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

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.

Density gradient enrichment of Escherichia coli lpxL mutants.

We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992-7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL.

Manganese is an additional cation that enhances colonial phase variation of Vibrio vulnificus.

Vibrio vulnificus, an inhabitant of marine and estuarine environments around the world, is the leading cause of reported seafood-related deaths in the United States. Disease is caused by opaque colony-forming strains that produce capsular polysaccharide, loss of which results in an unencapsulated translucent phenotype with diminished virulence potential. Rugose is a third phenotypic variant of V. vulnificus, and produces a separate exopolysaccharide that results in a dry, wrinkled appearance and the ability to form profuse biofilms. Phase variation among these three phenotypes is influenced by several environmental factors, including the presence of calcium in the medium (Garrison-Schilling et al.). In this study, we have identified a second cation, manganese, which substantially increases the propensity of opaque V. vulnificus strains to switch to translucent or rugose phenotypes. In comparative studies, manganese and calcium promoted switching to the same phenotype for some strains but to different phenotypes for others, results of which indicate that the two cations do not always promote the same changes in underlying gene expression. The data here provide further evidence that exposure of V. vulnificus to select cations results in phenotypic changes that impact both virulence capacity and ecology of the organism.

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.