Analysis of suppressors that alleviate the acute envelope stress phenotype of a ΔbamBΔdegP strain of Escherichia coli identified a novel protein MzrA and pleiotropic envZ mutations. Genetic evidence shows that overexpression of MzrA – formerly known as YqjB and EcfM – modulates the activity of EnvZ/OmpR similarly to pleiotropic EnvZ mutants and alter porin expression. However, porin expression in strains devoid of MzrA or overexpressing it is still sensitive to medium osmolarity, pH and procaine, all of which modulate EnvZ/OmpR activities. Thus, MzrA appears to alter the output of the EnvZ/OmpR system but not its ability to receive and respond to various environmental signals. Localization and topology experiments indicate that MzrA is a type II membrane protein, with its N-terminus exposed in the cytoplasm and C-terminus in the periplasm. Bacterial two-hybrid experiments determined that MzrA specifically interacts with EnvZ but not with OmpR or the related membrane sensor kinase, CpxA. This and additional genetic and biochemical evidence suggest that the interaction of MzrA with EnvZ would either enhance EnvZ's kinase activity or reduce its phosphatase activity, thus elevating the steady state levels of OmpR∼P. Furthermore, our data show that MzrA links the two-component envelope stress response regulators, CpxA/CpxR and EnvZ/OmpR.

Marine sponges are hosts to diverse and dense bacterial communities and thus provide a potential environment for quorum sensing. Quorum sensing, a key factor in cell-cell communication and bacterial colonization of higher animals, might be involved in the symbiotic interactions between bacteria and their sponge hosts. Given that marine Proteobacteria are known to produce N-acyl homoserine lactone (AHL) signal molecules, we tested the production of AHLs by Alpha- and Gammaproteobacteria isolated from marine sponges Mycale laxissima and Ircinia strobilina and the surrounding water column. We used three different AHL biodetection systems in diffusion assays: Chromobacterium violaceum, Agrobacterium tumefaciens and Sinorhizobium meliloti with optimal sensitivity to short-chain (C4-C6), moderate-chain (C8-C12) and long-chain (>or= C14) AHLs respectively. Thirteen of 23 isolates from M. laxissima and five of 25 isolates from I. strobilina were found to produce AHLs. Signals were detected from two of eight proteobacterial strains from the water column. Thin-layer chromatographic assays based on the A. tumefaciens reporter system were utilized to determine the AHL profiles of the positive isolates. The types and amounts of AHLs synthesized varied considerably among the strains. Small ribosomal rRNA gene sequencing revealed that the AHL-producing alphaproteobacterial isolates were mainly from the Silicibacter-Ruegeria subgroup of the Roseobacter clade. Two-dimensional gel electrophoresis (2DGE)-based proteomic analyses were congruent with phylogenetic relationships but provided higher resolution to differentiate these closely related AHL-producing strains.

Summary Bacteria isolated from marine sponges, including the Silicibacter–Ruegeria (SR) subgroup of the Roseobacter clade, produce N‐acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge‐associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI and sscI (sponge‐associated symbiont locus A, B and C, luxR or luxI homologue). SsaI produced predominantly long‐chain 3‐oxo‐AHLs and both SsbI and SscI specified 3‐OH‐AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild‐type KLH11. AHLs and the ssaI transcript were detected in M. laxissima extracts, suggesting that AHL signalling contributes to the decision between motility and sessility and that it may also facilitate acclimation to different environments that include the sponge host.

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB Salmonella infections occur in a wide variety of vertebrate hosts and continue to be a major health problem worldwide for humans. Salmonella infection requires at least two pathogenicity islands, Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), 3 both of which encode type III secretion systems as well as secreted effectors, chaperones, and regulatory proteins (1-3). Genes located on SPI-1 are required for initial adherence to and invasion of intestinal epithelial cells (4). SPI-2 genes are required for intracellular survival, replication, and systemic infection of Salmonella (5, 6). SPI-2 consists of a 40-kb region located at 31 centisomes on the chromosome and contains ϳ32 genes (7). SPI-2 genes are organized into functional clusters encoding regulatory, structural genes, effectors, and chaperones. To date 10 promoters of SPI-2 genes and SPI-2 co-regulated genes have been identified (8 -10). These promoters, located upstream of the ssrA, ssrB, ssaB, sseA, ssaG, ssaM, sseI, srfN, sifA, and sifB genes, transcribe genes either individually or in large operons. Transcription from these promoters is activated by the SPI-2 encoded two-component signal transduction system, SsrA/SsrB (8, 11-13).Two-component signal transduction systems regulate gene expression in response to specific environmental signals (for review see Ref. 14). These systems represent the major paradigm for signal transduction in prokaryotes. They are frequently involved in regulating expression of virulence genes in pathogenic bacteria and are also present in the archaea, lower eukaryotes, and plants. In its simplest form, a two-component system contains a sensor kinase, often a membrane protein that functions in trans-membrane signaling, and a response regulator, usually a DNA-binding protein that regulates transcription. In Salmonella enterica serovar typhimurium, the SsrA/SsrB tw...

A complex LuxR-LuxI type quorum sensing network in a roseobacterial marine sponge symbiont activates flagellar motility and inhibits biofilm formation. Mol Microbiol 85: 916-933.