Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.     

A tetracycline efflux gene on Bacteroides transposon Tn4400 does not contribute to tetracycline resistance.

Previously, we demonstrated that the Bacteroides transposon Tn4351, which confers tetracycline resistance only on aerobically grown Escherichia coli, carries a gene that codes for a tetracycline-inactivating enzyme (B. S. Speer and A. A. Salyers, J. Bacteriol. 170:1423-1429, 1988). However, Park et al. (B. H. Park, M. Hendricks, M. H. Malamy, F. P. Tally, and S. B. Levy, Antimicrob. Agents Chemother. 31:1739-1743, 1987) showed that E. coli carrying a closely related transposon, Tn4400, exhibits energy-dependent efflux of tetracycline as well as tetracycline-inactivating activity (B. H. Park and S. B. Levy, Antimicrob. Agents Chemother. 32:1797-1800, 1988). This result raised the question of whether efflux or inactivation or a combination of the two was necessary for resistance conferred by both transposons. We showed that cells carrying Tn4351 did not exhibit the clear-cut efflux activity seen with cells carrying Tn4400 but rather exhibited a tetracycline accumulation profile which could be explained solely on the basis of inactivation of tetracycline in the cytoplasm and rapid diffusion of altered tetracycline out of the cell. Additionally, we were able to clone the efflux and tetracycline-modifying genes of Tn4400 separately. The region carrying the efflux gene spanned one of the two regions in which Tn4400 differs from Tn4351. A clone containing the corresponding region of Tn4351 did not exhibit efflux. Thus, it appears that Tn4351 does not have the efflux gene and that efflux makes no contribution to the resistance conferred by Tn4351. The MIC for cells carrying the subclone from Tn4400 that contained only the gene for tetracycline inactivation was the same that for cells carrying both the inactivation and efflux genes. Cells carrying only the gene for tetracycline efflux were tetracycline sensitive. This was true even when the efflux gene was on a high-copy-number plasmid which increased the level of efflux to that associated with the Tcr gene on pBR328. These results indicate that efflux activity does not contribute significantly to the tetracycline resistance conferred by Tn4400.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

Transposition of Tn4351 in Porphyromonas gingivalis.

Genetic analysis of Porphyromonas gingivalis, an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751::*omega 4 has previously been used as a suicide vector to demonstrate transposition of Tn4351 in B. uniformis. The erythromycin resistance gene on Tn4351 functions in Bacteroides and Porphyromonas. Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 x 10(-7) from matings between Escherichia coli HB101 containing R751::*omega 4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn4351 and half contained both Tn4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn4351-mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis-Tn4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis.     

Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae.

Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.     

Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase.

Two transposons, Tn4351 and Tn4400, which were originally isolated from the obligate anaerobe Bacteroides fragilis, carry a tetracycline resistance (Tcr) gene that confers resistance only on aerobically grown Escherichia coli. This aerobic Tcr gene, designated tetX, has been shown previously to act by chemically modifying tetracycline in a reaction that appears to require oxygen. We have now obtained the DNA sequence of tetX and 0.6 kb of its upstream region from Tn4400. Analysis of the DNA sequence of tetX revealed that this gene encoded a 43.7-kDa protein. The deduced amino acid sequence of the amino terminus of the protein had homology with a number of enzymes, all of which had in common a requirement for NAD(P). In an earlier study, we had observed that disrupted cells, unlike intact cells, could not carry out the alteration of tetracycline. We have now shown that if NADPH (1 mM) is added to the disrupted cell preparation, alteration of tetracycline occurs. Thus, TetX appears to be an NADP-requiring oxidoreductase. Tn4400 conferred a fivefold-lower level of tetracycline resistance than Tn4351. This finding appears to be due to a lower level of expression of the tetX on Tn4400, because the activity of a tetX-lacZ fusion from Tn4400 was 10-fold lower than that of the same fusion from Tn4351. A comparison of the sequence of the tetX region on Tn4351 with that on Tn4400 showed that the only difference between the upstream regions of the two transposons was a 4-base change 350 bp upstream of the start of the tetX coding region. The 4-base change difference creates a good consensus -35 region on Tn4351 that is not present on Tn4400 and could be creating an extra promoter.     

Development of techniques to genetically manipulate members of the genera Cytophaga, Flavobacterium, Flexibacter, and Sporocytophaga.

The Bacteroides-Cytophaga-Flavobacterium branch of the eubacterial phylogenetic tree contains a diverse group of bacterial species. Techniques for the genetic manipulation of Bacteroides spp. are well developed (A. A. Salyers, N. B. Shoemaker, and E. P. Guthrie, Crit. Rev. Microbiol. 14:49-71, 1987). Recently we developed techniques to genetically manipulate the gliding bacterium Cytophaga johnsonae (M. J. McBride and M. J. Kempf, J. Bacteriol. 178:583-590, 1996). We now demonstrate that some of these techniques allow genetic manipulation of a number of environmentally or medically significant bacteria in this group. The Bacteroides transposon Tn4351 was introduced into Cytophaga hutchinsonii, Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadensis, Flexibacter sp. strain FS1, and Sporocytophaga myxococcoides by conjugation. Tn4351 integrated itself into the host chromosomes and conferred erythromycin resistance. We isolated several auxotrophic mutants of Flavobacterium meningosepticum following Tn4351 mutagenesis. The C. johnsonae-Escherichia coli shuttle vector pCP11 functioned in C. succinicans but not in the other bacteria. pLYL03 did not replicate in any of these bacteria and should function as a convenient suicide vector. The identification of a system of gene transfer, a selectable marker, a suicide vector, and a transposon that functions in these diverse bacteria allows genetic manipulations to be performed.     

Facilitated transfer of IncP beta R751 derivatives from the chromosome of Bacteroides uniformis to Escherichia coli recipients by a conjugative Bacteroides tetracycline resistance element.

The broad-host-range IncP beta plasmid R751 can mobilize itself from Escherichia coli to Bacteroides spp, but it is not maintained in Bacteroides spp. If R751 carries the Bacteroides transposon Tn4351, it can be integrated into the Bacteroides chromosome. Previously we showed that R751, integrated in the chromosome of Bacteroides uniformis, cannot mobilize itself out of B. uniformis into E. coli or isogenic B. uniformis strains. In this report, we showed that if the Bacteroides conjugative tetracycline resistance element Tcr ERL was coresident with the R751 insertion in B. uniformis, derivatives of R751 were transferred to E. coli, where they were recovered as plasmids. The most common derivatives were R751::Tn4351 and R751::IS4351, but some strains transferred R751 derivatives, containing additional DNA segments ranging in size from 10 to 23 kilobases. These DNA inserts cross-hybridized with chromosomal DNA from B. uniformis which did not carry the Tcr ERL element. Therefore, the inserts appeared to be segments of the wild-type B. uniformis chromosome and were not associated with the Tcr ERL element. The transfer of integrated R751 from B. uniformis was independent of the RecA phenotype of the E. coli recipients and did not appear to be due to transfer of B. uniformis chromosomal DNA, followed by RecA-dependent recombination between homologous IS4351 sequences to form the resultant R751 plasmid derivatives. Consistent with this, no transfer of Tn4351 (associated with the cointegrated R751) from B. uniformis donors to isogenic B. uniformis recipients was detected (< 10(-8)). Our data support the hypothesis that R751 excises from the B. uniformis chromosome by recombination involving flanking Tn4351 or IS4351 sequences and forms nonreplicating circles. The mobilization of these circular forms out of B. uniformis to E.coli is then facilitated by the Tcr ERL element.     

Identification of a second endogenous Porphyromonas gingivalis insertion element.

In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P. gingivalis was identified. Nucleotide sequence analysis of the Tn4351 insertion site in a P. gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351. PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence. Results of Southern hybridization of chromosomal DNA isolated from several P. gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P. gingivalis strains. Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

The Agrobacterium tumefaciens rnd homolog is required for TraR-mediated quorum-dependent activation of Ti plasmid tra gene expression

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58DeltaaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.     

Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome.

Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.     

Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene.

Mutants with insertion mutations in the Pseudomonas aeruginosa protein F (oprF) gene were created in vivo by Tn1 mutagenesis of the cloned gene in Escherichia coli and in vitro by insertion of the streptomycin resistance-encoding omega fragment into the cloned gene, followed by transfer of the mutated protein F gene back to P. aeruginosa. Homologous recombination into the P. aeruginosa chromosome was driven by a bacteriophage F116L transduction method in the oprF::Tn1 mutants or Tn5-instability in the oprF::omega mutants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting demonstrated that the resultant oprF insertion mutants had lost protein F, whereas restriction digestion and Southern blotting experiments proved that the mutants contained a single chromosomal oprF gene with either Tn1 or omega inserted into it. It has been proposed that protein F has a role in antibiotic uptake in P. aeruginosa. Measurement of antibiotic resistance levels showed small to marginal increases in resistance, compared with that of the parent P. aeruginosa strain, to a variety of beta-lactam antibiotics. Protein F-deficient mutants had altered barrier properties as revealed by a three- to fivefold increase in the uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine.     

Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics

In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.     

Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division

Flavobacterium johnsoniae is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn4351 mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to Escherichia coli ftsX. Two of the remaining 16 filamentous mutants also carried insertions in ftsX. Introduction of the wild-type F. johnsoniae ftsX gene restored motility and normal cell morphology to each of the three ftsX mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In E. coli, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in F. johnsoniae ftsX may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.     

阴沟肠杆菌B8拮抗活性基因‘admA’及上游调控序列的克隆与功能鉴定

为了阐明水稻白叶枯病拮抗菌阴沟肠杆菌B8的作用机理,文章采用转座子标签法和染色体步移技术克隆到突变株B8B中Tn5插入位点周边拮抗活性相关片段,并通过基因敲除验证了获得的拮抗相关片段admA’上游调控序列的功能。以转座子中Kan抗性基因为标签,克隆了B8B菌株中Tn5插入位点左侧2 608 bp序列,经两次染色体步移得到Tn5插入位点右侧的2 354 bp序列。序列拼接后获得B8菌株拮抗相关序列4 611 bp的Bcontig。生物信息学分析显示该序列含有7个ORF,分别对应于3-磷酸甘油醛脱氢酶(GADPH)基因的部分编码区、2个LysR家族转录调控因子、弧菌假设蛋白VSWAT3-20465及成团泛菌(Pantoea agglomerans)andrimid生物合成基因簇的admA、admB和部分admC基因序列。B8B菌株Tn5插入分别位于同源于弧菌假设蛋白的anrPORF及‘admA’基因上游200 bp和894 bp处。通过同源重组技术,借助敲除质粒pMB-BG,获得拮抗活性消失的突变株B-1和B-3。结果表明B8B突变株中Tn5的插入可能影响了anrP蛋白的转录和表达,进而调控拮抗物质编码基因簇的生物合成。B8菌株中拮抗物质相关基因是类似于andrimid生物合成基因簇的基因家族,其上游调控区对该抗生素的生物合成具有重要的作用。     

A glutamate-dependent acid resistance gene in Escherichia coli.

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.     

Survival and SOS induction in cisplatin-treated Escherichia coli deficient in Pol II,RecBCD and RecFOR functions

Cisplatin is a potent anticancer agent forming intrastrand-crosslinks in DNA. The efficacy of cisplatin in chemotherapy can be limited by the development of tumor resistances such as elevated DNA repair or damage tolerance. In Escherichia coli, cisplatin treatment causes induction of the SOS regulon resulting in elevated levels of DNA Pol II, DNA Pol IV, DNA Pol V, the cell division inhibitor SfiA (SulA), homologous recombination (HR) and DNA repair. In this work, the roles of Pol II and HR in facilitating resistance of E. coli to cisplatin are studied. SOS induction levels were measured by beta-galactosidase assays in cisplatin-treated and untreated E. coli PQ30 that has the lacZ gene fused to the sfiA promoter. Comparative studies were carried out with derivatives of PQ30 constructed by P1 transduction that have transposon insertions in the polB gene, the recB gene blocking the RecBCD pathway of HR and genes of the RecFOR pathway of HR. Resistance of E. coli strains to cisplatin as determined by plating experiments decreased in the following order: parent PQ30 strain, polB > recO, recR, recF > recB. Both the RecBCD and RecFOR pathways of HR are important for survival when E. coli is exposed to cisplatin, because treatment of double mutants deficient in both pathways reduced colony forming ability to 37% in 6-9min in comparison to 39-120min for single mutants. Pol II and RecF appear to function in two distinct pathways to initiate replication blocked due to damage caused by cisplatin because function of Pol II was required for survival in mutants deficient in the RecFOR pathway after 2h of cisplatin treatment. In contrast, Pol II was not required for survival in recB mutants. SOS induction was delayed in RecFOR deficient mutants but occurred at high levels in the recB mutant soon after cisplatin treatment in a RecFOR-dependent way. An SfiA independent, DNA damage dependent pathway is apparently responsible for the filamentous cells observed after cisplatin or MMC treatments of these SfiA defective strains.