Evidence for the horizontal transfer of an unusual capsular polysaccharide biosynthesis locus in marine bacteria.

The most intensely studied of the Vibrio vulnificus virulence factors is the capsular polysaccharide (CPS). All virulent strains produce copious amounts of CPS. Acapsular strains are avirulent. The structure of the CPS from the clinical isolate ATCC 27562 is unusual. It is serine modified and contains, surprisingly, N-acetylmuramic acid. We identified the complete 25-kb CPS biosynthesis locus from ATCC 27562. It contained 21 open reading frames and was allelic to O-antigen biosynthesis loci. Two of the genes, murA(CPS) and murB(CPS), were paralogs of the murA(PG) and murB(PG) genes of the peptidoglycan biosynthesis pathway; only a single copy of these genes is present in the strain CMCP6 and YJ016 genomes. Although MurA(CPS) and MurB(CPS) were functional when expressed in Escherichia coli, lesions in either gene had no effect on CPS production, virulence, or growth in V. vulnificus; disruption of 8 other genes within the locus resulted in an acapsular phenotype and attenuated virulence. Thus, murA(CPS) and murB(CPS) were functional but redundant. Comparative genomic analysis revealed that while completely different CPS biosynthesis loci were found in the same chromosomal region in other V. vulnificus strains, most of the CPS locus of ATCC 27562 was conserved in another marine bacterium, Shewanella putrefaciens strain 200. However, the average GC content of the CPS locus was significantly lower than the average GC content of either genome. Furthermore, several of the encoded proteins appeared to be of Gram-positive and archaebacterial origin. These data indicate that the horizontal transfer of intact and partial CPS loci drives CPS diversity in marine bacteria.

Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus.

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.

Identification of a Wzy polymerase required for group IV capsular polysaccharide and lipopolysaccharide biosynthesis in Vibrio vulnificus.

The estuarine bacterium Vibrio vulnificus is a human and animal pathogen. The expression of capsular polysaccharide (CPS) is essential for virulence. We used a new mini-Tn10 delivery vector, pNKTXI-SceI, to generate a mutant library and identify genes essential for CPS biosynthesis. Twenty-one acapsular mutants were isolated, and the disrupted gene in one mutant, coding for a polysaccharide polymerase (wzy), is described here. A wecA gene initiating glycosyltransferase was among the genes identified in the region flanking the wzy gene. This, together with the known structure of the CPS, supports a group IV capsule designation for the locus; however, its overall organization mirrored that of group I capsules. This new arrangement may be linked to our finding that the CPS region appears to have been recently acquired by horizontal transfer. Alcian Blue staining and immunoblotting with antisera against the wild-type strain indicated that the wzy::Tn10 mutant failed to produce CPS and was attenuated relative to the wild type in a septicemic mouse model. Interestingly, immunoblotting revealed that the mutant was also defective in lipopolysaccharide (LPS) production. However, the core-plus-one O-antigen pattern typical of wzy mutations was apparent. CPS production, LPS production, and virulence were restored following complementation with the wild-type wzy gene. Hence, Wzy participates in both CPS and LPS biosynthesis and is required for virulence in strain 27562. To our knowledge, this is the first functional demonstration of a Wzy polysaccharide polymerase in V. vulnificus and is the first to show a link between LPS and CPS biosynthesis.

Activators of cylindrical proteases as antimicrobials: identification and development of small molecule activators of ClpP protease.

ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.

Structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562.

The structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562 is presented. LPS hydrolysis gave two oligosaccharides, OS-1 and OS-2, as well as lipid A. NMR spectroscopic data corresponded to the presence of one Kdo residue, one beta-glucopyranose, three heptoses, one glyceric acid, one acetate, three PEtN, and one 5,7-diacylamido-3,5,7,9-tetradeoxynonulosonic acid residue (pseudaminic acid, Pse) in OS1. OS2 differed form OS 1 by the absence of glyceric acid, acetate, and Pse residues. Lipid A was analyzed for fatty acid composition and the following fatty acids were found: C14:0, C12:0-3OH, C16:0, C16:1, C14:0-3OH, C18:0, C18:1 in a ratio of 1:3:3:1:2.5:0.6:0.8.

Altered Expression of PERK Receptor Kinases in Arabidopsis Leads to Changes in Growth and Floral Organ Formation.

The proline-rich, extensin-like receptor kinase (PERK) family is characterized by a putative extracellular domain related to cell wall proteins, followed by a transmembrane domain and kinase domain. The original member, PERK1, was isolated from Brassica napus (BnPERK1) and 15 PERK1-related members were subsequently identified in the Arabidopsis thaliana genome. Ectopic expression and antisense suppression studies were performed using the BnPERK1 cDNA under the control of the 35S CaMV constitutive promoter and introduced into Arabidopsis. In the case of antisense suppression, the BnPERK1 cDNA shared sufficient sequence similarity to suppress several members of the At PERK family. In both sets of transgenic Arabidopsis, several heritable changes in growth and development were observed. Antisense BnPERK1 transgenic Arabidopsis showed various growth defects including loss of apical dominance, increased secondary branching, and floral organ defects. In contrast, Arabidopsis plants ectopically expressing BnPERK1 displayed a prolonged lifespan with increased lateral shoot production and seed set. Along with these phenotypic changes, aberrant deposits of callose and cellulose were also observed, suggestive of cell wall changes as a consequence of altered PERK expression.

A comprehensive expression analysis of the Arabidopsis proline-rich extensin-like receptor kinase gene family using bioinformatic and experimental approaches.

The Arabidopsis proline-rich extensin-like receptor kinase (PERK) family consists of 15 predicted receptor kinases. A comprehensive expression analysis was undertaken to identify overlapping and unique expression patterns within this family relative to their phylogeny. Three different approaches were used to study AtPERK gene family expression, and included analyses of the EST, MPSS and NASCArrays databases as well as experimental RNA blot analyses. Some of the AtPERK members were identified as tissue-specific genes while others were more broadly expressed. While in some cases there was a good association between these different expression patterns and the position of the AtPERK members in the kinase phylogeny, in other cases divergence of expression patterns was seen. The PERK expression data identified by the bioinformatics and experimental approaches were found generally to show similar trends and supported the use of data from large-scale expression studies for obtaining preliminary expression data. Thus, the bioinformatics survey for ESTs and microarrays is a powerful comprehensive approach for obtaining a genome-wide view of genes in a multigene family.