Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster (original) (raw)
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Journal of bacteriology, 1997
A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5alpha, and clones were screened for serum resistance. One clone was found resistant to serum, to bacteriocin 28b, and to bacteriophages TuIa and TuIb. This clone also showed O antigen in its lipopolysaccharide. Subcloning and sequencing experiments showed that a 2,124-bp DNA fragment containing the rmlD and wbbL genes was responsible for the observed phenotypes. On the basis of amino acid similarity, we suggest that the 288-residue RmlD protein is a dTDP-L-rhamnose synthase. Plasmid pJT102, containing only the wbbL gene, was able to induce O16-antigen production and serum resistance in E. coli DH5alpha. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rJb-50 mutation in E. coli K-12 derivatives, despite the low level of amino acid identity between WbbL and the E. coli rhamnosyltransferase (24.80%). S. marcescens N28b rmlD and wbbL...
Cloning and DNA sequence analysis of a bacteriocin gene of Serratia marcescens
Journal of General Microbiology, 1992
Serratia marcescens N28b synthesized and secreted a bacteriocin, with a molecular mass of 45 kDa, which was capable of inhibiting the growth of Escherichia coli. The expression of this bacteriocin was negligible unless induced with mitomycin C. The genes encoding the bacteriocin were cloned in plasmid pBR328. E. coli harbouring recombinant plasmid pBA189 or pBA289 expressed the Serratia marcescens N28b bacteriocin. The nucleotide sequence of the bss gene (Serratia marcescens N28b bacteriocin structural gene) was determined. The predicted amino acid sequence of the carboxy-terminal part of the bacteriocin 28b had a high degree of similarity to the pore-forming domains of colicins A, E1, B, N, Ia and Ib.
Journal of bacteriology, 1996
Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting th...
Molecular Microbiology, 1991
The TonB protein plays a key role in the energycoupled transport of iron siderophores, of vitamin B^z, and of colicins of the B-group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particuiar amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues {M^ 27389), which is unusually proline-rich and contains the tandem sequences (Glu-Pro)s and (Lys~Pro)5. In contrast to the TonB proteins of E coli and Salmonella fyphimurium., translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the A/-terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The 5. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages Tl and 'I'8O, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins la and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore Tl sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteoiytic degradation, it was stabilized in E. co//by the ExbBD proteins. In £. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.
Japanese Journal of Infectious Diseases, 2014
In this study, genetic environments of the transferable plasmid-mediated bla CTX-M-3 gene were characterized among 14 isolates of cefotaxime-resistant Serratia marcescens using PCR and BLAST DNA sequence analysis. A total of 3 types of genetic architectures in the regions surrounding this bla CTX-M-3 gene were identified. Type I architecture was characterized by the presence of a complete insertion sequence of tnpA-ISEcp1, identified as interrupting a reverse IS26 sequence in the upstream region of the bla CTX-M-3 gene. A reverse-directional orf477 fragment was located downstream of the bla CTX-M-3 gene, which was in the same direction of the mucA gene. A common region containing the orf513 element was located upstream of the mucA gene. Moreover, a copy of the 3?-CS2 element was located immediately upstream of the orf513 element. A novel complex class 1 integron was characterized by the presence of the dfrA19 gene, which was flanked by two copies of class 1 integrons. This is the first report to describe the dfrA19 gene within a novel complex class 1 integron in S. marcescens isolates from Taiwan. This novel complex class 1 integron structure was located distantly upstream of the bla CTX-M-3 gene.
FEMS Microbiology Letters, 2010
We report a Serratia marcescens and an Escherichia coli isolate simultaneously detected in the same patient. Both isolates showed susceptibility patterns suggestive of harbouring a plasmid-mediated AmpC beta-lactamase (pACBL) and a plasmid-encoded quinolone resistance (PMQR). PCR-based replicon, MOB typing, plasmid profile and Southern hybridization analyses revealed that both isolates coharboured bla(DHA-1) and qnrB genes on the same IncL/M-MOB(P13) plasmid approximately 70 kb in size. Together with the fact that both plasmids were conjugative in the laboratory, these results strongly suggest that a horizontal transfer event could take place in vivo. This is the first report of an isolate of S. marcescens harbouring a pACBL. The only phenotypic method that suggests the presence of a pACBL in an isolate harbouring an inducible chromosomal AmpC enzyme is the observation of scattered colonies near the edge of the inhibition zones of some beta-lactams. The presence of both resistance genes on the same plasmid and the reported increase in PMQR could perhaps explain the widespread distribution of bla(DHA-1) genes.
Microbiology, 1995
A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli and clones were screened for a bacteriocin 28b insensitive phenotype. One clone was found that showed partial resistance to bacteriocin 28b. By using Tn5tac1 insertions it was shown that this phenotype was due to the expression in E. coli of an outer-membrane protein of 17 kDa (Omp4). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 515 bp. The deduced amino acid sequence has 172 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues and, when compared to other enterobacterial outer-membrane proteins, most closely resembles a family of small outer-membrane proteins of Enterobacteriaceae whose known functions appear to be related with virulence. Immunoblotting experiments showed that Omp4 is present in 15 biotypes of S. marcescens. The bacteriocin 28b resistance phenotype conferred on E. coli by Omp4 appears to be pleiotropic since overexpression of the Omp4-encoding gene leads to a decrease in the amount of OmpA, OmpF and/or OmpC; OmpA and OmpF are the receptors for bacteriocin 28b in E. coli.
Bacteriocin 28b, a chromosomally encoded bacteriocin produced by most Serratia marcescens biotypes
Research in Microbiology, 1995
Twenty.dx ~atia marcescens strains belonging to fifteen different biotvpes were found to produce bactedccins ab-tive against E~ia coll. On the basis of the pattern of bacteriofin sensitivity of E. coil mutant strains, immunological assays and Southern blot hybridization with a probe for the S. marcescens bss (bacteriocin 28b ttmetural) gene, it was concluded that all these strains apperenth/prodeee chromosomalb/encoded baetedocins related to bectedocin 28b. To confirm thb conclusion, the genu encoding the bactldocin produced by one of these strains (S. marcesce~ JF246) were cloned in plasmkl pBR328. E. co//herboudng recombinant plmnnid pl)G301 prodluced a baetedocin aeti~ against E. co//and immunologically related to bacterioeio 2ab. Immunoblotti~ experiments showed that beetedocins 281) and L appear to have the same apparent molecular mass (45 kDa). Furthermore, recombinant plasmid pl)G301 DNA hybridized with a bss 9ene probe.
Expression of Serratia marcescens extracellular proteins requires recA
Journal of Bacteriology, 1990
A previously described regulatory mutation which abolishes expression of the extracellular nuclease of Serratia marcescens is shown to be a mutation of the Serratia recA gene. The defect in nuclease expression could be restored by introducing a plasmid carrying the recA gene of Escherichia coli. The DNA sequence of the Serratia gene is very similar to that of the E. coli gene. The putative LexA-binding site of the Serratia recA gene is almost identical to that of E. coli, along with the promoter. A similar LexA-binding site can also be found upstream of the nuclease gene. As expected from this finding, we show that nuclease expression can be induced by SOS-inducing agents such as mitomycin C. Although inducible in S. marcescens, the nuclease was expressed only at the uninduced levels in E. coli and could not be induced by mitomycin C. The extracellular chitinase and lipase were similarly affected by the mutations altering nuclease expression and were also induced by mitomycin C.
Replicon typing of 71 multiresistant Serratia marcescens strains
Research in Microbiology, 1994
Repticon typing is the identification of plasmids by hybridization with specific DNA probes which contain the genes involved in plasmid maintenance. This new method has been used to classify plasmids into replicon Irep) groups which can often be correlated with incompatibility (lnc} groups. We studied 71 multiresistant Serratia marceseens strains with 19 rap probes constructed from reference plasmid replicons belonging to known tnc groups. These probes are known to react with enteric bacterial plasmids, However. they did not repcesent the totality of the thirty known Inc groups. For 52 % of the studied strains, plasmids were identified and classified into groups FIB, FIC, FIIA, HI2, L/M, N, B/O, P, W, Y and Comg. Most (79 %l of the plasmid preparations hybridized with a single rep probe, and 21% hybridized with two different probes. Electrophoretie analysis of DNA suggested that double hybridization could result from the presence of either two different Inc plasmids in the same strain (e.g. S37] or one single plasmid with a multireplicon (e.g. $113).