Virulence of Pasteurella multocida recA mutants

Veterinary Microbiology 80 (2001) 53±61

Virulence of Pasteurella multocida recA mutants Maribel CaÂrdenasa, Antonio R. FernaÂndez de Henestrosaa, Susana Campoya, Ana M. Perez de Rozasb, Jordi BarbeÂa, Ignacio Badiolab, Montserrat Llagosteraa,* Molecular Microbiology Group. a

Departament de GeneÁtica i de Microbiologia, Edi®ci Cn, Universitat AutoÂnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Unitat de Sanitat Animal, Institut de Recerca i Tecnologia Agroalimentaria Barcelona, Barcelona, Spain Received 20 June 2000; received in revised form 22 September 2000; accepted 20 October 2000

Abstract In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a recA mutant was constructed and used in studies of virulence and competition in relation to wildtype strain. To achieve this, ®rstly, the recA gene was isolated and sequenced, showing an Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest identity with the Haemophilus in¯uenzae RecA protein. Further, the recA mutant was constructed, by inactivating this gene by single recombination of a suicide plasmid containing an internal region of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD50, the time of death of infected animals, and a failure to compete with the wild-type strain in mixed infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in competition were more a consequence of the long lag phase rather than a direct effect of the inactivation of the recA gene on genes involved in virulence. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Pasteurella multocida; recA gene; Virulence genes; DNA repair

1. Introduction Pasteurella multocida is responsible for infectious diseases in many species of mammals and birds, producing important economic losses. For this reason, development *

Corresponding author. Tel.: ‡34-93-5812615; fax: ‡34-93-5812387. E-mail address: [email protected] (M. Llagostera). 0378-1135/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 3 7 2 - 2

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of vaccine strains against this organism is an important goal. It has been largely demonstrated that live attenuated strains are more effective as vaccines than inactivated strains (Dougan, 1994; Marsden et al., 1996; Simmons et al., 1998; MaÈkelaÈ, 2000). It is essential that attenuated vaccine strains do not revert to virulence. To accomplish this objective, these vaccine strains usually present either deletion or insertion mutations in one gene of virulence, giving rise to attenuation. Nevertheless, these attenuated mutants are not totally safe since several recombinational processes (transduction, transformation and conjugation) could restore the wild-type phenotype. To avoid this, an important safety measurement is the introduction of mutations in the recA gene of vaccine strains (Fuchs et al., 1999). The RecA protein is a key component of homologous recombination in bacteria because it is involved in the early steps of this process, allowing the alignment of DNA molecules before strand exchange (Walker, 1984). This protein is also a positive regulator of the SOS system, one of the most important DNA repair-systems of bacteria. This network is made up of at least 20 genes whose products enable the cell to survive after DNA damage (Walker, 1984). The LexA protein is the repressor of all genes comprising the SOS response. It has been shown that, in E coli, the LexA repressor binds at a speci®c site at the 50 -ends of SOS genes. This site, known as the E. coli SOS box, is an imperfect palindrome comprising the sequence CTGTN8ACAG (Walker, 1984). Similar DNA repair-systems have been described in other bacteria, and several recA genes have been isolated and characterized (Miller and Kokjohn, 1990). Likewise, the SOS boxes of two other bacterial phylogenetic groups have been identi®ed (Gram-positive bacteria and alpha group of the Proteobacteria), with the sequences GAACN4GTTC and GTTCN7GTTC, respectively (Winterling et al., 1998; Labazi et al., 1999). The aim of this work was to isolate and sequence the P. multocida recA gene to facilitate the construction of RecAÿ derivatives, and to analyze the role of the P. multocida recA gene in the virulence of the organism. 2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains and plasmids used are listed in Table 1. E coli strains were grown in LB broth (Miller, 1992). P. multocida strains were cultured on brain-heart infusion (BHI) or on sheep blood agar (SBA) plates. Antibiotics were added to the culture media at the concentrations previously reported (Fernandez de Henestrosa et al., 1997). 2.2. Genetic methods The suicide plasmid pUA1002 was used to obtain the P. multocida RecAÿ mutant. This plasmid is a derivative of pGY2, which is unable to replicate in host strains devoid of the R6K-speci®ed p-protein product of the pir gene (Young and Miller, 1997) and which must be maintained in lysogenic strains for the lpir bacteriophage. Triparental mating, using pRK2013 as the mobilizing plasmid, was used to obtain the P. multocida RecAÿ

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Table 1 Bacterial strains and plasmids used in this work

Organism E. coli AB2463 DH5a HB101 MC1061 (lpir) P. multocida PM25 PM1002 PM1028 Plasmids pRK2013 pBBR1MCS pUA520 pUA811 pGY2 pUA826 pUA1002 a

Relevant features

Source or reference

recA13, supE44, thr1, leu6, proA2, his4, argE3, thi1, galK2, ara14, xyl5, mtl1, tsx33, rpsL31 supE4, DlacU169 (f80 lacZDM15), hsdR17, recA1, endA1, gyrA96, thi1, relA1 supE4, hsdS20 (rÿBmÿB), recA13, ara14, proA2, lacY1, galK2, rpsL20, xyl5, mtl1 hsdR, mcrB, araD139, D (araABC-leu) 7679, DlacX74, gal1, galK, rpsL, thi, lysogenized with lpir bacteriophage

ECGSCa

Wild-type As PM25, but RifR, SpcR As PM1002, but recA::pUA1002

This laboratory This laboratory This work

Tra‡ of RK2, ColE1 replicon, KmR A broad-host-range cloning vector, CmR As pBBR1MCS, but KmR As pUA520 but carrying a 2.2 kb chromosomal fragment of P. multocida containing the recA gene Mob‡, R6K replicon, ApR StrR SpcR

D.R. Helinski Kovach et al., 1994 This work This work

As pGY2, but lacking the 800 bp SalI fragment containing the cat gene As pUA826, but carrying a 548 bp internal fragment of the P. multocida recA gene

Clontech J. Frey This laboratory

Young and Miller, 1997 This laboratory This work

ECGSC, E. coli Genetic Stock Center.

mutant as previously reported (Fernandez de Henestrosa et al., 1997). Brie¯y, conjugation was performed by mixing 1 ml aliquots of early-stationary-phase cultures of donor (E. coli MC1061 (lpir) carrying the plasmid pUA1002), recipient (P. multocida PM1002) and E. coli HB101, carrying pRK2013 plasmid. This mixture was ®ltered through a sterile swinex ®lter unit equipped with a 0.45 mm Millipore ®lter, which was put on a BHI agar plate and incubated overnight at 308C. Afterwards, the ®lter was suspended in 3 ml of BHI liquid medium and dilutions were plated on BHI agar plates, supplemented with streptomycin, rifampicin and ampicillin. 2.3. Biochemical methods and DNA techniques DNA methodology and sequence computer analyses were as published (Fernandez de Henestrosa et al., 1997). A library of P. multocida PM25 chromosomal DNA was constructed by inserting size-fractionated, Sau3AI restriction fragments of 4 kb average size from a partial digest into the BamHI site of the pUA520 broad host-range plasmid, and transforming the RecAÿ E. coli strain DH5a. From this library, a clone carrying a 2.2 kb SacII±KpnI fragment putatively containing the P. multocida recA gene was

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Table 2 Oligonucleotide primers used in this work Primer

Sequence

Position

PosRec1a PosRec2a Rec1c Rec2c RecA2d Aadd

50 -TTTGCGATGCGTTAGTGC-30 50 -TTCTAACCATTTCATCGCG-30 50 -GCTCTATTATGAAATTGGGCG-30 50 -AAGGGTTAAGGTGGTTTTACCG-30 50 -CGCGAGCAACTCATTGCG-30 50 -CGGCGATCACCGCTTCCC-30

‡386b ‡933b ‡74b ‡235b ‡990b ‡22e

a

Primers used to obtain the 548 bp internal fragment of the P. multocida recA gene. Position of the 50 -end of the oligonucleotide with respect of the translational starting point of the P. multocida recA gene. c Primers used to obtain the 161 bp internal fragment of the P. multocida recA gene, used as probe. d Primers used to determine the presence of the 548 bp internal fragment of P. multocida recA gene into the plasmid pUA1002. e Position of the 50 -end of the oligonucleotide with respect the translational starting point of aad gene of the plasmid pGY2. b

identi®ed through its ability to confer methyl methanesulfonate resistance to the DH5a cells. The plasmid carrying this 2.2 kb SacII±KpnI fragment was designated pUA811 and was transformed into E. coli AB2463, which carries the mutant recA13 allele. The presence of the plasmid was found to restore UV resistance in AB2463 to parental strain levels (data not shown). The entire nucleotide sequence of P. multocida recA gene was determined for both DNA strands by the dideoxy method on an ALF Sequencer (Pharmacia Biotech). The nucleotide sequence of this gene and its ¯anking regions appears in the EMBL/GenBank/ DDBJ Nucleotide Sequence Data Libraries under Accession Number X99294. Oligonucleotide primers used in this work are listed in Table 2. 2.4. Virulence and competition assays Swiss mice, 3±5 weeks old, were used for studies of virulence. The LD50 of strains was determined by triplicate as reported (Fernandez de Henestrosa et al., 1997). Basically, groups of four mice were injected with serial 10-fold dilutions of bacteria in buffered peptone water. The concentration of the original bacterial suspensions was determined by the plate-count method. For competition assays, PM1002 and PM1028 strains were grown separately on SBA, and mixed before injection to obtained the desired concentration of each. Three mice were inoculated intraperitoneally with 0.1 ml of a suspension containing 121 CFU of PM1002 and 150 CFU of PM1028 as determined by plating. In both assays, bacteria were recovered of the hearts of dead animals to study their characteristics. 3. Results The ®rst experimental approach of this work was the isolation and sequence of the recA gene of P. multocida. Following procedures above mentioned (Section 2.3), a 2.2 kb

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SacII±KpnI fragment of the plasmid pUA811 was identi®ed to contain the recA gene of P. multocida. The nucleotide sequence of this fragment revealed one large open-reading frame, which was located seven nucleotides downstream of a typical Shine-Dalgarno sequence (GAGGA). The P. multocida recA ORF of 1065 nucleotides codes for a polypeptide of 354 amino acids whose calculated molecular mass is 37,912 Da. P. multocida RecA showed the highest similarity with the RecA protein of Haemophilus in¯uenzae (79%). The greatest degree of differences between both proteins was found primarily at the C-termini. Amino acid residues in E. coli RecA known to be associated with functional activities which include protease activity, and homologous recombination (Karlin and Brocchieri, 1996), were all highly conserved in the RecA protein of P. multocida. Likewise, upstream of the P. multocida recA gene was the sequence CTGTN8ACAG, which was identical to the E. coli LexA binding site (SOS box), suggesting that the regulation of this P. multocida gene is the same as that of the E. coli gene. This fact is in agreement with the high resistance against UV irradiation shown by the E. coli RecAÿ cells carrying the pUA811 plasmid (data not shown). The second purpose of this work was to obtain a RecAÿ mutant of P. multocida for studying the role of this gene in the virulence of this organism. Firstly the recA gene was disrupted by cloning a kanamycin-resistance cassette in an internal point of the recA gene. However, all attempts to introduce this mutated copy of P. multocida recA by marker exchange into the P. multocida chromosome were unsuccessful. For this reason, we decided to use an alternative approach inactivating the P. multocida recA gene by single recombination of a suicide plasmid containing an internal region of this gene. A 548 bp internal region of the P. multocida recA gene was isolated through PCRampli®cation using PosRec1 and PosRec2 oligonucleotide primers (Table 2), corresponding to nucleotides 386±403 and 933±915 of this gene, respectively. This 548 bp fragment was afterwards cloned in the pUA826 suicide-plasmid, giving rise to the pUA1002 plasmid. The presence of this fragment in pUA1002 plasmid was con®rmed through PCR-ampli®cation using the oligonucleotide primers RecA2 and Aad (Table 2). In addition, the fact that an ampli®cation product was obtained with these primers (data not shown) allowed us to predict the result of the single recombination with the recA chromosomal gene of P. multocida (Fig. 1). The pUA1002 plasmid was introduced by triparental matting into P. multocida PM1002. Eighteen ampicillin-resistant transconjugants were obtained and screened for UV sensitivity. Among these clones only one, the PM1028 strain, showed a higher UV sensitivity than the wild-type strain. Thus, at a dosage of 6 Jmÿ2, the fraction surviving of PM1028 was lower than 4  10ÿ4 . Moreover, wild-type cells UV irradiated at the same doses presented a 105-fold higher survival. In addition, a product of PCR-ampli®cation was obtained with oligonucleotide primers RecA2 and Aad (data not shown). To con®rm that P. multocida PM1028 was a recA mutant, chromosomal DNA from this strain and also from the parental cells was digested with EcoRI and probed by Southern blotting with a 161 bp internal fragment of the recA gene, obtained by PCR-ampli®cation with oligonucleotide primers Rec1 and Rec2 (Table 2). Fig. 2 shows that the probe hybridized to a 22 kb fragment in the parental strain PM1002 (lane 1) and to a 3 kb fragment in the PM1028 strain (lane 2), indicating that the recA gene had been disrupted in this transconjugant.

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Fig. 1. Schematic representation of single recombination between the 548 bp internal fragment of the P. multocida recA, cloned in pUA1002 plasmid, and the P. multocida chromosomal recA gene. Primers used to con®rm the presence of the 548 bp internal fragment of P. multocida recA gene into the plasmid pUA1002, and in the RecAÿ strain are shown.

The role of recA gene in virulence of the P. multocida was studied by determining the LD50 of both the recA mutant and the parental strain in a mouse model. Values (CFU/ animal) obtained were 54.9 (S:D: ˆ 5) and 11.75 (S:D: ˆ 2:6), respectively, with a statistical signi®cance P < 0:05. To determine stability during the infection with the P. multocida recA cells, several clones recovered from the heart of intraperitoneally infected mice were analyzed by both UV sensitivity and PCR ampli®cation with the oligonucleotide primers RecA2 and Aad. It is worth noting that all of these clones showed the same pattern for both parameters, as did the recA mutant (data not shown). In the course of virulence assays, we observed that mortality produced by the recA mutant was slightly delayed (72 h after infection) in relation to the parental strain (48 h after infection). This delay could re¯ect differences in growth parameters between both strains. When growth kinetics (data not shown) were studied, a similar growth rate for both strains was found, but signi®cant differences in the duration of the lag phase (10 min for PM1002 versus 90 min for PM1028) were seen. The next step was to perform competition assays between PM1002 and PM1028 strains in mixed infections. Death of all animals was at 48 h from infection. Clones (500), recovered from the heart of each animal, were replicated in the absence and in the presence of streptomycin and ampicillin. All clones were sensitive to both antibiotics indicating that all were the wildtype strain.

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Fig. 2. Southern analysis of chromosomal DNA of wild-type (lane 1) and RecAÿ (lane 2) strains of P. multocida PM1002. A 161 bp fragment containing an internal region of the P. multocida recA gene was used to hybridize with the EcoRI-digested genomic DNA. Molecular size markers corresponding to HindIII-digested lDNA are shown in the left.

4. Discussion Little is known about the importance of the recA gene in bacterial virulence. Some studies indicate that this gene is involved in the infection process by several bacteria by either: (i) increasing the expression of phage encoded-virulence genes as in enterohemorrhagic E. coli O157:H7 and Vibrio cholerae strains (Fuchs et al., 1999; Faruque et al., 2000) (ii) promoting DNA rearrangements as has been shown in enteroinvasive strains of E. coli and Shigella ¯exnerii (Zagaglia et al., 1991), and in Campylobacter fetus and Neisseria gonorrhoeae (Dworkin et al., 1997; Ilver et al., 1998) or (iii) enhancing cell survival against host-factors damaging bacterial DNA (Buchmeier et al., 1995). Thus, it has been shown that the presence of a recA mutation gives rise to an attenuated phenotype in Vibrio cholerae, Staphylococcus aureus and Salmonella typhimurium (Buchmeier et al., 1993; Kumar et al., 1994; Mei et al., 1997). Nevertheless, this mutation did not seem to modify the virulence of Brucella abortus and Corynebacterium pseudotuberculosis (Tatum et al., 1993; Pogson et al., 1996). These opposite behaviors indicate that the role of the recA gene is not the same for all pathogenic bacteria. Values of LD50 obtained in this work clearly indicate that the recA mutation only provokes a marginal reduction of the P. multocida virulence, especially in comparison

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with the effect on virulence described for the galE mutation (Fernandez de Henestrosa et al., 1997). However, concerning competition assays, the recA mutant has a clear disadvantage in front to wild-type strain. We hypothesize that non-success in competition assays could be more a consequence of a long lag phase than a direct effect of recA gene on genes related to virulence. Additionally, this delay in growing could also explain the marginally diminished virulence of the P. multocida recA mutant. In fact it is known that E. coli recA mutants produce anucleate cells disturbing signi®cantly the culture growth (Zyskind et al., 1992). This behavior seems to be attributed to the role of RecA protein in the partition of the bacterial chromosome between daughter cells (Mao et al., 1991). In this line, our hypothesis also agrees with the relationship found between generation time and virulence in E. coli, S. typhimurium and Actinobacillus pleuropneumoniae pathogenic strains (Byrd and Hooke, 1997; Linde et al., 1998). In summary, we conclude that recA gene has not an speci®c role in the virulence of P. multocida, although its de®ciency gives rise to a delay in the process of infection and to a marginal reduction of virulence. The P. multocida recA mutant obtained in this work will be useful in both the development of a stable host/vector system which will facilitate genetic studies to characterize virulence factors of this organism as well as in the construction of safe live vaccines against it. Acknowledgements This work was partially funded by grant BIO99-0779 of the ComisioÂn Interdepartamental de Ciencia y TecnologõÂa of Spain (CICYT), and by the Comissionat per Universitats i Recerca de la Generalitat de Catalunya (1999SGR-106). Susana Campoy was a recipient of a predoctoral fellowship from the Direccio General d'Universitats de la Generalitat de Catalunya. We are deeply indebted to Joan Ruiz and M. Mar LoÂpez for their excellent technical assistance.

References Buchmeier, N.A., Lipps, C.J., So, M.Y., Heffron, F., 1993. Recombination-de®cient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages. Mol. Microbiol. 7, 933±936. Buchmeier, N.A., Libby, S.J., Xu, Y., Loewen, P.C., Switala, J., Guiney, D.G., Fang, F.C., 1995. DNA repair is more important than catalase for Salmonella virulence in mice. J. Clin. Invest. 95, 1047±1053. Byrd, W., Hooke, A.M., 1997. Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice. Infect. Immun. 65, 2206±2210. Dougan, G., 1994. The molecular basis for the virulence of bacterial pathogens: implications for oral vaccine development. Microbiology 140, 215±224. Dworkin, J., Shedd, O.L., Blaser, M.J., 1997. Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent. J. Bacteriol. 179, 7523±7529. Faruque, S.M., Asadulghani, A.M.M.R., Waldor, M.K., Sack, D.A., 2000. Sunlight-induced propagation of the lysogenic phage encoding cholera toxin. Infect. Immun. 68, 4795±4801. Fernandez de Henestrosa, A.R., Badiola, I., Saco, M., Perez de Rozas, A.M., Campoy, S., BarbeÂ, J., 1997. Importance of the galE gene on the virulence of Pasteurella multocida. FEMS Microbiol. Lett. 154, 311± 316.

M. CaÂrdenas et al. / Veterinary Microbiology 80 (2001) 53±61

61

Fuchs, S., MuÈhldorfer, I., Donohue-Rolfe, A., KereÂnki, M., EmoÈdy, L., Alexiev, R., Nenkov, P., Hacker, J., 1999. In¯uence of RecA on in vivo virulence and Shiga toxin 2 production in Escherichia coli pathogens. Microb. Pathog. 27, 13±23. Ilver, D., Kallstrom, H., Normak, S., Jonsson, A.B., 1998. Transcellular passage of Neisseria gonorrhoeae involves pilus phase variation. Infect. Immun. 66, 469±473. Karlin, S., Brocchieri, L., 1996. Evolutionary conservation of recA genes in relation to protein structure and function. J. Bacteriol. 178, 1881±1894. Kovach, M.E., Phillips, R.W., Elzer, P.H., Roop, R.M., Peterson, K.M., 1994. pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16, 800±802. Kumar, K.K., Srivastava, R., Sinha, V.B., Michalski, J., Kaper, J.B., Srivastava, B.S., 1994. recA mutations reduce adherence and colonization by classical and El Tor strains of Vibrio cholerae. Microbiology 140, 1217±1222. Labazi, M., del Rey, A., Fernandez de Henestrosa, A.R., BarbeÂ, J., 1999. A consensus sequence for the Rhodospirillaceae SOS operators. FEMS Microbiol. Lett. 171, 37±42. Linde, K., Fthenakis, G.C., Fichtner, A., 1998. Bacterial live vaccines with graded level of attenuation achieved by antibiotic resistance mutations: transduction experiments on the functional unit of resistance, attenuation and further accompanying markers. Vet. Microbiol. 62, 121±134. MaÈkelaÈ, P.H., 2000. Vaccines, coming of age after 200 years. FEMS Microbiol. Rev. 24, 9±20. Mao, Y.M., Sho, Q., Li, Q.G., Sheng, J., 1991. RecA dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined site. Mol. Gen. Genet. 225, 234±240. Marsden, M.J., Vaughan, L.M., Foster, T.J., Secombe, C.J., 1996. A live (DaroA) Aeromonas salmonicida vaccine for furunculosis preferentially stimulates T-cell responses relative to B-cell responses in rainbow trout (Oncorhynchus mykiss). Infect. Immun. 64, 3863±3869. Mei, J.M., Nourbakhsh, F., Ford, C.W., Holden, D.W., 1997. Identi®cation of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol. Microbiol. 26, 399±407. Miller, J.H., 1992. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Miller, R.V., Kokjohn, T.A., 1990. General microbiology of recA. Environmental and evolutionary signi®cance. Annu. Rev. Microbiol. 44, 365±394. Pogson, C.A., Simmons, P.C., Strugnell, R.A., Hodgson, A.L.M., 1996. Cloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development. FEMS Microbiol. Lett. 142, 139±145. Simmons, C.P., Dunstan, S.J., Tachedjian, M., Krywult, J., Hodgson, A.L.M., Strugnell, R.A., 1998. Vaccine potential of attenuated mutants of Corynebacterium pseudotuberculosis in sheep. Infect. Immun. 66, 474± 479. Tatum, F.M., Mor®tt, D.C., Halling, S.M., 1993. Construction of a Brucella abortus RecA mutant and its survival in mice. Microb. Pathog. 14, 177±185. Walker, G.C., 1984. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48, 60±93. Winterling, K.W., Cha®n, D., Hayes, J.J., Sun, J., Levine, A.S., Yasbin, R.E., Woodgate, R., 1998. The Bacillus subtilis DinR binding site: rede®nition of the consensus sequence. J. Bacteriol. 180, 2201±2211. Young, G.M., Miller, V.L., 1997. Identi®cation of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis. Mol. Microbiol. 25, 319±328. Zagaglia, C., Casalino, M., Colonna, B., Conti, C., Calconi, A., Nicoletti, M., 1991. Virulence plasmids of enteroinvasive Escherichia coli and Shigella ¯exnerii integrate into a speci®c site on the host chromosome: integration greatly reduces expression of plasmid-carried virulence genes. Infect. Immun. 59, 792±799. Zyskind, J.W., Svitil, A.L., Stine, W.B., Biery, M.C., Smith, D.W., 1992. RecA protein of Escherichia coli and chromosome partitioning. Mol. Microbiol. 6, 2525±2537.