATATAAGGATGCATTAAAGGTA

1630 1640 1650 1660 1670 1680AGAACACTATGAGATTGTATATCGTTACCGAAAACACTTACTTCTTTGCAGGAATGAAAT

1690 1700 1710 1720 1730 1740GTATTTTCCGGACATCCAATATTATCTTTGTCTAATATCAAACAGTGATATACATGATAC

1750 1760 1770 1780 1790 1800CTACCAGACTCAAACACAATATTTTTATTAGATGGTGTGAATCATAAATTTTCGATAAAA

1810 1820 1830 1840 1850 1860GAGTATAGCCATCTAAAAAAGAGACAATMTTAAGATAATCGGTTAATTCAGCCATTTTAC

1870 1880 1890 1900 1910 1920CGAATTGTACTGCCAGTTTATTTAATCACAGTTCGTCCTCTGATGAAAAACGGGAAAATG

1930 1940 1950 1960 1970 1980CGGGGCTGTCTATGTCCAGAGCACCAACGACGGTATTATTAATCCGGACAGGTAAAACGA

1990 2000 2010 2020 2030 2040TTTCCGAGTTACTGGCTGTATCACAGACAATATGACCATTAAAACTATGGACATTATTTA

2050 2060 2070 2080 2090 2100TGCACAQSATCCGTTCCTCTGCAACTGCCGTCCCGCAAACGCCGGAGCCTACCGCGAAM

BamHI EcoRI

FIG. 1. (A) DNA sequence and restriction sites of a 2.1-kb Hin-dIII-EcoRI fragment encompassing rmpA2 and IS3-like sequence(opposite orientation) cloned from K pneumoniae Chedid (01:K2).The deduced amino acids encoded by rmpA2 are shown. A possiblepromoter region of rmpA2 is boxed. The reverse complementarysequence of IS3 from Shigella dysenteriae (bp 1 to 340) (SDIS3),which shows the highest homology to the IS3-like sequence of Kpneumoniae K2, is shown below bp 5 to 342. An asterisk denotes anidentical base. (B) HarrPlot analysis comparing DNA sequencesencompassing rmpA2 (horizontal) and rmnpA (vertical). A restrictionmap of the 2.1-kb HindIII-EcoRI fragment carried on pROJ31,showing the positions and transcriptional direction of rmpA2 andIS3-like sequence, is shown corresponding to the HarrPlot.

on the 2.1-kb HindIII-EcoRI fragment (sequence shown inFig. 1A) carried by plasmid pROJ31, with the 414 bp on the3' side 80% homologous to the 411-bp rmpA gene encoding137 amino acids reported by Nassif et al. (18). HarrPlot

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EXTRACAPSULAR POLYSACCHARIDE SYNTHESIS BY RmpA2 3167

A 1

RmpA2 NH2-_100 200 212

FCOOH

K. pneumoniae NtrC

E. coli NtrC

E. coli ArcB

E. coli RcsA2

N.o%

KK

127 aa/ 16.5%

127 aa / 15.0%

108 aa/ 20.4%

E. coli Omp pR3a

E. coli UhpAa17.4

E. col YbcA17aa/ 2.8

R. meliloti FixJ aa/17.2

E. coli FhlAViaac/27.0R

32 a

V. fischeniLuxRR

1 MEKYIYFMCNKDVTLVLTDDYYFYFGLKQLTGLPLVYITYEGSMDKPIVI 50

198 AAIPKDLIESELFGHEKGAFTGANTVRQGRFEQADGGTLFLDEIGDMPLD 247{{* . *..

497 STFTLTIHAPSVAEEVDDAFDEDDMPLPALNVLLVEDIELNVIVARSVLE 546*. 0E.{. . { {.0 {0 . .{.

1 MSTIIMDLCS-YTRLGLT-GYLLSRGVKKREINDIETVDDLAIACDSQRP 48

2 TDYTVHIVDDEEPVRKSLAFMLTMNGFAVKMHQSAEAFLAFAPDVRNGVL 51

24 DINQCLSDMTKMVHCEYYLLAIIYPHSMVKSDISILDNYPKKWRQYYDDA 73

RmpA2 51 KQKRNIRVLVDSRIFYSG-KWDGYKMLRKTLNMISQWMWLDISGGG--EK 97. { . {.{ .... . .. { .. . * { .

NtrC 248 VQTRLLRVLADGQFYRVG-GYAPVKVDVRIIAATHQNLELRVQEGKFRED 296{ ... { .... . { ..{ ... .... .. .

ArcB 547 KLGNSVDVAMTGKAALEMFKPGEYDLVLLDIQL-PDMTGLDISREL--TK 593

RcsA 49 SVVFINEDCFIHDASNSQRIKLIINQHPNTLFIVFMAIANVHFDEYLLVR 98

FixJ 52 VTDLRMPDMSGVELLRNL-GDLKINIPSIVITGHGDVPMAVEAMKA--GA 98

LuxR 74 NLIKYDPIVDYSNSNHSP-INWNIFENNAVNKKSPNVIKEAKSSGL--IT 120

RmpA2 145 EQIILQCLLSRREGIHELKSRLKIEEKTLSCHRCKITRKFGC-KRFIRFM 193..NtrC 344 TEMALT-RLAWPGNVRQLENTCRWLTVMAAGQEVLTQDLPSE-LFETAIP 391

ArcB 640 KKFWDTQDDEESTVTTEENSKSEALLDIPMLEQYLELVGPKL-ITDGLAV 688* *.0{.......0.{ .. {0{.0{.0{. . {. { .

RcsA 142 ESSMLRMWMAGQGTIQ-ISDQMNIKAKTVSSHKGNIKRKIKT-HNKQVIY 189{.... . .. 0{.0{. .0 .{.. { {~0.. .

FixJ 146 ERQVLSAVVAGLPN-KSIAYDLDISPRTVEVHRANVMAKMKA-KSLPHLV 193{ .. *0{ {

LuxR 168 NYRKINIANNKSNNDLTKREKECLAWACEGKSSWDISKILGCSKRTVTF- 216

RmpA2 194 YLYNLNKEITDEKWCTSNT

NtrC 392 DNPTQMLPDSWATLLGQWA

ArcB 689 FEKMMPGYVSVLESNLTAQ........ {..

RcsA 190 HVVRLTDNVTNGIFVNMR

FixJ 194 RMALAGGFGPS{ ..... { {

LuxR 217 HLTNAQMKLNTTNRCQSIS

212

410

707

207

204

235

RmpA2 98 FYPKGCDYDIYVNMQGNLKKNIEELYYAYLKKNV- - SRIGNHYPQLTKK 144

NtrC 297 IFHRLNVIRVHLPPLRERREDIPRLARHFLQIAA- - -RELGVEAKQLHPE 343{{0.. { {0.. .0{. . { 0F .. . .

ArcB 594 RYPRE-DLPPLVALTANVLKDKQEYLNAGMDDVL---SKPLSVPALTAMI 639{{{ { .0.. {S

RcsA 99 KNLLISSKSIKPES---LDDILGDI----LKKETTITSFLNMTLSLSRT 141

FixJ 99 VDFIEKPFEDTVIIEAIERASEHLVAAEADVDDA--- NDIRARLQTLSER 145

LuxR 121 GFSFPIHTANNGFGMLSFAHSEKDNYIDSLFLHA---CMNIPLIVPSLVD 167

FIG. 2. (A) Illustration of similarities (length of amino acid [aa] sequence which shows similarity/percent identical residues) of variousparts of RmpA2 with various regulatory proteins. (B) Alignments of the deduced amino acid sequence of RmpA2 and those of similarregulatory proteins. An asterisk (*) or dot (-) above each amino acid denotes an amino acid which is identical or similar to that in RmpA2,respectively. Alignments were done by eye-matching with the GENETYX peptide homology data. The positions of the amino acid residuesin each protein are shown before and after each amino acid sequence.

analysis (Fig. 1B) shows that 222 bp of the 5' side of rmpA2are also homologous to the sequence upstream of rnpA. Acomputer-aided search for homologous sequences by theGENETYX system (SDC, Tokyo, Japan) found that a puta-

tive 212-amino-acid peptide deduced from the 636-bp rmpA2shows considerable homology in its central part to thecentral domains of the NtrC proteins of E. coli and Kpneumoniae (SWISSPROT accession numbers: E. coli

BRmpA2

NtrC

ArcB

RcsA

FixJ

LuxR

68 aa / 23. 1%

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1 9 4 5 f 7 9 10 1 1

.P

23.1 kb

lP

23.1 kb

FIG. 3. Southern hybridization analysis of K pneumoniae K2strains. Large plasmids were prepared by the method of Kado andLiu (11), electrophoresed, and Southern blotted (21). The 422-bpDraI fragment carrying a part of rmpA2 was used as the probe. Theprobe hybridized strongly to a large plasmid in all highly mucovis-cous virulent strains (K2-324, K2-350, K2-410, K2-694, B5055, andK2-Chedid); faint hybridization was seen with K2-112, which is lessmucoviscous than the strains mentioned above. The probe did nothybridize at all to slightly mucoviscous avirulent strains (K2-215 andK2-277). Lanes: 1 and 11, HindIII-digested A DNA size markers; 2,K2-112; 3, K2-215; 4, K2-277; 5, K2-324; 6, K2-350; 7, K2-410; 8,K2-694; 9, B5055; 10, Chedid. LP, large plasmid.

NtrC, P06713; K pneumoniae NtrC, P03029), E. coli UhpA(SWISSPROT P10940) and other transcriptional regulatorsbelonging to the two-component regulatory system, and tothe C terminus of pilin proteins from Pseudomonas aerugi-nosa (SWISSPROT: P08015, P04739, and P17837). More-over, the amino acid sequence between the N terminus andcentral region of RmpA2 is similar to the central part ofthe ArcB protein of E. coli (SWISSPROT P22763), which isa sensor-regulator protein for anaerobic repression of thearc modulon. Thirty-two amino acid residues and 64 resi-dues near the C-terminal region showed considerable homol-ogy to the putative helix-turn-helix motifs of Vibrio fischeriLuxR (SWISSPROT P12746) and Rhizobium meliloti FixJ

FIG. 4. Double-immunodiffusion analysis of K pneumoniae K2strains. Capsular polysaccharide preparations from various strainswere reacted with anti-K2 serum (center well). (a) Wells: 1 and 4,Chedid; 2, K2-112; 3, K2-215; 5, K2-277; 6, K2-324. (b) Wells: 1 and4, Chedid; 2, K2-350; 3, K2-410; 5, K2-694; 6, B5055. (c) Wells: 1,Chedid; 2, K2-215(pROJ31); 3, K2-215.

4 1 a 3 1 4 1- Glc * Glc * Man

a GIcA

FIG. 5. Structure of K2 capsular polysaccharide. Abbreviations:Glc, glucose; GlcA, glucuronic acid; Man, mannose.

(SWISSPROT P10958), two transcriptional regulators alsobelonging to the two-component regulatory system, respec-tively. Furthermore, RmpA2 showed similarity at both the Nand C termini to RcsA, a positive regulator of colanic acidsynthesis in E. coli (25) which is homologous to KiebsiellaRcsA, reported to be involved in serotype-specific K antigenproduction (29) (Fig. 2).The sequence upstream of rmpA2 (bp 5 to 342) was found

to be highly homologous (79.5 to 86.5%) to the 5' end of theinsertion sequence IS3 of Shigella dysenteriae, Shigellaflexneri, E. coli, E. fergusonii, and Serratia odonifera(EMBL accession numbers: Z11609, Z11751, X02311,Z11607, and Z11752, respectively), to insertion elements ofE. coli traX (EMBL X57429), and to the ArgU-tRNA geneoverlap of the prophage DLP12 integrase gene (GenBankM27155). Although the 2.1-kb HindIII-EcoRI fragment en-abled strain HB101 carrying CPSK* to produce Kiebsiella K2capsular polysaccharide, the 1.5-kb EcoRI-EcoRI fragmentcarrying the rmpA2 coding frame did not. Moreover, the0.5-kb HindIII-EcoRI fragment upstream of rmpA2 by itselfhas no ability to confer the mucoid phenotype.

Southern hybridization. The existence of DNA sequenceshomologous to the rmpA2 region in various K pneumoniaeK2 strains was investigated by Southern hybridization withthe 422-bp Dral fragment containing a part of the rmpA2gene as the DNA probe. We found that the rmpA2 probehybridized to the large plasmid found in all the highlymucoviscous virulent strains, K2-324, K2-350, K2-410, K2-694, B5055, and Chedid. Faint hybridization was detected instrain K2-112, which is less mucoviscous than those men-tioned above, while no hybridization was detected at all inthe two slightly mucoviscous avirulent strains K2-215 andK2-277 (Fig. 3). Moreover, the probe also hybridized to thechromosomal DNAs of mucoviscous virulent K2 strains.

Transformation of K. pneumoniae K2-215 with rmpA2.When plasmid pROJ31 carrying rmpA2 was introduced intothe slightly mucoviscous K pneumoniae K2 strain K2-215,the transformants became highly mucoviscous. The viscos-ity of the colony was even higher than that of Chedid,probably because rmpA2 was carried on a multicopy vector,pHSG398. The colonies of K2-215(pROJ31) adhered sotightly to the surface of the agar that it was very difficult topick the colonies from the agar plate. However, the convexand mucoviscous colonies of K2-215(pROJ31) turned flat

TABLE 2. Neutral-sugar components of surface polysaccharidesa

Neutral-sugar content (,ug)Strain

Mannose Galactose Glucose

K2-215 267 438 690K2-215(pROJ31) 454 867 1,307Chedid 1,314 2,416 3,796

a Surface polysaccharides were prepared from approximately 1.2 x 1010cells of K pnewnoniae K2 strains K2-215, K2-215(pROJ31), and Chedid.More than 90% of the cells in an overnight culture of each strain were viable.

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FIG. 6. FITC staining ofK pneumoniae K2 strains. The bacterial cells were stained with FITC-labeled goat anti-rabbit immunoglobulinG serum after treatment with rabbit anti-K2 serum. (a) K2-277; (b) B5055; (c) K2-694; (d) K2-Chedid; (e) K2-215; (f) K2-215(pROJ31). All themucoviscous strains (b, c, and d) showed extracapsular polysaccharide. The more viscous the colony is, the more extracapsularpolysaccharide was observed. The slightly mucoviscous strains (a and e) showed only capsules. Magnification, x 10,000.

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FIG. 7. Freeze substitution electron microscopy ofK pneumoniae K2 strains. (a) Chedid, (b) K2-215, and (c) K2-215(pROJ31). Bars, 500 nm.

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EXTRACAPSULAR POLYSACCHARIDE SYNTHESIS BY RmpA2 3171

FIG. 8. Freeze substitution electron microscopy of KK72(pROJ31). Bars, 500 nm.

and dry after short periods at temperatures above 25°C. Incontrast, the wild-type strains K2-215 and Chedid could bekept for a week without loss of the mucoviscous phenotype.

Double-immunodiffusion analysis. In double-immunodiffu-sion analysis, the precipitation bands produced by K2 cap-sular polysaccharides prepared from all the K pneumoniaeK2 strains tested fused with each other (Fig. 4a and b).Strain K2-215(pROJ31) also gave a precipitation band thatfused with those of strains K2-215 and Chedid (Fig. 4c).These results suggested a similar antigenicity among thecapsular polysaccharides produced by the highly mucovis-cous virulent and the slightly mucoviscous avirulent K2strains of K pneumoniae and that of the mucoviscoustransformant harboring rmpA2.

Analysis of neutral-sugar composition of bacterial surfacepolysaccharide by HPLC. A quantitative analysis of threemonosaccharides, mannose, galactose, and glucose, in thesurface polysaccharide preparation fromK pneumoniae K2strains Chedid, K2-215, and K2-215(pROJ31) was done.Mannose and a part of glucose are components of K2

v:v,

*a42 X * tb _ r~~~~~~~~~~~~~~~A

Ze@B>w~~~~~~~~z x; Ss Z) vs _~~~~~~~ftt

pneumoniae K9 and K72 strains. (a) K9; (b) K9(pROJ31); (c) K72; (d)

capsular polysaccharide (Fig. 5) (20), while galactose andsome amount of glucose are from 01 lipopolysaccharide(10). We found no significant difference in the ratio ofneutral-sugar components between strains K2-215 and K2-215(pROJ31). Moreover, no other kinds of neutral sugarwere detected in the surface polysaccharide of K2-215(pROJ31). However, K2-215(pROJ31) produced 1.5 to 2times more K2 capsular polysaccharide than did an equiva-lent number of cells of strain K2-215 as quantitated by theamount of mannose, indicative of K2 capsular polysaccha-ride (Table 2).FITC labeling of K2 capsular polysaccharide. All the mu-

coviscous K pneumoniae K2 strains tested showed a thickK2 capsule together with the extracapsular K2-positivereticulated structure. The more viscous the colony, the moreextracapsular 12-positive substance was found (Fig. 6b, c,and d). The slightly mucoviscous strains K2-215 and K2-277showed only the K2 capsule without detectable extracapsu-lar K2-positive substance (Fig. 6a and e). K2-215(pROJ31)showed the thick K2 capsule with a large amount of extra-

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capsular K2-positive reticulated structure (Fig. 6f), evenmore than the other mucoviscous strains, including Chedid,which is the strain from which rmpA2 was isolated.

Phase-contrast microscopy of antibody-treated cells. Tosettle the question of whether the extracapsular K2-positivereticulated structure detected by FITC staining is an artifactof dehydration during flame fixation, we observed cellswhich were treated with rabbit anti-K2 serum and kept inwet conditions without any dehydration treatment under thephase-contrast microscope. Phase-contrast microscopy gaveresults similar to those of FITC labeling. The extracapsularsubstance was found in all mucoviscous K2 strains when thecell suspension was incubated with a low concentration ofrabbit anti-K2 serum. If the concentration of antiserum wastoo high, the fibril structure resulted very soon after incuba-tion with antiserum, and a large amount of dark precipitatewas found instead. Moreover, under these conditions, thecontrast between the capsule and the cell was so low that itwas hard to distinguish the capsule layer. Avirulent strainsK2-215 and K2-277 also showed thick capsules but not asthick as that of Chedid. As in FITC staining, no extracap-sular substance was found in K2-215 (data not shown).K2-215(pROJ31) showed a large amount of extracapsularsubstance, as if cells were embedded in the reticulatedground substance. The capsule of K2-215(pROJ31) seemednot to be much thicker than that of K2-215.

Introduction ofrmpA2 into K. pneumonuze serotypes K9 andK72. K pneumoniae serotype K9 and K72 reference strains(19), which are slightly mucoviscous, were also transformedwith pROJ31 carrying rmpA2. Both K pneumoniae K9 andK72 strains harboring pROJ31 became highly mucoviscous,and the colonies adhered tightly to the agar surface, as wasobserved with K2-215(pROJ31). Neither transformant wasstained by FITC with anti-K2 serum as the primary antibodyor gave any precipitation band in double-immunodiffusionanalysis with anti-K2 serum (data not shown). They did giveprecipitation bands against anti-K9 and anti-K72 sera, re-spectively, in double-immunodiffusion analysis (data notshown).

Freeze substitution electron microscopic study. Strain Che-did, a mucoviscous and highly virulent strain, showed adense, thick capsule (approximately 180 nm), which is a bitthicker than that reported before (15) (Fig. 7a). The finestructure of the capsule was found to be the same asdescribed previously (15), with an inner domain of parallelfilaments vertical to the outer membrane and an outerdomain of interlacing filaments that was sometimes seen as alamellar arrangement around cells when observed in thicksections (data not shown). The dense interlacing extracap-sular polysaccharide was nearly as dense as the outerdomain of the capsule. This material was observed betweencells and prevented resolution of each capsule.

Strain K2-215 showed discrete cells covered with a cap-sule with a clear boundary as thick as 120 nm, a little thinnerthan that of Chedid (Fig. 7b). The arrangement of capsularfibrils was the same as that of Chedid but looser (Fig. 7b,inset). The extracapsular polysaccharide was very loose instrain K2-215.

In contrast to K2-215 and Chedid, most K2-215(pROJ31)cells did not show a well-defined capsule. Large amounts ofextracellular fibrils were observed between cells (Fig. 7c).The K pneumoniae K9 strain, which is slightly mucovis-

cous, showed a thin capsule composed of short (less than 10nm) filaments. The details of the capsule could not be seenclearly. The cells were close together, and there was verylittle extracapsular polysaccharide (Fig. 8a). K9(pROJ31) did

not express a capsule structure. The cells were well sepa-rated by the presence of a large amount of dense reticulatedfibrils, which did not adhere tightly to the cell surface (Fig.8b).The K pneumoniae K72 strain showed cells with a

discrete, well-bound capsule of approximately 120 nm (Fig.8c). Two domains could be observed. Extracapsular poly-saccharide was hardly seen. K72(pROJ31), like K2-215(pROJ31) and K9(pROJ31), did not express a clear capsulestructure but showed a large amount of reticulated extracel-lular fibrils (Fig. 8d).

DISCUSSION

RmpA2 consists of 212 amino acid residues, and itsmolecular mass was calculated to be 25.49 kDa. Its centralpart was found to show considerable homology to the centraldomains of NtrC, UhpA, and ArcB. These domains arespeculated to interact with the sigma factor of the core RNApolymerase. Furthermore, residues near the C terminus ofRmpA2 are similar to the putative helix-turn-helix motifs ofLuxR and FixJ and to the central domain of FhlA protein, atranscriptional activator for formate hydrogenlyase. Thesehelix-turn-helix motifs have been speculated to be a DNA-binding domain. From these results, together with the find-ings that RmpA2 confers the mucoid phenotype on E. coliand K pneumoniae via production of different capsularpolysaccharides (2, 18), it is possible to speculate thatRmpA2 is a member of the family of transcriptional regula-tors.

It was reported that rmpA might be involved in thevirulence ofK pneumoniae K2 by positively controlling themucoid phenotype as distinct from capsule production (18).Although RmpA was shown to behave like E. coli RcsA, apositive regulator for colanic acid production (25), in a Lon-background, no considerable homology was detected be-tween them (18). However, we found homology betweenRmpA2 and RcsA in both the N- and C-terminal regions(Fig. 2). This suggests that RmpA2 can probably be activatedand function similarly to RcsA even though some differencesin their functions were found (unpublished data). Seventy-three amino acid residues in the N-terminal domain ofRmpA2 were deleted in RmpA, but RmpA was reported tohave the ability to render E. cQli HB101 mucoid by enhance-ment of colanic acid production. If so, the N-terminaldomain of RmpA2 may not be essential for its activity,although this domain shows considerable homology to thecentral region of NtrC. However, we found significanthomology in nucleotide sequence between 222 bp in the5'-terminal region of nnpA2 and the upstream region ofrmpA (Fig. lb). These results strongly suggest that the222-bp sequence upstream of nnpA may be essential for itsactivity, although no coding frame was identified in thisregion by Nassif et al. (18).The sequence upstream of rmpA2 (bp 5 to 342) was found

to be highly homologous to the IS3 insertion sequences ofmany members of the family Enterobacteriaceae. Togetherwith the fact that rmpA2 has a low GC content, this suggeststhat rmpA2 might be derived from another organism andmight have been introduced into K pneumoniae by thetransposon-like element IS3. Moreover, rmpA2 might beduplicated in the chromosome of each mucoviscous K2strain, because the rmpA2 probe hybridized with their chro-mosomal DNAs. To our knowledge, no report of an IS3-likesequence from K pneumoniae has been published before.This joins the list of IS3 sequences found in Enterobacteri-

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aceae (13). However, the total sequence of IS3 which mightbe deleted during subcloning ofrmpA2 has now been deter-mined.Although rmpA2 conferred the mucoviscous phenotype on

the slightly mucoviscous strain K2-215, the antigenicities ofthe capsular polysaccharides produced by strains K2-215and K2-215(pROJ31) were similar in a double-immunodiffu-sion analysis. This result was consistent with the double-immunodiffusion analysis of capsular polysaccharides pre-

pared from various K2 strains, which showed that highlymucoviscous and slightly mucoviscous strains were antigen-ically similar. From quantitative analysis of the neutral-sugarcompositions of bacterial surface polysaccharides, strainsK2-215 and K2-215(pROJ31) are not significantly different.These results suggested that rmpA2 might not change thechemical structure of the polysaccharide chain, at least inthe neutral-sugar components, of strain K2-215's surfacepolysaccharide but may simply enable strain K2-215 tooverproduce the K2 capsular polysaccharide. This specula-tion is different from that reported by Nassif et al. (18) thatrmpA conferred the mucoid phenotype onK pneumoniae byproducing a substance distinct from K2 capsular polysaccha-ride. However, we also considered the pleiotropic effects ofrmpA2.By FITC staining with rabbit anti-K2 serum as the primary

antibody, the capsule found in K2-215(pROJ31) seemed tobe slightly thicker than that of K2-215, and K2-215(pROJ31)produced more extracapsular polysaccharide than strainChedid. The results of phase-contrast microscopy of anti-K2-treated bacteria confirmed the FITC staining results,although the sensitivity is lower. The exopolysaccharide or

extracapsular polysaccharide which we observed by FITCstaining, phase-contrast microscopy, and freeze-substitutedelectron microscopy might be very similar or identical to theso-called external slime or cell-free capsular polysaccharidedescribed by Beveridge and Graham (4) and Whitfield (29),in that it is the extracellular polymer produced by bacteriaand does not anchor securely to the surface. K pneumoniaecan produce both slime and capsule (30), but to our knowl-edge, there has been no report on the fine structure of slimefrom this organism. Fibrils of slime between cells of Entero-bacter aerogenes were previously demonstrated by freeze-etched scanning electron microscopy (24). These fibrilslooked similar to the exopolysaccharide in this study. Themore viscous the colony of theK pneumoniae K2 strain is,the farther the bundle of fine fibrils could be stretched andthe more of this K2 reticulated exopolysaccharide is present.Thus, the exopolysaccharide or extracapsular polysaccha-ride might be the major determinant for mucoviscosity inKpneumoniae K2 strains. We analyzed the molecular mass ofexopolysaccharides prepared from Chedid, K2-215, andK2-215(pROJ31) by gel electrophoresis and immunoblotting,but we could not detect significant differences among them(data not shown). Therefore, other physical properties, suchas ionic interactions among polysaccharide chains, may beinvolved in the formation of the structure of extracapsularpolysaccharide observed in mucoviscous K2 strains.The introduction of rmpA2 into K pneumoniae strains

belonging to serotypes K9 and K72, which are normallyslightly mucoviscous, also made these bacteria highly mu-

coviscous. Freeze substitution electron microscopy alsoshowed that neither K9(pROJ31) nor K72(pROJ31) ex-

pressed well-defined capsules, whereas they did producelarge amounts of exopolysaccharide, similar to K2-215(pROJ31). The polysaccharides produced by these transfor-mants were proved not to be the K2 capsular polysaccharide

(data not shown) but were K9 and K72 positive, respec-tively, by double-immunodiffusion analysis. However, twoprecipitation bands were formed in both transformants, withone of each fused to that of the parental strain. Thus, wespeculate that rnpA2 enhances capsular polysaccharideproduction generally in various serotypes ofK pneumoniaeas well as enhancing colanic acid synthesis in E. coli.The production of extracapsular polysaccharide may be

closely related to the virulence of these organisms. Possibly,this highly hydrated K2 extracapsular polysaccharide couldefficiently protect the bacteria from hazardous environmentsin the host, including phagocytosis by polymorphonucleatedneutrophils and serum killing by complements. In the Kpneumoniae K2 strains tested, the rmpA2 region was presenton a large plasmid in all of the mucoviscous virulent strainsbut not in the slightly mucoviscous avirulent ones. This time,the rnpA2 probe hybridized weakly to the chromosome ofK2-112, suggesting that K2-112 has some differences in thecorresponding rnpA2 region, which showed less than 60%DNA homology with the probe. However, when we intro-duced nnpA2 into K2-215, the transformants became evenmore mucoviscous than Chedid, from which rmpA2 wasoriginally cloned. It was reported that rmpB positivelycontrols rnpA expression (18). However, in our experi-ments, the rmpA2 region without rmpB was sufficient toenhance capsular polysaccharide synthesis. This may bebecause of multicopy expression of rmpA2 in bacterial cells.However, multicopy rmpA2 seemed to have adverse effectson bacterial growth. The colonies of K2-215(pROJ31) turnedflat and dry much sooner than did those of K2-215 andChedid. Therefore, the virulence of K2-215(pROJ31) couldnot be assayed exactly. Further analysis of bacterial viru-lence with scientific exactitude is in progress.

ACKNOWLEDGMENTS

We thank Worawit Wajwalku for. suggestions on FITC stainingand Ikuyo Mizuguchi for her skillful technique in electron micros-copy.

This work was supported by grants-in-aid 62304036 and 01480117for scientific research from the Ministry of Education, Science, andCulture of Japan.

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