Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis.

A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.

LysR-type transcriptional regulator ChiR is essential for production of all chitinases and a chitin-binding protein, CBP21, in Serratia marcescens 2170.

To identify the genes required for chitinase production by Serratia marcescens 2170, various Tn5 mutants somehow defective in chitinase production were isolated in a previous study. In order to identify the mutated gene in one of the chitinase-deficient mutants, N1, DNA regions flanking the Tn5 insertion were cloned and sequenced. Sequence comparison showed that the mutation occurred in the ORF located between chiB and cbp, which encode chitinase B and chitin-binding protein CBP21, respectively. The ORF encodes a 313-amino acid polypeptide which has significant similarity with various LysR-type transcriptional regulators, and thus the gene was designated chiR. Targeted mutagenesis confirmed that disruption of the chiR gene results in the phenotype of N1. Gel mobility shift assays using partially purified ChiR protein demonstrated that this protein specifically binds to the intergenic region between chiR and cbp. These results strongly suggest that ChiR is a LysR-type transcriptional regulator which is essential for production of all chitinases and CBP21.

Cloning and sequencing of the Klebsiella pneumoniae O5 wb gene cluster and its role in pathogenesis.

One representative recombinant clone encoding Klebsiella pneumoniae O5-antigen lipopolysaccharide (LPS) was found upon screening for serum resistance in a cosmid-based genomic library of K. pneumoniae KT769 (O5:K57) introduced into Escherichia coli DH5alpha. A total of eight open reading frames (wb(O5) gene cluster) were necessary to produce K. pneumoniae O5-antigen LPS in E. coli K-12. The enzymatic activities proposed for the wb(O5) gene cluster are in agreement with the activities proposed for the biosynthesis of K. pneumoniae O5-antigen LPS. Using the complete DNA sequence of the K. pneumoniae wb(O5) gene cluster, we obtained (by single or double recombination) genetically well-characterized mutants devoid only of this O5-antigen LPS. Finally, using these O5(-) mutants and the corresponding wild-type strains or complemented mutants with the wb(O5) gene cluster (O5(+) strains), we found that the presence of K. pneumoniae O5-antigen LPS is essential for some pathogenic features like serum resistance, adhesion to uroepithelial cells, and colonization (experimental infections) of the urinary tract in rats.

Two genes from the capsule of Aeromonas hydrophila (serogroup O:34) confer serum resistance to Escherichia coli K12 strains.

The Escherichia coli DH5alpha strain as well as other K12-derived strains are unable to produce O-specific lipopolysaccharide and are thus rough and serum-sensitive. One representative recombinant clone (COS-SR1) containing Aeromonas hydrophila (serogroup O:34) chromosomal DNA conferred serum resistance to E. coli K12 strains. Genetic, biochemical, and immunological studies suggested that the two genes (orf1 and wcaJ) identified in a subclone (pAC-SR9) of COS-SR1 are necessary for the production of the colanic acid capsule at 37 degrees C on E. coli DH5alpha, rendering the strain serum-resistant. A. hydrophila strains from serogroup O:34 are able to produce capsule when they grow both in synthetic medium and in an autolysate of fish viscera. However, defined wcaJ insertion mutants of A. hydrophila 1051-88 (serogroup O:34) are unable to produce capsule on these media. This strongly suggests that both genes belong to the gene cluster responsible for capsule production (wca) of A. hydrophila 1051-88 (serogroup O:34).

Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process.

Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster.

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, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster.

A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

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 mutants were constructed by mobilization of suicide plasmids containing a portion of rmlD or wbbL. These insertion mutants were unable to produce O antigen; since strain N28b produces O4 antigen, these results suggest that both genes are involved in O4-antigen biosynthesis.

Genetic analysis of the chitinase system of Serratia marcescens 2170.

To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.

Molecular characterization of a 17-kDa outer-membrane protein from Klebsiella pneumoniae.

A cosmid-based genomic library of Klebsiella pneumoniae 52145 (O1:K2) was introduced into Escherichia coli, and clones were screened for the bacteriocin 28b resistance phenotype. One clone was found which conferred 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 (OMP) with an apparent molecular mass of 17 kDa (OmpK17). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 510 bp. The deduced amino acid sequence has 170 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues. When compared with other enterobacterial OMPs, OmpK17 most closely resembles members of a family of small OMPs of Enterobacteriaceae the known functions of which appear to be related to virulence. Immunoblotting experiments showed that OmpK17 is also present in various K. pneumoniae strains belonging to different O and K serotypes.

Taxonomic changes in tailed phages of enterobacteria.

Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species beta 4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

The role of the capsular polysaccharide of Aeromonas hydrophila serogroup O:34 in the adherence to and invasion of fish cell lines.

The ability of Aeromonas hydrophila serogroup O:34 strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared. The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation. However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade the fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation. Isogenic unencapsulated mutants grown under conditions promoting capsule formation showed a lower ability to invade the fish cell lines than the parental capsulated strains. From these results, we concluded that the capsular polysaccharide is an important factor in intracellular invasion.

Taxonomic changes in tailed phages of enterobacteria.

Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species b4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

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 that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.

Genetic evidence for an activator required for induction of colicin-like bacteriocin 28b production in Serratia marcescens by DNA-damaging agents.

Bacteriocin 28b production is induced by mitomycin in wild-type Serratia marcescens 2170 but not in Escherichia coli harboring the bacteriocin 28b structural gene (bss). Studies with a bss-lacZ transcriptional fusion showed that mitomycin increased the level of bss gene transcription in S. marcescens but not in the E. coli background. A S. marcescens Tn5 insertion mutant was obtained (S. marcescens 2170 reg::Tn5) whose bacteriocin 28b production and bss gene transcription were not increased by mitomycin treatment. Cloning and DNA sequencing of the mutated region showed that the Tn5 insertion was flanked by an SOS box sequence and three genes that are probably cotranscribed (regA, regB, and regC). These three genes had homology to phage holins, phage lysozymes, and the Ogr transcriptional activator of P2 and related bacteriophages, respectively. Recombinant plasmid containing this wild-type DNA region complemented the reg::Tn5 regulatory mutant. A transcriptional fusion between a 157-bp DNA fragment, containing the apparent SOS box upstream of the regA gene, and the cat gene showed increased chloramphenicol acetyltransferase activity upon mitomycin treatment. Upstream of the bss gene, a sequence similar to the consensus sequence proposed to bind Ogr protein was found, but no sequence similar to an SOS box was detected. Our results suggest that transcriptional induction of bacteriocin 28b upon mitomycin treatment is mediated by the regC gene whose own transcription would be LexA dependent.

Detección de Escherichia coli toxigénica (LT) mediante la reacción en cadena de la polimerasa [The detection of toxigenic Escherichia coli (LT) by the polymerase chain reaction].

In this paper it is described the detection enteroxigenic Escherichia coli LT (+). This method is based on the amplification of a DNA fragment of 400 pairs of bases by polymerase chain reaction (PRC). The oligonucleotides were designed by the authors and the characteristic patterns were observed when the samples were submitted to an electrophoresis in an Agarose gel at 2%. The PCR had positive results with the strains of Escherichia coli 0:149 K; 88 (LT+) collection and with 20 strains isolated from patients with acute diarrhea. Negative results were found in Escherichia coli 0:101 K:99 NM (ST+), Vibrio cholerae 01 and Aeromonas hydrophila.

Bacteriocin 28b from Serratia marcescens N28b: identification of Escherichia coli surface components involved in bacteriocin binding and translocation.

Serratia marcescens N28b produces bacteriocin 28b, active against Escherichia coli. Bacteriocin sensitivity tests performed on a collection of E. coli envelope mutants, and isolation and characterization of E. coli bacteriocin-28b-insensitive mutants, showed that the core lipopolysaccharide, outer membrane proteins OmpA and OmpF, and TolQ, TolA, and TolB proteins are involved in bacteriocin 28b lethal activity. These mutants are assayed for bacteriocin 28b sensitivity under normal and bypass conditions, and their bacteriocin-binding ability was determined. The results obtained suggest that the core lipopolysaccaride and outer membrane proteins OmpA and OmpF are involved in bacteriocin 28b binding. Furthermore, bacteriocin 28b translocation requires proteins TolA, TolB, and TolQ.

A 17 kDa outer-membrane protein (Omp4) from Serratia marcescens confers partial resistance to bacteriocin 28b when expressed in Escherichia coli.

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.

Twenty-six Serratia marcescens strains belonging to fifteen different biotypes were found to produce bacteriocins active against Escherichia coli. On the basis of the pattern of bacteriocin sensitivity of E. coli mutant strains, immunological assays and Southern blot hybridization with a probe for the S. marcescens bss (bacteriocin 28b structural) gene, it was concluded that all these strains apparently produce chromosomally encoded bacteriocins related to bacteriocin 28b. To confirm this conclusion, the genes encoding the bacteriocin produced by one of these strains (S. marcescens JF246) were cloned in plasmid pBR328. E. coli harbouring recombinant plasmid pDG301 produced a bacteriocin active against E. coli and immunologically related to bacteriocin 28b. Immunoblotting experiments showed that bacteriocins 28b and L appear to have the same apparent molecular mass (45 kDa). Furthermore, recombinant plasmid pDG301 DNA hybridized with a bss gene probe.

Protection against bacteriocin 28b in Serratia marcescens is apparently not related to the expression of an immunity gene.

The gene encoding bacteriocin 28b from Serratia marcescens N28b (bss gene) has been cloned in Escherichia coli and its nucleotide sequence has been determined. The genetic determinants coding for other well-characterized bacteriocins from enterobacteria (colicins) are located in plasmids and they have always been shown to contain a gene responsible for immunity located downstream from the bacteriocin structural gene. In some cases there is another gene located downstream from the immunity gene, which is responsible for bacteriocin release. Analysis of bacteriocin 28b release and the sensitivity to this bacteriocin of E. coli strains harbouring recombinant plasmids containing the bss gene showed that bacteriocin 28b is not released from the cell in these strains and that their phenotypic insensitivity is not associated with any region close to the structural gene. The nucleotide sequence of the region downstream from the bss gene contains two putative open reading frames transcribed in the opposite direction to the bss gene. These open reading frames apparently encode proteins that seem not to be involved in bacteriocin immunity or release. Moreover, a S. marcescens N28b genomic library was screened and no immunity gene was found. Therefore, bacteriocin 28b differs greatly from the bacteriocins from other enterobacteria, and in the following senses it is unique: firstly, the gene encoding bacteriocin 28b seems to be located on the chromosome, and secondly, insensitivity to this bacteriocin in S. marcescens N28b is not associated with the expression of an immunity gene.

Cloning and DNA sequence analysis of a bacteriocin gene of Serratia marcescens.

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.