BackgroundBacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context.ResultsHerein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer.ConclusionsOur data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5439-1) contains supplementary material, which is available to authorized users.

Background Utilizing unique carbohydrates or utilizing them more efficiently help bacteria expand and colonize new niches. Horizontal gene transfer (HGT) of catabolic systems is a powerful mechanism by which bacteria can acquire new phenotypic traits that can increase survival and fitness in different niches. In this work, we examined carbon catabolism diversity among Vibrio parahaemolyticus , a marine species that is also an important human and fish pathogen. Results Phenotypic differences in carbon utilization between Vibrio parahaemolyticus strains lead us to examine genotypic differences in this species and the family Vibrionaceae in general. Bioinformatics analysis showed that the ability to utilize d -galactose was present in all V. parahaemolyticus but at least two distinct transporters were present; a major facilitator superfamily (MFS) transporter and a sodium/galactose transporter (SGLT). Growth and genetic analyses demonstrated that SGLT was a more efficient transporter of d -galactose and was the predominant type among strains. Phylogenetic analysis showed that d -galactose gene galM was acquired multiples times within the family Vibrionaceae and was transferred between distantly related species. The ability to utilize d -gluconate was universal within the species. Deletion of eda (VP0065), which encodes aldolase, a key enzyme in the Entner-Doudoroff (ED) pathway, reached a similar biomass to wild type when grown on d -gluconate as a sole carbon source. Two additional eda genes were identified, VPA1708 ( eda2 ) associated with a d -glucuronate cluster and VPA0083 ( eda3 ) that clustered with an oligogalacturonide (OGA) metabolism cluster. EDA2 and EDA3 were variably distributed among the species. A metabolic island was identified that contained citrate fermentation, l -rhamnose and OGA metabolism clusters as well as a CRISPR-Cas system. Phylogenetic analysis showed that CitF and RhaA had a limited distribution among V. parahaemolyticus , and RhaA was acquired at least three times. Within V. parahaemolyticus , two different regions contained the gene for L-arabinose catabolism and most strains had the ability to catabolism this sugar. Conclusion Our data suggest that horizontal transfer of metabolic systems among Vibrionaceae is an important source of metabolic diversity. This work identified four EDA homologues suggesting that the ED pathway plays a significant role in metabolism. We describe pre...

Factor for inversion stimulation is a global regulator that is highly expressed during exponential phase growth and undetectable in stationary phase growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to changes in cell density and growth phase. In Vibrio parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, are expressed during exponential growth and negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation, which is required for biofilm formation, and is a repressor of lateral flagella required for swarming motility. In V. parahaemolyticus, we show that Fis is a positive regulator of the qrr sRNAs expression. In an in-frame fis deletion mutant, qrr expression was repressed and opaR expression was induced. The Δfis mutant produced CPS and biofilm, but swarming motility was abolished. Also, the fis deletion mutant was more sensitive to polymyxin B. Swarming motility requires expression of both the surface sensing scrABC operon and lateral flagella laf operon. Our data showed that in the Δfis mutant both laf and scrABC genes were repressed. Fis controlled swarming motility indirectly through the QS pathway and directly through the surface sensing pathway. To determine the effects of Fis on cellular metabolism, we performed in vitro growth competition assays, and found that Δfis was outcompeted by wild type in minimal media supplemented with intestinal mucus as a sole nutrient source. The data showed that Fis positively modulated mucus components L-arabinose, D-gluconate and N-acetyl-D-glucosamine catabolism gene expression. In an in vivo colonization competition assay, Δfis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a global regulatory role for Fis in V. parahaemolyticus that includes QS, motility, and metabolism.

Here, we characterized the roles of CRP and CyaA in V. parahaemolyticus , showing that cAMP-CRP is an activator of metabolism, motility, capsule production, and biofilm formation. These results are in contrast to cAMP-CRP in V. cholerae , which represses capsule and biofilm formation.

Fis (Factor for Inversion Stimulation) is a global regulator that is highly expressed during exponential growth and undetectable in stationary growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to cell density and growth phase. In V. parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation required for biofilm formation and a repressor of swarming motility. In Vibrio parahaemolyticus, we showed, using genetics and DNA binding assays, that Fis bound directly to the regulatory regions of the qrr genes and was a positive regulator of these genes. In the Δfis mutant, opaR expression was induced and a robust CPS and biofilm was produced, while swarming motility was abolished. Expression analysis and promoter binding assays showed that Fis was a direct activator of both the lateral flagellum laf operon and the surface sensing scrABC operon, both required for swarming motility. In in vitro growth competition assays, Δfis was outcompeted by wild type in minimal media supplemented with intestinal mucus, and we showed that Fis directly modulated catabolism gene expression. In in vivo colonization competition assays, Δfis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a direct role for Fis in QS, motility, and metabolism in V. parahaemolyticus.IMPORTANCEIn this study, we examined the role of Fis in modulating expression of the five-quorum sensing regulatory sRNAs, qrr1 to qrr5, and showed that Fis is a direct positive regulator of QS, which oppositely controls CPS and swarming motility in V. parahaemolyticus. The Δfis deletion mutant was swarming defective due to a requirement for Fis in lateral flagella and surface sensing gene expression. Thus, Fis links QS and surface sensing to control swarming motility and, indirectly, CPS production. Fis was also required for cell metabolism, acting as a direct regulator of several carbon catabolism loci. Both in vitro and in vivo competition assays showed that the Δfis mutant had a significant defect compared to wild type. Overall, our data demonstrates that Fis plays a critical role in V. parahaemolyticus physiology that was previously unexamined.

CRP (cyclic AMP receptor protein), encoded by crp, is a global transcription factor that is activated by cyclic AMP (cAMP), a second messenger synthesized from ATP by adenylate cyclase, cyaA in Escherichia coli. In this species, growth on the preferred carbon source glucose, results in low levels of cAMP, and inactive CRP. When glucose is absent, cAMP levels are high which activates CRP to regulate 100s of genes. To investigate the role of CRP in Vibrio parahaemolyticus, we constructed non-polar deletions of the crp and cyaA homologs. The Dcrp mutant did not grow in minimal media supplemented with non-glucose carbon sources, however the DcyaA mutant grew similar to wild type suggesting the presence of an additional adenylate cyclase. Bioinformatics analysis identified a homolog of a class IV adenylate cyclase, CyaB present in V. parahaemolyticus. Both V. parahaemolyticus cyaA and cyaB functionally complemented an E. coli DcyaA mutant indicating adenylate cyclase activity. Growth pattern analysis showed that cyaA is controlled by CRP, but cyaB is not. cAMP-CRP was required for motility with the Dcrp and DcyaB/DcyaA mutants having a defect in swimming and swarming motility. The DcyaA mutant produces capsule polysaccharide (CPS) and biofilm similar to wild type. However, the DcyaB mutant was defective in CPS production and biofilm formation, similar to the Dcrp and DcyaB/DcyaA mutants, indicating that CyaB, but not CyaA, is required for these phenotypes. Phylogenetic analysis of cyaB showed broad distribution within the Vibrionaceae family and was acquired by horizontal gene transfer in several species.