Quorum sensing in bacteria associated with marine sponges Mycale laxissima and Ircinia strobilina

Sponges can form close associations with microbes that in some cases comprise up to 30% of the biomass of the sponge, which we hypothesized would provide an ideal environment for quorum sensing. I isolated 420 bacterial strains from two marine sponge species and screened these isolates for acyl-homoserine lactone (AHL) production. Results showed that isolates from the Silicibacter- Ruegeria (SR) subclade of the Roseobacter group are the dominant AHL producers. Production of these signaling compounds was consistently observed in isolates obtained from different sponge individuals during different seasons. The SR-type strain Ruegeria sp. KLH11 was isolated from tissue of the sponge Mycale laxissima. Chemical analysis of the AHLs produced by Ruegeria sp. KLH11 showed them to be predominantly composed of a mixture of long chains AHLs with 3-OH substitutions. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated as ssaRI, ssbRI and sscI (sponge-associated symbiont locus A, B and C, luxR or luxI homologue). SsaI directs synthesis of predominantly 3-oxo-AHLs whereas SsbI and SscI specify 3-OH-AHL derivatives. Wild type Ruegeria sp. KLH11 cultures are dominated by SsbI or SscI-specified AHLs. Mutation of either ssaR or ssaI results in loss of swimming motility, flagellar production and flagellin synthesis whereas mutation of ssbR or ssbI had no effect on these characteristics and no detectable phenotype. In wild type cultures, flagella are produced only in late stage growth. The non-essential phosphorelay cckA-chpT-ctrA system acts downstream of ssaRI to control flagellar motility. Mutants of ssaI and ssaR showed increased biofilm formation while mutants of ssbI and ssbR were not affected in biofilm formation, and this is not due solely to the loss of motility. The results showed the presence of AHL molecules similar to those specified by SsaI in sponge tissues and that the ssaI gene is actively expressed in situ, revealed by RT-PCR. We have established Ruegeria sp. KLH11 as a model to study the complex symbiotic relationships between sponges and microbes

Detection of ESKAPE bacterial pathogens at the point of care using isothermal DNA-based assays in a portable degas-actuated microfluidic diagnostic assay platform

An estimated 1.5 billion microbial infections occur globally each year and result in ~4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridgebased system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuumdegassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/ reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ~10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting

Acyl-Homoserine Lactone Quorum Sensing in the Roseobacter Clade

Members of the Roseobacter clade are ecologically important and numerically abundant in coastal environments and can associate with marine invertebrates and nutrient-rich marine snow or organic particles, on which quorum sensing (QS) may play an important role. In this review, we summarize current research progress on roseobacterial acyl-homoserine lactone-based QS, particularly focusing on three relatively well-studied representatives, Phaeobacter inhibens DSM17395, the marine sponge symbiont Ruegeria sp. KLH11 and the dinoflagellate symbiont Dinoroseobacter shibae. Bioinformatic survey of luxI homologues revealed that over 80% of available roseobacterial genomes encode at least one luxI homologue, reflecting the significance of QS controlled regulatory pathways in adapting to the relevant marine environments. We also discuss several areas that warrant further investigation, including studies on the ecological role of these diverse QS pathways in natural environments

Genome Sequence of Ruegeria sp. Strain KLH11, an N-Acylhomoserine Lactone-Producing Bacterium Isolated from the Marine Sponge Mycale laxissima ▿

Ruegeria sp. strain KLH11, isolated from the marine sponge Mycale laxissima, produces a complex profile of N-acylhomoserine lactone quorum-sensing (QS) molecules. The genome sequence provides insights into the genetic potential of KLH11 to maintain complex QS systems, and this is the first genome report of a cultivated symbiont from a marine sponge

The CckA-ChpT-CtrA Phosphorelay System Is Regulated by Quorum Sensing and Controls Flagellar Motility in the Marine Sponge Symbiont <i>Ruegeria</i> sp. KLH11

<div><p>Bacteria respond to their environment via signal transduction pathways, often two-component type systems that function through phosphotransfer to control expression of specific genes. Phosphorelays are derived from two-component systems but are comprised of additional components. The essential <i>cckA-chpT-ctrA</i> phosphorelay in <i>Caulobacter crescentus</i> has been well studied and is important in orchestrating the cell cycle, polar development and flagellar biogenesis. Although <i>cckA, chpT</i> and <i>ctrA</i> homologues are widespread among the <i>Alphaproteobacteria</i>, relatively few is known about their function in the large and ecologically significant <i>Roseobacter</i> clade of the <i>Rhodobacterales</i>. In this study the <i>cckA-chpT-ctrA</i> system of the marine sponge symbiont <i>Ruegeria</i> sp. KLH11 was investigated. Our results reveal that the <i>cckA, chpT</i> and <i>ctrA</i> genes positively control flagellar biosynthesis. In contrast to <i>C. crescentus</i>, the <i>cckA</i>, <i>chpT</i> and <i>ctrA</i> genes in <i>Ruegeria</i> sp. KLH11 are non-essential and do not affect bacterial growth. Gene fusion and transcript analyses provide evidence for <i>ctrA</i> autoregulation and the control of motility-related genes. In KLH11, flagellar motility is controlled by the SsaRI system and acylhomoserine lactone (AHL) quorum sensing. SsaR and long chain AHLs are required for <i>cckA</i>, <i>chpT</i> and <i>ctrA</i> gene expression, providing a regulatory link between flagellar locomotion and population density in KLH11.</p></div

Suppression of motility defects in Δ<i>ssaI</i> and Δ<i>ssaR</i> mutants by CtrA.

<p><i>P_lac-ctrA</i> plasmid (pJZ008) was conjugated into Δ<i>ssaI</i> and Δ<i>ssaR</i> mutants, respectively. The conjugants were selected and inoculated on swim agar plates for 8 days at 28°C. 200 µM IPTG was added to the media. The Δ<i>ssaI</i> mutant complemented with <i>P_lac-ssaI</i> (pEC108) and the Δ<i>ssaR</i> mutant with <i>P_lac-ssaR</i> (pEC112) were used as positive controls. Wild type KLH11 (EC1) was used as a positive and the Δ<i>ssaI</i> and Δ<i>ssaR</i> strains were used as negative control. The results were representatives of several independent experiments each with three biological replicates.</p

Phylogeny of the CtrA protein cross-complementation between KLH11 and <i>A. tumefaciens</i>.

<p> <b>A) Phylogenetic analyses of CtrA from members of alpha-</b><b><i>Proteobacteria</i></b><b>.</b> CtrA sequences from bacterial species in which CtrA has been studied were chosen for phylogenetic anaylsis. The sequences used were <i>A. tumefaciens</i> C58 (GenBank Accession No. NP_355385), <i>B. abortus</i> (AAL86376), <i>C. crescentus</i> (NP_421829), <i>E. chaffeensis</i> (YP_507798), <i>Magnetospirillum magneticum</i> AMB-1 (YP_419992), <i>Rhodopseudomonas palustris</i> (NP_946978), <i>Rhodobacter capsulatus</i> (AAF13177), <i>Rhodospirillum centenum</i> (YP_002297962), <i>Ruegeria</i> sp. KLH11 (ZP_05124475), <i>Silicibacter</i> sp. TM1040 (YP_613394) and <i>Sinorhizobium meliloti</i> (NP_386824). The star indicates the divergence between organisms in which CtrA is essential or implied to be essential and in which CtrA does affect viability, which was originally proposed by Green et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066346#pone.0066346-Greene1" target="_blank">[37]</a> a = motility-related genes are enriched in putative CtrA binding sites (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066346#pone.0066346-Brilli1" target="_blank">[12]</a>; b = unable to obtain a ctrA deletion mutant without providing an extra copy of the <i>ctrA</i> gene (J.E. Heindl and C. Fuqua, unpublished) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066346#pone.0066346-Mercer1" target="_blank">[36]</a>; c = gene target (<i>ccrM</i>) of CtrA is essential <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066346#pone.0066346-Robertson1" target="_blank">[50]</a>. The scale bar indicates the number of amino acid substitutions per site. <b>B) Cross complementation of motility between KLH11 and </b><b><i>A. tumefaciens</i></b><b> homologues.</b> Wild-type KLH11 (EC1) and derivatives were inoculated on MB2216 (supplemented with 0.25% agar) swim agar plates for about 8 days at 28°C. 200 µM IPTG was added to the media. The diameter of the swim ring was measured. Parentheses indicate from which species the relevant homologue is used (At stands for <i>A. tumefaciens</i>). Values are averages of assays performed in triplicate and error bars are standard deviations.</p

Exogenous AHLs complement for the lack of a functional <i>ssaI</i> gene in the indirect activation of the <i>cckA</i> and <i>ctrA</i> genes.

a<p>β-Galactosidase activity was expressed in Miller unit. Average of three biological replicates (standard deviation).</p>b<p>3-oxo-C16:1 Δ11-HSL (2 µM) was added.</p>c<p>P<0.05 when the expression level with AHL is compared to that without AHL in the Δ<i>ssaI</i> strain.</p

Regulation of <i>cckA, chpT</i> and <i>ctrA</i> gene expression by the <i>ssaRI</i><b> system.</b>

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066346#s3" target="_blank">Results</a> of β-galactosidase assays in detecting the expression <i>of ctrA–lacZ</i> (A), <i>chpT-lacZ</i> (B) and <i>cckA-lacZ</i> (C) in Δ<i>ssaI</i> and Δ<i>ssaR</i> mutants. Plasmids <i>P_lac-ssaI</i> (pEC108) and <i>P_lac-ssaR</i> (pEC112) were conjugated into the Δ<i>ssaI</i> and Δ<i>ssaR</i> mutants, respectively, to restore the expression of the <i>ctrA</i>, <i>chpT</i> and <i>cckA</i> genes. 2 µM 3-oxo-C16:1 Δ11-HSL was added into Δ<i>ssaI ctrA-lacZ</i>, Δ<i>ssaI chpT-lacZ and</i> Δ<i>ssaI cckA-lacZ</i> strains, respectively. Filled asterisks indicated statistically significant differences between the indicated strain and wild-type quorum sensing strain. Unfilled asterisks indicated statistically significant differences between the quorum sensing complemented strains and quorum sensing mutants for the expression of the <i>ctrA</i>, <i>chpT</i> and <i>cckA</i> genes. Representative results of several independent experiments each with three biological replicates are presented. Values are averages of assays performed in triplicate and error bars are standard deviations.</p

Structure, function and regulation of CckA, ChpT and CtrA.

<p><b>A) Diagram of the predicted CckA protein, ChpT protein and CtrA protein.</b> The N-terminus is shown at the left and the C-terminus is shown at the right. TM = transmembrane domain, HisKA = histidine kinase A dimerization/phosphoacceptor domain, HATPase_c = histidine kinase-like ATPase domain, REC = signal receiver domain. DUF2328 is a Pfam domain with unknown function. DBD = DNA binding domain. The length of line was drawn according to scale. The partial promoter regions of the <i>cckA</i> and <i>ctrA</i> genes are shown on top of the lines. CtrA full recognition site (TTAAN7TTAAC) is in bold and both the CtrA half recognition site (TTAACCAT) and the region that has one mismatch are in grey. The start codon is boxed. <b>B). Swimming motility assays.</b> Wild-type KLH11 and derivatives were inoculated on MB2216 (supplemented with 0.25% agar) swim agar plates for 8 days at 28°C. 200 µM IPTG was added to the media. The results are representative of several independent experiments each with three biological replicates. <b>C).. </b><b>CtrA autoregulates its own expression.. </b><i>P_lac-ctrA</i> plasmid (pJZ008) was conjugated into the <i>ctrA</i>⁻ mutant (JZ06) and the expression of <i>ctrA</i>-<i>lacZ</i> was monitored by β-galactosidase assay. The pSRKGm was conjugated into the <i>ctrA</i>⁻ mutant as a negative control. Representative results of several independent experiments each with three biological replicates are presented. Values are averages of assays performed in triplicate and error bars are standard deviations.</p