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.The number of studies dealing with bacterial chitinasestheir biochemical properties, the structure of the genes encoding them, the catalytic mechanism involved, and their tertiary structures-has been increasing rapidly. The hydrolysis of chitin by chitinases is the most critical step in the degradation and utilization of chitin by bacteria. However, the study of chitinases is not sufficient to elucidate the process by which chitin is degraded and utilized by bacteria. The process involves a number of steps, including the recognition of chitin outside of the cell, the induction of chitinases, the maintenance of proper levels of chitinase production, and the incorporation and catabolism of degradation products. In this study our intent was to identify the genes involved in the degradation and utilization of chitin. Our long-term goal is to answer the following questions. How do bacteria recognize chitin? How is chitinase production induced and regulated? Why do chitinolytic bacteria produce multiple chitinases? How are degradation products processed? Our ultimate goal in these studies is a general understanding of how bacterial cells degrade and utilize chitin.We have studied the chitinase system of Bacillus circulans WL-12 and have provided comprehensive findings on biochemical properties, structure-function relationships, the identification of essential amino acid residues for catalytic activity, and the mechanisms by which multiple chitinases are generated (1, 2, 4, 23, 31-35). However, this bacterium is not a suitable...

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 ␤-D-Glc to the O-4 position of L-glycero-D-manno-heptose

Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide ␤-Glcp(1-6)-␣-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide ␣-Hep(1-4)-␣-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-D-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model.

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to ␣-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an ␣-D-galactopyranosyluronic acid (␣-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.In gram-negative bacteria the lipopolysaccharide (LPS) is one of the major structural and immunodominant molecules of the outer membrane. LPS consists of three domains: lipid A, core oligosaccharide, and O-specific antigen or O side chain. In smooth LPS, the core region is conceptually divided into two regions: a lipid A proximal inner core and an outer core that provides the attachment site for the O antigen (21). Comparison of the known core LPS structures from Enterobacteriaceae organisms reveals that the first outer core residue might be either glucose (Glc) or a galacturonic acid (GalA) residue. In the four known Escherichia coli core types and in Salmonella enterica, a substitution of the L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position for a Glcp residue was found (12). For Klebsiella pneumoniae, Proteus mirabilis, and Yersinia enterocolitica, a substitution of the L,D-HeppII at the O-3 position for an ␣-D-galacturonic acid residue (␣-DGalpA) residue has been described (20,29,30). On the other hand, in most of the Enterobacteriaceae studied, the core LPS contains inner core phosphoryl modifications (21), but K. pneumoniae core LPS is devoid of such modifications (29) (Fig. 1).Important roles in outer membrane permeability and in pathogenesis have been shown for the outer core and for the negative charges contributed by phosphoryl inner core modification in E. coli and/or S. enterica serovar Typhimurium (32,33,34). In view of the peculiarities of the K. pneumoniae core LPS, we sought in this work to determine the importance of the outer core LPS in K. pneumoniae outer membrane permeability and in pathogenesis. The previous knowledge of the K. pneumoniae waa gene cluster (the nomenclature proposed by Reeves et al. [23] for proteins and genes involved in core LPS biosynthesis is used in this work) and t...

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5␣, 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 DH5␣. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rfb-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.Serratia marcescens N28b is able to produce bacteriocin 28b (6,35). In studies aimed at identifying a putative bacteriocin 28b immunity system, a gene coding for a 17-kDa outer membrane protein (Omp4) has been previously characterized by us (11). The Omp4 protein was similar to a family of small outer membrane proteins of Enterobacteriaceae. Two members of this protein family, Ail from Yersinia enterocolitica and Rck from Salmonella typhimurium, have been associated with complement resistance, and when the genes encoding these proteins were expressed in Escherichia coli HB101, both conferred serum resistance (3, 13). Despite the high level of similarity among Omp4, Ail, and Rck proteins, E. coli HB101 or DH5␣ producing Omp4 protein remained serum sensitive but became bacteriocin 28b resistant (11).S. marcescens strains have been classified as serum sensitive, delayed serum sensitive, and serum resistant (32). As a general rule in gram-negative bacteria, with many exceptions, smooth strains (O ϩ ) are serum resistant while rough strains (O Ϫ ) are serum sensitive (30). S. marcescens N28b is a serum-resistant strain, and probably this phenotype is related to O-antigen production. We screened a previously constructed cosmidbased genomic library of this strain (11) introduced into E. coli DH5␣ (which is O Ϫ and serum sensitive) for serum resistance. In this work we report the characterization of a gene from S. marcescens N28b that confers the ability to produce E. coli O16-antigen lipopolysaccharide (LPS) and serum resistance on E. coli DH5␣ and other K-12-derived strains.S. marcescens N28b (7) and E. coli strains used in this study were grown in Luria-Bertani (LB)-Miller broth and LB-Miller agar (18) supplemented with ampicillin (50 g/ml), chloramphenicol (50 g/ml), kanamycin...

Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O ؊ :K ؊ phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

Comparison between the lipopolysaccharide (LPS) core structures of Aeromonas salmonicida subsp. salmonicida A450 and Aeromonas hydrophila AH-3 shows great similarity in the inner LPS core and part of the outer LPS core but some differences in the distal part of the outer LPS core (residues LD-Hep, D-Gal, and D-GalNAc). The three genomic regions encoding LPS core biosynthetic genes in A. salmonicida A450, of which regions 2 and 3 have genes identical to those of A. hydrophila AH-3, were fully sequenced. A. salmonicida A450 region 1 showed seven genes: three identical to those of A. hydrophila AH-3, three similar but not identical to those of A. hydrophila AH-3, and one without any homology to any well-characterized gene. A. salmonicida A450 mutants with alterations in the genes that were not identical to those of A. hydrophila AH-3 were constructed, and their LPS core structures were fully elucidated. At the same time, all the A. salmonicida A450 genes identical to those of A. hydrophila AH-3 were used to complement the previously obtained A. hydrophila AH-3 mutants for each of these genes. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. salmonicida A450. Furthermore, hybridization studies with internal probes for the A. salmonicida-specific genes using different A. salmonicida strains (strains of different subspecies or atypical strains) showed a unique or prevalent LPS core type, which is the one fully characterized for A. salmonicida A450.