A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants

ELSEVIER

FEMS MicrobiologyLetters 125 (1995) 23-30

A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants Gordon Stevenson

*, Annette Kessler, Peter R. Reeves

Department of Microbiology, Bldg. G08, University of Sydney, Sydney, New South Wales 2006, Australia

Received31 August 1994; revised 24 October 1994; accepted 1 November 1994

Abstract We identify a function-controlling 0 antigen chain length for a plasmid-borne gene, cZd,,,_,, harboured by Flexneri strains of Escherichia coli known to cause reactive arthritis. The predicted amino acid sequence of the gene product is very similar to those of other cld genes and that of fepE, thought to be part of the enterobactin iron uptake system of E. coli. The predicted proteins are compared with @-associated chain length determinants as a family of related genes Keywords:

cld;

fepE; Reactive arthritis; Lipopolysaccharide; 0 antigen; Flexneri; Escherichia

1. Introduction The repeating oligosaccharide units (0 units) of lipopolysaccharide (LPS) constitute the major 0 antigens of many enterobacterial species and are attached through a core oligosaccharide to a proximal lipid A moiety. The 0 units are polymerised and subsequently ligated to the core by two integral inner membrane proteins, Rfc and RfaL, respectively. The chain length determinant (Cld) is the essential element in defining the number of 0 units present on the majority of LPS molecules on the outer membrane. The distribution of LPS molecules seen under the influence of this protein is that of a modal (i.e. preferred length) or multimodal pattern. The gene has been named cld by us [1,2] and rol by others in an independent report [3]. In a previous paper we

’ Corresponding author. Tel.: (+ 61-2) 692 2536; Fax: (+ 61-2) 692 4571; e-mail: [email protected] 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(94)00485-4

of European

Microbiological

coli

showed that the chain length distribution depends on the source of the Cld as complimentation could occur if the cld gene of one form was present with the fi genes of another, resulting in a chain length distribution resembling that of the form providing the cld gene [2]. This paper describes a gene present in an Escherichiu coli Flexneri plasmid which resembles cld genes in sequence and can alter the 0 antigen chain length distribution of LPS. It is very similar to the fepE gene of E. coli K-12 believed to be involved in iron uptake. A related cld sequence from Yersinia pseudotuberculosis IIA is also presented. In this paper we name Shigella strains as members of the species E. coli, because of their relatedness as determined by hybridisation of chromosomal DNA. Thus, Shigella names are not italicised and the first letter of the old species name is capitalised in accordance with the tradition used for old species names of Salmonella enterica. Societies. All rights reserved

I

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fepE, FepE of E. coli K-12; cldpHS-2, Cld encoded on pHS-2 of E. coli Flexneri strain SSU6335; cldypIIA, Cld from Y cob 075; cldLT2, Cld from 5. entericn sv. typhimurium LT2; cldOll1, Cld from E. coli 0111; rfeORF2, predicted Cld for downstream of rfe gene of E. coli K-12). Shaded areas represent regions of > 50% consensus for each position in the alignment referred to in the text. Underlined regions indicate predicted trans-membrane LYhelices. Numbers refer to amino acid position.

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Fig. 1. Alignment of Cld protein sequences: pseudotuberculosis Iti, cld075, Cld from E. Enterobacterial Common Antigen (immediately whilst boxed regions contain conserved motifs

d&RF2

CldOll 1

:I%?;:

fepE c!dpHS-2 CldypllA

fepf cldpHS-2 CldvpllA cldO75 cldLT2 cldOll1 rfeORF2

cldOIll rfeORf2

cMLT2 CM0111 IfeORF2

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cldLT2 ddOll1 rfeORF2

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20 10 UYPLASPSNN KQGSDAHI’P MKI S KVS DE Hf:S NYNMLI’NGNE MSuKQAKNNl.DNl~~lPN8YDFSNVSSS~N M R V I: \’ N.5 V S Ci C_lN H Brl’ MrVUJNTSSC;KtiNZJPP;. MS I 5 DNN MS G R S Hal’% ~~QPMPGKPAE*,~~~N

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fepE cldpHS-2 cldy IIA old875 cfdLT2 cldO111 rleOAF2

G. Stevenson et al. / FEMS Microbiology Letters 125 (1995) 23-30

2. Materials and methods

2.1. Bacterial strains and plasmids The strains and plasmids used are described in Table 1. Bacteria were grown in nutrient broth containing (per litre of deionised water): yeast extract (Oxoid), 5 g; peptone (Amy1 Media), 10 g; NaCl, 5 g. 2.2. LPS analysis Proteinase K-digested whole membranes [2] were analysed by glycine-SDS-PAGE [4] with lo-20% gradient gels. Silver staining of LPS was performed according to Hitchcock and Brown [51. Carrier-free [ 32PIphosphoric acid (specific activity 9000 Ci mmol-’ > from Bresatec, Adelaide, South Australia, was added when needed at a final concentration of 10 mCi ml-’ to a lo-ml mid-log phase culture in nutrient broth at 37°C and growth was continued for 60 min. D-[U-‘4C]galactose (spec. act. 200 mCi mmoll ’ ) from Amersham was used when needed at a final concentration of 0.2 PCi ml-’ as above followed by a 20-min chase of cold galactose (final concentration 0.1%). The chain length distribution of radiolabelled LPS in PAGE gels was determined using a Molecular Dynamics PhosphoImager as described previously [2]. 2.3. DNA methods Restriction and ligation enzymes were obtained from Boehringer Mannheim and Pharmacia and used according to the manufacturers’ recommendations. Restriction enzyme digestion, DNA ligation and transformation as well as plasmid DNA preparation were carried out as described by Sambrook et al. [6]. 2.4. Sequencing and computer analysis The chain termination method of Sanger et al. [7]

was employed using an Applied (AB373A) sequencer with dye-labelled plementary to the universal or reverse and using a Perkin Elmer Cetus DNA cler. Analysis of open readign frames

Biosystems primers compriming sites Thermal Cy(ORFS) was

25

carried out using NIP from the Staden programs [8]. Several GCG package [9] programs were used, including PILEUP (for aligning the sequences), BESTFIT (for determining levels of sequence identities), also PEPPLOT and PLOTSTRUCTURE (for determining transmembrane segments and secondary structure elements respectively). Synthetic oligomers complimentary to the regions at positions 514 and 1891 (corrected sequence, see below) of cld,,,, and containing the universal or reverse priming sites were used to re-sequence this area. The phylogenetic tree was generated using PROTDIST and NEIGHBOR from the PHYLIP package (version 3.4 written by Joseph Felsenstein, Department of Genetics, University of Washington, Seattle, WA).

3. Results and discussion 3.1. Characterisation of a plasmid-borne gene of E. coli Flexneri 2a

The discovery of a plasmid-borne cld gene occurred as a result of a computer-based search using the Genbank sequence of fepE (accession X74129) which we realised had a significant homology to existing cld genes (see below). A gene of unknown function on plasmid pHS-2 [lo] in E. coli Flexneri 2a strain SSU6335 had 66% identity to fepE. Plasmid pHS-2 is found in certain strains of E. coli Flexneri 2a associated with reactive arthritis [lo]. The cld gene resident on it is additional to the cld gene in the chromosome of E. coli Flexneri 2a reported to be in the usual location close to the rfb cluster [l&12]. An amino acid sequence alignment of the published pHS-2 ORF (Fig. 1) with the cld gene of E. coli 0111 strain showed a substantial similarity after partial re-sequencing to confirm a suspected sequencing error (the absence of a guanosine residue following position 939 was confirmed). We cloned the gene by extracting the plasmid DNA from an agarose gel and then inserted into pT7T3,19U a fragment between unique SmaI and XbaI sites at positions 593 and 1966, respectively. This plasmid (pPR1580) was transformed into P4766 which lacks a functional chromosomal $J region but carries pPR1231 which contains the rfb region (but

26

G. Stevenson et al. /FEMS

Microbiology Letters 125 (1995) 23-30

not the cld gene) of E. coli 0111; its LPS has a non-modal chain length distribution (Fig. 2A). The presence of pPR1580 changed the distribution of the 0111 0 antigen of strain P4766 into a distinctly modal pattern (Fig. 2B). We conclude that the ORF on pHS-2 encodes a chain length determinant and name it cld,,,.,. The LPS profile from SSU6335 itself featured a pattern of dual modality with modal values of 15 and approx. 90-100 (measured upward from the core) (Fig. 2C). The higher value was the same as that observed for LPS from P4766 carrying pPR1580. We conclude that the modal value of 15 is due to the chromosomally encoded cld gene and the modal value of 90-100 is due to cldpHs_2. Note that the modal length specificity of the cldpHs_2is maintained when introduced into P4766 with the LPS modal pattern, reflecting that seen in SSU6335 although the 0 antigen is that from E. cob 0111 (Fig. 2B). A plasmid, pPR1585, containing the other SmaI/XbaI fragment from the digestion of pHS-2 in pT7T3,19U and assumed to contain the replication region of pHS-2, was able to displace pHS-2 when transformed into SSU6335. As expected, the LPS profile of the resulting strain, M851, lacked the mode at about 90-100, but there was an increase in the number of molecules with a modal value of 15 and some with a modal value of approx. 30 (Fig. 2D). A bi-modal distribution, with a second modal value about double that of the first, has been observed in other E. coli [13-151 and we have suggested that it could be due to some molecules undergoing a second round of elongation [2]. We put pPR1580 into SSU6335 to give cld,,s.2 in multicopy form and observed another LPS profile in which the lower mode at 15 0 units was still

Fig. 2. 0 antigen chain length distribution of LPS molecules as revealed by PAGE. Plots show relative pixel values for radioactively labelled LPS obtained from a Phospholmager. Small peaks represent individual LPS bands differing by one 0 antigen repeat with the (fastest running) core region at the left hand side. [r4C]galactose-labelled profiles are: A, P4766 (K-12 cld mutant containing an Olllrfb clone); B, P5110 (P4766 + pPR1580); C, SSU6335; D, MS51 (SSU6335 cured of pHS-2) with arrow indicating the secondary mode attending the lower peak; and E, M499 (SSU6335 + pPR1580). 32P-labelled LPS profiles shown are: F, SSU6335 with an arrow indicating a mode of approx 90-100; and G, M851.

G. Stevenson et al. / FEMS Microbiology Letters 125 (I 995) 23-30

present but with a lesser number of molecules whilst the upper (90-100) mode became a broader peak, indicating an increased number of high molecular mass LPS molecules (Fig. 2E). Since SSU6335 is not a gulE strain, the [r4C]galactose would be incorporated into LPS components other than galactose, making estimation of the relative number of molecules per peak difficult. A more accurate estimate of the number of molecules in each modal peak was obtained by labelling the LPS with [ ‘*PIphosphoric acid which labels LPS core only (of the molecules running on the gel). As can be seen in Fig. 2F, only relatively few molecules are in the range of 90-100 repeat units. It is likely therefore that the Cld,,s_2 does not compete effec-

27

tively with the chromosomal Cld in influencing the polymerisation process as even in the presence of multiple copies of the plasmid-borne cld,,,,there are still far more LPS molecules with lengths determined by the chromosomal Cld than by Cld,,,,. This contrasts with previous observations [2] where a foreign cld gene on a high copy number plasmid introduced into a strain with a functional, chromosomally located cld gene out-competed the resident cfd gene to give a modal number typical of the strain contributing the cld gene. In the present case, cld,,,, does not dominate the chromosomal cld gene so that modal patterns reflecting the influence of both Cld proteins are visible (Fig. 2C). Note that although only a small proportion of LPS molecules have the

Table 1 Laboratory stock number

Characteristics

Reference or source

CGSC5789

M515 M851 M499 P4051

Centers for Disease Control, Atlanta, This study This study Parkinson [251

CGSC5935

P4052

C600

PSllO P4825 P334

Wild-type SSU6335 cured of pHS-2 but containing pPR1585 M515 containing pPR1580 hpA9761, del(his-g&)81, gyrA12, rec41, thyA25, gaIE66, rpsL229, hlacZ2286, trp-49, del(sbc+)86, upp-12, relA1, rpsL150, hP4051 containing pPR1231 P4766 containing pPR1580 P4052 containing pPR1440 Fthr-1, leu36, tonAl, lacYI, ton.421, glnV44, rjbD, hYersinia pseudotuberculosis IL4 Cloning/expression

Pharmacia

Strains or plasmids

strains SSIJ6335

MS5

Neuhard and Thomassen

GA

[26]

Bastin et al. [2] This study This study Appleyard [27] D. Hughes, NSW Dairy Corp. lab Australia

Plasmids PT7T3, 19u pHS-2 pPR1580 pPR1585 pPR981 pPR1197 pPR1440 pPR1228 pPR1229 pPR1441

vector

Naturally occurring 3.048-kb plasmid resident in SSU6335 1.373-kb SmaI/XbaI fragment from pHS-2 in pT7T3, 19u 1.675-kb SmaI/XbaI fragment from pHS-2 in pT7T3, 19u M85 DNA cosmid clone including entire rjb region of Y. pseudotuberculosis Il.4 25-kb MS5 rfb DNA clone of pPR981 MS5 DNA from position 0.0 kb (SalI) to 23.6 kb (BarnHI) M85 DNA from position 16.5 kb (&I) to 21.5 kb (EarnHI) of pPR1197 in pl7T3, 19U As pPR1228 but insert reversed in orientation M85 DNA from position 21.5 to 23.0 kb downstream from pPR1197 in pUC18

LKB Biotechnology,

CDC (as above) This study This study Kessler et al. [16] Kessler et al. [16] This study This study This study This study

Sweden

G. Sleuenson et al. / FEMS Microbiology Letters 125 (1995) 23-30

28 S kb0

fib I 4

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, 8

I 12

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-

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I 32

1 36

I 40

I 44

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pPR1441 pPRI228,pPRl229 pPR1197

c!

8

Y

Fi

!

$ N

Fig. 3. Map of Yersinia pseudotuberculosis IL4 clone pPR981 [16] and sub-clones used in sequencing the cld gene. An expanded view from pPR1440 shows the region sequenced. Relevent restriction sites are shown. Abbreviations are as follows: S, SalI; B, EarnHI; P, PSI. The thickened line for pPR981 represents vector DNA. gsk is the gene for inosine-guanosine kinase.

very long 0 antigens, they nevertheless contain a substantial proportion of the total 0 units (Fig. 2C). 3.2. Sequence and function of Y. pseudotuberculosis Ila cld The rfb cluster of Y. pseudotuberculosis M85 has been cloned on plasmid pPR981 and the cld gene is known to be adjacent to the rfb region [16]. Plasmid pPR1197 contains the rfb cluster, but not the cld gene, on a SalI/BamHI fragment [16] and has a non-modal distribution of 0 antigen chain lengths. We cloned into pPR1197 the 1.5kb BarnHI fragment which in the chromosome is adjacent to the insert of pPR1197, in such a way as to extend the length of the insert. The resulting plasmid, pPR1440 (Fig. 3), in CGSC5935 was found to encode LPS identical to the wild-type LPS of this strain carrying pPR981 (data not shown but see [16]). The additional 1.5-kb BamHI fragment must therefore contain all or part of a cld gene. The sequence is reported here because of its similarity to cld,,,, and fepE. Sequencing was performed by creating an Exonuclease III deletion family from pPR1228 and pPR1229 (Table 1) but only deletion clones which gave sequence information near the BamHI site were used. The 1.5-kb BamHI fragment (above) was cloned into pUC18 to give pPR1441 and sequenced in the same way. The 1950-bp sequence contained one incomplete ORF and an ORF of 1149 bp with predicted protein product showing considerable ho-

mology to other Clds (Fig. 1). (This sequence has been deposited in Genbank under the accession number U13865.) All chain length determinant genes previously identified [2,3], including that for Y. pseudotuberculosis IIA (published here), and that reported for the E. coli Flexneri 2a strain investigated by other workers 111,121, are in close proximity to their respective rfb regions. 0RF2, the putative cld for the enterabacterial common antigen (ECA), is also very close to the ECA rff gene cluster [17]. It is interesting to note, however, that the rfb cluster is located at quite different positions on the chromosomes of E. coli and Y. pseudotuberculosis. The gene upstream of the rfb cluster of Y. pseudotuberculosis IIA is very similar (68% amino acid identity) to the hemH gene of E. coli [18]. The incomplete ORF downstream of the cld gene has 80.6% amino acid identity to gsk. The hemH and gsk genes are separated by only one gene in E. coli K-12 at 11 min [19]. We suggest that the rfb cluster has been inserted between the hemH and gsk genes during the evolution of Yersinia, or translocated from there to a position next to gnd during the evolution of the Enterobacteriaceae. Despite the different chromosomal location, the associated cld gene remained with its rfb cluster, confirming a general pattern. 3.3. Structural comparison of Clds An alignment of the predicted proteins from four cld genes associated with an rfb cluster, cld,,,, fepE and ORF 2 from the rfe locus of E. coli K-12 is shown in Fig. 1 and a phylogenetic tree in Fig. 4. It can be seen that these homologous genes essentially fall into three groups comprising: firstly, fepE, cld,,, and cldPw and cldyPIN; secondly, cld,,, and thirdly, rfeORF2. The three groups CldOlll; differ not only in amino acid sequence but also in the presence of substantial insertions or deletions (Fig. 1). A study of the seven sequences shown in Fig. 1 reveals both an overall similarity and a number of commonly conserved residues and motifs. All sequences include two stretches of hydrophobic amino acids towards the N- and C-termini, putatively representing transmembrane (TM) (Y helices (as indicated

G. Stevenson et al. / FEMS Microbiology Letters 125 (1995) 23-30

86.1

‘ldLT2

c’do75 c’dorll 99.8 100 loo

f-II.? Fig. 4. Phylogenetic (milking PROTDIST amino acid sequences bootstrap confidence proteins are as in Fig.

FepE

chain lengths of 90-100, a much higher value than that reported for other cld genes. The extreme lengths produced under the agency of this Cld (which seems to act independently of the chromosomal Cld) may confer special properties and our cured strain h&51 offers a way to investigate this possibility experimentally.

C’dpHS-2 C’dypm RfeORF2

tree generated by the PHYLIP program and NEIGHBOR) for cld genes using the of deduced Cld proteins. Numbers represent limits. The abbreviations for the various 1.

by PEPPLOT and by hydrophobic moment [20]. Two strongly conserved motifs occur in proximity to the N-terminus TM segment, namely (Q(E)IDL) and (Q(E)KWTS) which, on the basis of our model [2], could speculatively be points of interaction with either Rfc (0 antigen polymerase) or RfaL (0 antigen ligase), both of which are integral membrane proteins. The other, YxxxPxxPxR(K)R(K)DxP at the C-terminal end would be expected to lie close to the inner membrane in this scheme. 3.4. cld,,_,

29

as a virulence determinant

The function of the Cld is presumably to ensure that a significant proportion of 0 antigen chains is distributed around a modal value, commonly about 12-20. There have been several reports on the effects of 0 antigen chain length on interaction with complement and on serum sensitivity of the organism [21,22], and bacteria lacking 0 antigen are killed by serum complement [23]. The arthritogenic effect seen in the presence of pHS-2 has been attributed [lo] to a short peptide (predicted from the DNA sequence) but this does not seem likely to confer an advantage for the bacteria and, if true, is probably an unfortunate side effect of its similarity to human antigens. pHS-2 contains a single (non-plasmid maintenance) ORF making a candidate for a role in the arthritogenic CldpIiW effect. The primary effect of cld,,s_2 is in the production of a small proportion of LPS molecules with

3.5. Evolutionary origin of cld,,,_,

and fepE

Most of the pHS-2 DNA shows considerable similarity to the replication/transfer regions of E. coli plasmids ColEl and F, and cldp,s_2 appears to be the only gene not involved in plasmid maintenance. The replication region has a G + C content of about 0.5, but cld,,,, has a G + C content of 0.37, suggesting that this gene has its origins outside E. coli. FepE is considered a strong candidate for a component of an enterobactin permease, as a transposon insertion in this region disrupts iron uptake [24]. The good homology of FepE with other Clds and in particular its close similarity to cld,,,, leads us to suspect that this does encode a Cld protein, although it is not clear how it could affect ferric enterobactin uptake. Interestingly, fepE has a G + C content of 0.46, which is within the range expected for E. coli, but it is in a gene cluster concerned with enterobactin synthesis and enterobactin uptake with G + C contents ranging from 0.55 to 0.61. It certainly seems that fepE is a late addition to the gene cluster as the G + C content of the other fep genes and for the ent genes indicates an origin outside E. coli with a different origin, perhaps E. coli itself, for fepE.

Acknowledgements The assistance of Dr. Matthew Hobbs in the preparation of this manuscript is gratefully acknowledged. Funding from the Australian Research Council is also acknowledged.

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G. Stevenson et al. /FEMS Microbiology Letters 125 (1995) 23-30

[2] Bastin, D.A., Brown, P.K., Haase, A., Stevenson, G. and Reeves, P.R. (1993) Repeat unit polysaccharides of bacteria: a model for polymerisation resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length. Mol. Microbial. 7, 725-734. [3] Batchelor, R.A., Ahfano, P., Biffali, E., Hull, S.I. and Hull, R.A. (1992) Nucleotide sequences of the genes regulating 0-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: Protein homology and functional complementation. J. Bacterial. 174, 5228-5236. [4] Lugtenberg, - B., Meiiers, J., Peters, R., van der Hoek, P. and van Alphen, L. (1975) Electrophoretic resolution of the major outer membrane proteins of Escherichia cofi K12 into four bands. FEBS Len. 58, 254-258. [51 Hitchcock, P.J. and Brown, T.M. (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacterial. 154, 269-277. if51Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 171 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 14, 5463-5467. [81Staden, R. (1984) Computer methods to locate signals in nucleic acid sequences. Nucleic Acids Res. 12, 505-519. 191 Devereux, J., Haeberli, P. and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. [lOI Stieglitz, S., Fosmire, S. and Lipsky, P. (1989) Identification of a 2-Md plasmid from Shigella flexneri associated with reactive arthritis. Arthr. Rheum. 32, 937-946. [ill Macpherson, D.F., Morona, R., Beger, D.W., Cheah, K. and Manning, P.A. (1991) Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O-antigen specificity. Mol. Microbial. 5, 1491-1499. [121 Morona, R., Mavris, M., Fallarino, A. and Manning, P.A. (19941 Characterisation of the rfc region of Shigella flexneri. J. Bacterial. 176, 133-747. (131 Kusecek, B., Wloch, H., Mercer, A., Vaisanen, V., Pluschke, G., Korhonen, T. and Achtman, M. (1984) Lipopolysaccharide capsule and fimbriae as virulence factors among 01, 07, 016, 018, 075, and Kl, K5 or KlOO Escherichia coli. Infect. Immun. 43, 368-379. [I41 Peterson, A.A. and McGroarty, E.J. (1985) High-molecularweight components in lipopolysaccharide of Salmonella ty phimurium, Salmonella minnesota, and Escherichia coli. J. Bacterial. 162, 738-745. [15] Valvano, M.A (1992) Pathogenicity and molecular genetics

of O-specific side-chain lipopolysaccharides of Escherichia coli. Can. J. Microbial. 38, 711-719. [16] Kessler, A., Brown, P.K., Romana, L.K. and Reeves, P.R. (1991) Molecular cloning and genetic characterization of the r& region from Yersinia pseudotuberculosis serovar IIA, which determines the formation of the 3,6-dideoxyhexose abequose. J. Gen. Microbial. 137, 2689-2695. [17] Meier-Dieter, U., Barr, K., Stannan, R., Hatch, L. and Rick, P.D. (1992) Nucleotide sequence of the Escherichia coh tfe gene involved in the synthesis of enterobacterial common antigen. J. Biol. Chem. 267, 746-753. [18] Kessler, A., Haase, A. and Reeves, P.R. (1993) Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIa. J. Bacterial. 175, 14121422. 1191 Rudd, K.E. (1993) Maps, genes, sequences, and computers: an Escherichia coli case study. Am. Sot. Microbial. News 59, 335-341. La Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R. (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179, 125142. El1 Joiner, K.A., Schmetz, M.A., Goldman, R.C., Leive, L. and Frank, M.M. (1984) Mechanism of bacterial resistance to complement-mediated killing: inserted C5b-9 correlates with killing for Escherichia coli 0111B4 varying in O-antigen capsule and 0-polysaccharide coverage of lipid A core oligosaccharide. Infect. Immun. 45, 112. [221Joiner, K.A., Grossman, N., Schmetz, M. and Leive, L. (1986) C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella monteuideo. J. Immunol. 136, 710-715. [231Mushel, L.H. and Larsen, I.J. (1970) The sensitivity of smooth and rough gram-negative bacteria to the immune bactericidal reaction. Proc. Sot. Exp. Biol. Med. 133, 345352. [241Ozenberger, B.A., Nahlik, M.S. and McIntosh, M.A. (1987) Genetic organization of multiple fep genes encoding ferric enterobactin transport functions in Escherichia coli. J. Bacteriol. 169, 3638-3646. 1251Parkinson, J.S. (1976) cheA, chel?, and cheC genes of Escherichia coli and their role in chemotaxis. J. Bacterial 126, 758-770. 1261Neuhard, J. and Thomassen, E. (1976) Altered deoxyribonucleotide pools in P2 educants of Escherichia coli K-12 due to deletion of the dcd gene. J. Bacterial. 126, 999-1001. [271Appleyard, R.K. (1953) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K-12. Genetics 39, 440-452.