Comparative sequence analysis of bacterial symbionts from the marine sponges Geodia cydonium and Ircinia muscarum

Marine sponges (Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Recently, several studiesindicated that sponges are the most prolific source of biologically-active compounds produced by symbiotic microorganisms ratherthan by the sponges themselves. In the present study we characterized the bacterial symbionts from two Demospongiae, Irciniamuscarum and Geodia cydonium. We amplified 16S rRNA by PCR, using specific bacterial-primers. The phylogenetic analysisrevealed the presence of nine bacterial clones from I. muscarum and ten from G. cydonium. In particular, I. muscarum resultedenriched in Bacillus species and G. cydonium in Proteobacterium species. Since these bacteria were able to produce secondarymetabolites with potential biotechnological and biopharmaceutical applications, we hypothesized that I. muscarum and G. cydoniumcould be a considered as a “gold mine” of natural products.     

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO₂ and N₂, have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)—the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules—led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.     

Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria

Quorum sensing describes the ability of bacteria to sense their population density and respond by modulating gene expression. In the plant soft-rotting bacteria, such as Erwinia, an arsenal of plant cell wall-degrading enzymes is produced in a cell density-dependent manner, which causes maceration of plant tissue. However, quorum sensing is central not only to controlling the production of such destructive enzymes, but also to the control of a number of other virulence determinants and secondary metabolites. Erwinia synthesizes both N-acylhomoserine lactone (AHL) and autoinducer-2 types of quorum sensing signal, which both play a role in regulating gene expression in the phytopathogen. We review the models for AHL-based regulation of carbapenem antibiotic production in Erwinia. We also discuss the importance of quorum sensing in the production and secretion of virulence determinants by Erwinia, and its interplay with other regulatory systems.     

Sterols and related metabolites from five species of sponges

Free sterol fractions were isolated from the marine sponges Phyllospongia madagascarensis, Scalarispongia sp., Oceanapia sp., Monanchora clathrata and studied by GLC, GLC–MS, and spectroscopy NMR. P. madagascarensis and Scalarispongia sp. contained common Δ⁵-sterols; cholesterol was shown to be a main sterol of both the sponges. Oceanapia sp. contained stanols and minor Δ⁵-sterols with 24R-24,25-methylene-5α-cholestan-3β-ol as a main constituent. Many free sterols from M. clathrata were Δ⁷-series compounds, and latosterol was a main sterol. Δ⁴-3-Ketosteroids and Δ⁵-sterol esters were found in the Antarctic sponge Haliclona sp., but free sterols were practically absent except for trace amount of cholesterol. A chemotaxonomic application of sterols in relation to the genera Phyllospongia, Oceanapia and the family Crambeidae is provided. The known cases of the absence of sterols in sponges and probable reasons of the phenomenon are discussed.     

Genome sequence of Phaeobacter caeruleus type strain (DSM 24564T), a surface-associated member of the marine Roseobacter clade

In 2009 Phaeobacter caeruleus was described as a novel species affiliated with the marine Roseobacter clade, which, in turn, belongs to the class Alphaproteobacteria. The genus Phaeobacter is well known for members that produce various secondary metabolites. Here we report of putative quorum sensing systems, based on the finding of six N-acyl-homoserine lactone synthetases, and show that the blue color of P. caeruleus is probably due to the production of the secondary metabolite indigoidine. Therefore, P. caeruleus might have inhibitory effects on other bacteria. In this study the genome of the type strain DSM 24564^T was sequenced, annotated and characterized. The 5,344,419 bp long genome with its seven plasmids contains 5,227 protein-coding genes (3,904 with a predicted function) and 108 RNA genes.     

Computational identification and analysis of functional polymorphisms involved in the activation and detoxification genes implicated in endometriosis

Endometriosis is a complex disorder of the female reproductive system where endometrial tissue embeds and grows at extrauterine location leading to inflammation and pain. Hundreds of polymorphisms in several genes have been studied as probable risk factors of this debilitating disease. Bioinformatics tools have come a long way in augmenting the search for putative functional polymorphisms in human diseases. In this study we have explored 16 genes involved in the detoxification of xenobiotic chemicals that are implicated in endometriosis by utilising publically available programs like SIFT, Polyphen, Panther, FastSNP, SNPeffect and PhosSNP. The variations among different ethnic populations of the SNPs were studied. We then calculated the extent to which bioinformatics based predictions are concurrent with real world epidemiological, genotyping studies using a set of SNPs that have been studied in endometriosis case–control studies. Our study shows that there is a significant positive correlation (r = 0.569, p < 0.005) between the summary of the predicted scores taken from 4 different servers and the odds ratio found from epidemiological studies. This report has identified and catalogued various deleterious SNPs that could be important in endometriosis and could aid in further analysis by in vitro and in vivo methods for the better understanding of the disease pathophysiology.     

Diversity,abundance and expression of nitrite reductase (nirK)-like genes in marine thaumarchaea

Ammonia-oxidizing archaea (AOA) are widespread and abundant in aquatic and terrestrial habitats and appear to have a significant impact on the global nitrogen cycle. Like the ammonia-oxidizing bacteria, AOA encode a gene homologous to copper-containing nitrite reductases (nirK), which has been studied very little to date. In this study, the diversity, abundance and expression of thaumarchaeal nirK genes from coastal and marine environments were investigated using two mutually excluding primer pairs, which amplify the nirK variants designated as AnirKa and AnirKb. Only the AnirKa variant could be detected in sediment samples from San Francisco Bay and these sequences grouped with the nirK from Candidatus Nitrosopumilus maritimus and Candidatus Nitrosoarchaeum limnia. The two nirK variants had contrasting distributions in the water column in Monterey Bay and the California Current. AnirKa was more abundant in the epi- to mesopelagic Monterey Bay water column, whereas AnirKb was more abundant in the meso- to bathypelagic California Current water. The abundance and community composition of AnirKb, but not AnirKa, followed that of thaumarchaeal amoA, suggesting that either AnirKa is not exclusively associated with AOA or that commonly used amoA primers may be missing a significant fraction of AOA diversity in the epipelagic. Interestingly, thaumarchaeal nirK was expressed 10–100-fold more than amoA in Monterey Bay. Overall, this study provides valuable new insights into the distribution, diversity, abundance and expression of this alternative molecular marker for AOA in the ocean.     

Signal diffusion and the mitigation of social exploitation in pneumococcal competence signalling

Quorum sensing (QS) in bacteria is thought to enable populations of cells to coordinately and cooperatively regulate gene expression for traits that confer group benefits. While this view has strong empirical and theoretical support, it is increasingly appreciated that QS under natural conditions may be incapable of monitoring bacterial numbers and, furthermore, that QS is evolutionarily unstable owing to conflicts of interest among competing cells. An alternative hypothesis, termed diffusion sensing (DS), proposes that autoinducer secretion monitors the diffusive properties of the local environment, with benefits that are directly realized by individual cells rather than populations. Here, we test central predictions of this hypothesis using the competence signalling system of Streptococcus pneumoniae as our model, which regulates the induction of natural transformation by the secretion and detection of a small diffusible peptide, CSP (competence-stimulating peptide). By experimentally manipulating the diffusive properties of the growth medium, we found that there is no fixed quorum for competence induction. Instead, induction cell density scales with diffusivity. In agreement with QS and DS expectations, we show that the benefit of signal exploitation by mutant cells that can use but not secrete CSP is strongly frequency-dependent. However, we also find that the magnitude of this benefit declines significantly as diffusion is reduced, a result more consistent with the predictions of DS. Together, these data provide strong support for the DS hypothesis for autoinducer response systems. More specifically, our results imply that autonomous rather than group benefits should be sought in order to more completely understand the role and evolution of CSP signalling in pneumococci.     

Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas

The life cycle of Vibrio parahaemolyticus has been conventionally associated with estuarine areas characterized by moderate salinity and warm seawater temperatures. Recent evidence suggests that the distribution and population dynamics of V. parahaemolyticus may be shaped by the existence of an oceanic transport of communities of this organism mediated by zooplankton. To evaluate this possibility, the presence of V. parahaemolyticus in the water column of offshore areas of Galicia was investigated by PCR monthly over an 18-month period. Analysis of zooplankton and seawater showed that the occurrence of V. parahaemolyticus in offshore areas was almost exclusively associated with zooplankton and was present in 80% of the samples. The influence of environmental factors assessed by generalized additive models revealed that the abundance and seasonality of V. parahaemolyticus in zooplankton was favoured by the concurrence of downwelling periods that promoted the zooplankton patchiness. These results confirm that offshore waters may be common habitats for V. parahaemolyticus, including strains with virulent traits. Additionally, genetically related populations were found in offshore zooplankton and in estuaries dispersed along 1500 km. This finding suggests that zooplankton may operate as a vehicle for oceanic dispersal of V. parahaemolyticus populations, connecting distant regions and habitats, and thereby producing impacts on the local community demography and the spread of Vibrio-related diseases.     

Inhibition of Quorum Sensing Mediated Virulence Factors Production in Urinary Pathogen Serratia marcescens PS1 by Marine Sponges

The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.     

Single-cell genomics reveals the lifestyle of Poribacteria,a candidate phylum symbiotically associated with marine sponges

In this study, we present a single-cell genomics approach for the functional characterization of the candidate phylum Poribacteria, members of which are nearly exclusively found in marine sponges. The microbial consortia of the Mediterranean sponge Aplysina aerophoba were singularized by fluorescence-activated cell sorting, and individual microbial cells were subjected to phi29 polymerase-mediated ‘whole-genome amplification''. Pyrosequencing of a single amplified genome (SAG) derived from a member of the Poribacteria resulted in nearly 1.6 Mb of genomic information distributed among 554 contigs analyzed in this study. Approximately two-third of the poribacterial genome was sequenced. Our findings shed light on the functional properties and lifestyle of a possibly ancient bacterial symbiont of marine sponges. The Poribacteria are mixotrophic bacteria with autotrophic CO₂-fixation capacities through the Wood–Ljungdahl pathway. The cell wall is of Gram-negative origin. The Poribacteria produce at least two polyketide synthases (PKSs), one of which is the sponge-specific Sup-type PKS. Several putative symbiosis factors such as adhesins (bacterial Ig-like domains, lamininin G domain proteins), adhesin-related proteins (ankyrin, fibronectin type III) and tetratrico peptide repeat domain-encoding proteins were identified, which might be involved in mediating sponge–microbe interactions. The discovery of genes coding for 24-isopropyl steroids implies that certain fossil biomarkers used to date the origins of metazoan life on earth may possibly be of poribacterial origin. Single-cell genomic approaches, such as those shown herein, contribute to a better understanding of beneficial microbial consortia, of which most members are, because of the lack of cultivation, inaccessible by conventional techniques.     

In Vitro Characterization of a Recombinant AHL-Lactonase from Bacillus cereus Isolated from a Striped Catfish (Pangasianodon hypophthalmus) Pond

aiiA gene encoding AHL-lactonase was isolated from Bacillus cereus strain N26.2, originating from a striped catfish (Pangasianodon hypophthalmus) pond in Vietnam. This gene, abbreviated as aiiA(N26.2), was cloned and expressed in a competent Escherichia coli strain BL21(DE3)pLysS. The resulting protein, abbreviated as AiiA_N26.2, was highly active in the pH range of 6–8 and could retain 80 % of the maximum activity under storage for 5 days at 4 °C or for 3 days at 20 °C. These properties of AiiA_N26.2 protein confers its future application via feed supplementation, with the purpose of controlling aquaculture pathogens which regulate the virulence via a quorum sensing system.     

Genome sequence of the Leisingera aquimarina type strain (DSM 24565T), a member of the marine Roseobacter clade rich in extrachromosomal elements

Leisingera aquimarina Vandecandelaere et al. 2008 is a member of the genomically well characterized Roseobacter clade within the family Rhodobacteraceae. Representatives of the marine Roseobacter clade are metabolically versatile and involved in carbon fixation and biogeochemical processes. They form a physiologically heterogeneous group, found predominantly in coastal or polar waters, especially in symbiosis with algae, in microbial mats, in sediments or associated with invertebrates. Here we describe the features of L. aquimarina DSM 24565^T together with the permanent-draft genome sequence and annotation. The 5,344,253 bp long genome consists of one chromosome and an unusually high number of seven extrachromosomal elements and contains 5,129 protein-coding and 89 RNA genes. It was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2010 and of the activities of the Transregional Collaborative Research Centre 51 funded by the German Research Foundation (DFG).     

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.     

Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18

The extreme variability of the mitochondrial (mito) genomes of bivalves makes it difficult to understand their evolutionary dynamics, given that species from different families do not share comparable features. We compared the mitogenomes from four Paphia clams (three of them were firstly sequenced) and found that mitogenome reorganization among the four congeneric species is not random but follows phylogenetic trends. Start/stop codon variations are species-correlated rather than gene-correlated, and bear useful phylogenetic information. Unique start/stop codon usage in P. euglypta and A+T content in P. amabilis indicates that these mitogenome-level characters, usually considered to be conservative features in other lineages, may not be phylogenetically evolved, but may have evolved via species-specific mitogenomic maintenance mechanisms. Variable divergence of two trnM genes in different lineages may demonstrate differences in mechanisms by which paralogous trnM genes are maintained. Sequence alignment analysis indicates that the VNTRs in the four mitogenomes have a common origin. The rationale of the subgenus Neotapes Kuroda and Habe, 1971 was supported by evidence from morphological characters, mitogenomic features, as well as phylogenetic analyses using cox1 and rrnS genes. The data suggest that the taxonomic basis of the subgenus should be “smooth surface” but not “undulated lines,” and P. textile should be classified to the Neotapes subgenus.     

Growth phase-differential quorum sensing regulation of anthranilate metabolism in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas quinolone signal (PQS) plays a role in the regulation of virulence genes and it is intertwined in the las/rhl quorum sensing (QS) circuits of Pseudomonas aeruginosa. PQS is synthesized from anthranilate by pqsA-D and pqsH whose expression is influenced by the las/rhl systems. Since anthranilate can be degraded by functions of antABC and catBCA, PQS synthesis might be regulated by the balance between the expression of the pqsA-D/phnAB, pqsH, antABC, and catBCA gene loci. antA and catA are repressed by LasR during log phase and activated by RhlR in late stationary phase, whereas pqsA-E/phnAB is activated by LasR in log phase and repressed by RhlR. QscR represses both but each repression occurs in a different growth phase. This growth phase-differential regulation appears to be accomplished by the antagonistic interplay of LasR, RhlR, and QscR, mediated by two intermediate regulators, AntR and PqsR, and their cofactors, anthranilate and PQS, where the expressions of antR and pqsR and the production of anthranilate and PQS are growth phase-differentially regulated by QS systems. Especially, the anthranilate level increases in an RhlR-dependent manner at late stationary phase. From these results, we suggest that RhlR and LasR regulate the anthranilate metabolism in a mutually antagonistic and growth phase-differential manner by affecting both the expressions and activities of AntR and PqsR, and that QscR also phase-differentially represses both LasR and RhlR functions in this regulation.     

Rate heterogeneity in six protein-coding genes from the holoparasite Balanophora (Balanophoraceae) and other taxa of Santalales

Background and Aims

The holoparasitic flowering plant Balanophora displays extreme floral reduction and was previously found to have enormous rate acceleration in the nuclear 18S rDNA region. So far, it remains unclear whether non-ribosomal, protein-coding genes of Balanophora also evolve in an accelerated fashion and whether the genes with high substitution rates retain their functionality. To tackle these issues, six different genes were sequenced from two Balanophora species and their rate variation and expression patterns were examined.

Methods

Sequences including nuclear PI, euAP3, TM6, LFY and RPB2 and mitochondrial matR were determined from two Balanophora spp. and compared with selected hemiparasitic species of Santalales and autotrophic core eudicots. Gene expression was detected for the six protein-coding genes and the expression patterns of the three B-class genes (PI, AP3 and TM6) were further examined across different organs of B. laxiflora using RT-PCR.

Key Results

Balanophora mitochondrial matR is highly accelerated in both nonsynonymous (d_N) and synonymous (d_S) substitution rates, whereas the rate variation of nuclear genes LFY, PI, euAP3, TM6 and RPB2 are less dramatic. Significant d_S increases were detected in Balanophora PI, TM6, RPB2 and d_N accelerations in euAP3. All of the protein-coding genes are expressed in inflorescences, indicative of their functionality. PI is restrictively expressed in tepals, synandria and floral bracts, whereas AP3 and TM6 are widely expressed in both male and female inflorescences.

Conclusions

Despite the observation that rates of sequence evolution are generally higher in Balanophora than in hemiparasitic species of Santalales and autotrophic core eudicots, the five nuclear protein-coding genes are functional and are evolving at a much slower rate than 18S rDNA. The mechanism or mechanisms responsible for rapid sequence evolution and concomitant rate acceleration for 18S rDNA and matR are currently not well understood and require further study in Balanophora and other holoparasites.