Phylogenomic analysis of the understudied Neisseriaceae species reveals a poly- and paraphyletic Kingella genus.

IMPORTANCE: Understanding the evolutionary relationships between the species in the Neisseriaceae family has been a persistent challenge in bacterial systematics due to high recombination rates in these species. Previous studies of this family have focused on Neisseria meningitidis and N. gonorrhoeae. However, previously understudied Neisseriaceae species are gaining new attention, with Kingella kingae now recognized as a common human pathogen and with Alysiella and Simonsiella being unique in the bacterial world as multicellular organisms. A better understanding of the genomic evolution of the Neisseriaceae can lead to the identification of specific genes and traits that underlie the remarkable diversity of this family.

Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella.

The bacterial genus Kingella includes two pathogenic species, namely Kingella kingae and Kingella negevensis, as well as strictly commensal species. Both K. kingae and K. negevensis secrete a toxin called RtxA that is absent in the commensal species. Here we present a phylogenomic study of the genus Kingella, including new genomic sequences for 88 clinical isolates, genotyping of another 131 global isolates, and analysis of 52 available genomes. The phylogenetic evidence supports that the toxin-encoding operon rtxCA was acquired by a common ancestor of the pathogenic Kingella species, and that a preexisting type-I secretion system was co-opted for toxin export. Subsequent genomic reorganization distributed the toxin machinery across two loci, with 30-35% of K. kingae strains containing two copies of the rtxA toxin gene. The rtxA duplication is largely clonal and is associated with invasive disease. Assays with isogenic strains show that a single copy of rtxA is associated with reduced cytotoxicity in vitro. Thus, our study identifies key steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer, co-option of an existing secretion system, and gene duplication.

Pathogenic determinants of Kingella kingae disease.

Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a leading etiology of septic arthritis, osteomyelitis, and bacteremia and an occasional cause of endocarditis in young children. The pathogenesis of K. kingae disease begins with colonization of the upper respiratory tract followed by breach of the respiratory epithelial barrier and hematogenous spread to distant sites of infection, primarily the joints, bones, and endocardium. As recognition of K. kingae as a pathogen has increased, interest in defining the molecular determinants of K. kingae pathogenicity has grown. This effort has identified numerous bacterial surface factors that likely play key roles in the pathogenic process of K. kingae disease, including type IV pili and the Knh trimeric autotransporter (adherence to the host), a potent RTX-family toxin (epithelial barrier breach), and multiple surface polysaccharides (complement and neutrophil resistance). Herein, we review the current state of knowledge of each of these factors, providing insights into potential approaches to the prevention and/or treatment of K. kingae disease.

CosR Is a Global Regulator of the Osmotic Stress Response with Widespread Distribution among Bacteria.

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2 Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.IMPORTANCEVibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness.IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.

CRISPR-Cas systems are present predominantly on mobile genetic elements in Vibrio species.

BACKGROUND: Bacteria 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. RESULTS: Herein, 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. CONCLUSIONS: Our data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.