A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives (original) (raw)
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Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster
Journal of bacteriology, 1999
The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigí, N. Piqué, N. Climent, S. Merino, S. Albertí, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581-7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5alpha by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, sugg...
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.
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.
FEMS Microbiology Letters, 2021
Serratia marcescens SCH909 is a multidrug resistant strain isolated in 1988 harboring three class 1 integrons. We wondered if these integrons were retained over time and if there were other antimicrobial resistant determinants contributing to its multidrug resistant profile. Genomic analysis showed a fourth multidrug resistance integron, a Tn7 transposon with dfrA1-sat2-ybeA-ybfA-ybfB-ybgA gene cassettes in the variable region. Insertion sequences were involved in the genesis of novel composite transposons in the L4 subtype plasmid pSCH909, such as Tn6824 carrying an arsenic regulon and two head to head class 1 integrons surrounded by two complete IS1. Remarkably, a novel chromosomal genomic island, SmaR, was identified, closely related to Multiple Antimicrobial Resistance Regions (MARR), usually found in AbaR0-type and AbGRI2-0 from global clones of Acinetobacter baumannii, and in M-type plasmids circulating in Enterobacteriaceae. Maintenance studies showed that the three class 1 i...
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.
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).
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, 2004
The gene cluster (waa) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis was identified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae led to the identification of five genes coding for products involved in the biosynthesis of a shared inner core structure: [L,D-HeppIII␣(137)-L,D-HeppII␣(133)-L,D-HeppI␣(135)-KdopI(442)␣KdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marcescens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K. pneumoniae, indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additional common features. Chemical and structural analysis of the major oligosaccharide from the core region of LPS of an O-antigen-deficient mutant of S. marcescens N28b as well as complementation analysis led to the following proposed structure: -Glc-(136)-␣-Glc-(134))-␣-D-GlcN-(134)-␣-D-GalA-[(241)-␣-D,D-Hep-(241)-␣-Hep]-(133)-␣-L,D-Hep[(741)-␣-L,D-Hep]-(133)-␣-L,D-Hep-[(441)--D-Glc]-(135)-Kdo. The D configuration of the -Glc, ␣-GclN, and ␣-GalA residues was deduced from genetic data and thus is tentative. Furthermore, other oligosaccharides were identified by ion cyclotron resonance-Fourier-transformed electrospray ionization mass spectrometry, which presumably contained in addition one residue of D-glycero-D-talooct-2-ulosonic acid (Ko) or of a hexuronic acid. Several ions were identified that differed from others by a mass of ؉80 Da, suggesting a nonstoichiometric substitution by a monophosphate residue. However, none of these molecular species could be isolated in substantial amounts and structurally analyzed. On the basis of the structure shown above and the analysis of nonpolar mutants, functions are suggested for the genes involved in core biosynthesis.
Journal of Bacteriology, 2004
The gene cluster (waa) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis was identified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae led to the identification of five genes coding for products involved in the biosynthesis of a shared inner core structure: [L,D-HeppIII␣(137)-L,D-HeppII␣(133)-L,D-HeppI␣(135)-KdopI(442)␣KdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marcescens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K. pneumoniae, indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additional common features. Chemical and structural analysis of the major oligosaccharide from the core region of LPS of an O-antigen-deficient mutant of S. marcescens N28b as well as complementation analysis led to the following proposed structure: -Glc-(136)-␣-Glc-(134))-␣-D-GlcN-(134)-␣-D-GalA-[(241)-␣-D,D-Hep-(241)-␣-Hep]-(133)-␣-L,D-Hep[(741)-␣-L,D-Hep]-(133)-␣-L,D-Hep-[(441)--D-Glc]-(135)-Kdo. The D configuration of the -Glc, ␣-GclN, and ␣-GalA residues was deduced from genetic data and thus is tentative. Furthermore, other oligosaccharides were identified by ion cyclotron resonance-Fourier-transformed electrospray ionization mass spectrometry, which presumably contained in addition one residue of D-glycero-D-talooct-2-ulosonic acid (Ko) or of a hexuronic acid. Several ions were identified that differed from others by a mass of ؉80 Da, suggesting a nonstoichiometric substitution by a monophosphate residue. However, none of these molecular species could be isolated in substantial amounts and structurally analyzed. On the basis of the structure shown above and the analysis of nonpolar mutants, functions are suggested for the genes involved in core biosynthesis.