Structure, function, and regulation of the essential virulence factor capsular polysaccharide of vibrio vulnificus

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen

Genetic analysis and prevalence studies of the brp exopolysaccharide locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus. © 2014 Garrison-Schilling et al

Evidence that thaxtomin C is a pathogenicity determinant of Streptomyces ipomoeae, the causative agent of streptomyces soil rot disease of sweet potato

Streptomyces ipomoeae is the causal agent of Streptomyces soil rot of sweet potato, a disease marked by highly necrotic destruction of adventitious roots, including the development of necrotic lesions on the fleshy storage roots. Streptomyces potato scab pathogens produce a phytotoxin (thaxtomin A) that appears to facilitate their entrance into host plants. S. ipomoeae produces a less-modified thaxtomin derivative (thaxtomin C) whose role in pathogenicity has not been examined. Here, we cloned and sequenced the thaxtomin gene cluster (txt) of S. ipomoeae, and we then constructed targeted txt mutants that no longer produced thaxtomin C. The mutants were unable to penetrate intact adventitious roots but still caused necrosis on storage-root tissue. These results, taken in context with previous histopathological study of S. ipomoeae infection, suggest that thaxtomin C plays an essential role in inter- and intracellular penetration of adventitious sweet potato roots by S. ipomoeae. Once inside the plant host, the pathogen uses one or more yet-tobe- determined factors to necrotize root tissue, including that of any storage roots it encounters. © 2012 The American Phytopathological Society

A novel phase variant of the cholera pathogen shows stress-adaptive cryptic transcriptomic signatures

© 2016 The Author(s). Background: In a process known as phase variation, the marine bacterium and cholera pathogen Vibrio cholerae alternately expresses smooth or rugose colonial phenotypes, the latter being associated with advanced biofilm architecture and greater resistance to ecological stress. To define phase variation at the transcriptomic level in pandemic V. cholerae O1 El Tor strain N16961, we compared the RNA-seq-derived transcriptomes among the smooth parent N16961, its rugose derivative (N16961R) and a smooth form obtained directly from the rugose at high frequencies consistent with phase variation (N16961SD). Results: Differentially regulated genes which clustered into co-expression groups were identified for specific cellular functions, including acetate metabolism, gluconeogenesis, and anaerobic respiration, suggesting an important link between these processes and biofilm formation in this species. Principal component analysis separated the transcriptome of N16961SD from the other phase variants. Although N16961SD was defective in biofilm formation, transcription of its biofilm-related vps and rbm gene clusters was nevertheless elevated as judged by both RNA-seq and RT-qPCR analyses. This transcriptome signature was shared with N16961R, as were others involving two-component signal transduction, chemotaxis, and c-di-GMP synthesis functions. Conclusions: Precise turnarounds in gene expression did not accompany reversible phase transitions (i.e., smooth to rugose to smooth) in the cholera pathogen. Transcriptomic signatures consisting of up-regulated genes involved in biofilm formation, environmental sensing and persistence, chemotaxis, and signal transduction, which were shared by N16961R and N16961SD variants, may implicate a stress adaptation in the pathogen that facilitates transition of the N16961SD smooth form back to rugosity should environmental conditions dictate

Genome content and phylogenomics reveal both ancestral and lateral evolutionary pathways in plant-pathogenic Streptomyces species

© 2016, American Society for Microbiology. All Rights Reserved. Streptomyces spp. are highly differentiated actinomycetes with large, linear chromosomes that encode an arsenal of biologically active molecules and catabolic enzymes. Members of this genus are well equipped for life in nutrient-limited environments and are common soil saprophytes. Out of the hundreds of species in the genus Streptomyces, a small group has evolved the ability to infect plants. The recent availability of Streptomyces genome sequences, including four genomes of pathogenic species, provided an opportunity to characterize the gene content specific to these pathogens and to study phylogenetic relationships among them. Genome sequencing, comparative genomics, and phylogenetic analysis enabled us to discriminate pathogenic from saprophytic Streptomyces strains; moreover, we calculated that the pathogen-specific genome contains 4,662 orthologs. Phylogenetic reconstruction suggested that Streptomyces scabies and S. ipomoeae share an ancestor but that their biosynthetic clusters encoding the required virulence factor thaxtomin have diverged. In contrast, S. turgidiscabies and S. acidiscabies, two relatively unrelated pathogens, possess highly similar thaxtomin biosynthesis clusters, which suggests that the acquisition of these genes was through lateral gene transfer

Further characterization of Vibrio vulnificus rugose variants and identification of a capsular and rugose exopolysaccharide gene cluster

Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 μg/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus, designated the wcr (capsular and rugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen. Copyright © 2008, American Society for Microbiology. All Rights Reserved

Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related Streptomyces Plasmid pSB24.2

A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24.2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems

The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon

Conjugal transfer of circular plasmids in Streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded DNA by an as yet uncharacterized intercellular route. Efficient transfer of the circular streptomycete plasmid pIJ101 requires only two plasmid loci: the pIJ101 tra gene, and as a cis-acting function known as clt. Here, we compared the ability of the pIJ101 transfer apparatus to promote conjugal transfer of circular versus linear versions of the same replicon. While the pIJ101 tra locus readily transferred the circular form of the replicon, the linear version was transferred orders of magnitude less efficiently and all plasmids isolated from the transconjugants were circular, regardless of their original configuration in the donor. Additionally, relatively rare circularization of linear plasmids was detectable in the donor cells, which is consistent with the notion that this event was a prerequisite for transfer by TraB(pIJ101). Linear versions of this same replicon did transfer efficiently, in that configuration, from strains containing the conjugative linear plasmid SLP2. Our data indicate that functions necessary and sufficient for transfer of circular DNA were insufficient for transfer of a related linear DNA molecule. The results here suggest that the conjugation mechanisms of linear versus circular DNA in Streptomyces spp. are inherently different and/or that efficient transfer of linear DNA requires additional components