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Salmonellosis is one of the most common foodborne illnesses in the world. The irrational use of antibiotics in medicine and in animal nutrition has greatly favored the emergence and spread of resistant strains of non-typhoid Salmonella. This study aims the determination of the prevalence of Salmonella in bivalve mollusks in Northern Morocco, as well as the molecular typing and antibiotic susceptibility testing of the strains isolated from positive samples. In total, 150 samples from shellfish composed of mussels (Mytilus galloprovincialis), clams (Callista chione and Ruditapes descussatus) and oysters (Magallana gigas). Isolated Salmonella were characterized by Molecular techniques PCR, MLST and MLVA, phylogenetically grouped by MLSA, and susceptibilities were determined for 30 antimicrobial drugs using microdilution method by the BD Phoenix Automated Microbiology System. Prevalence of Salmonella enterica subsp. enterica was 12.67%, grouped in four serovars identified as Chester, Hadar, Typhimurium and Kentucky. Five different MLST STs (sequence types) were detected, ST1954 being the most common, which was mostly found in Chester isolates. Forty-two percent of the isolates showed resistance to more than one antibiotic, especially trimethoprim, sulfa drugs, quinolones and β-lactam. There was a marked change in the serovars and antimicrobial resistance profiles of the Salmonella isolates in this study compared to those in previous studies., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Vibrio parahaemolyticus is the most common pathogen causing seafood-borne illnesses in Korea. The present study evaluated the occurrence, virulence, and antimicrobial resistance of V. parahaemolyticus in seawater and bivalves obtained in 2016 from the southern coast of Korea, an important region for commercial aquaculture industries, especially the Korean raw seafood culture. V. parahaemolyticus was detected in 87 of 160 (54.4%) bivalve samples and in 32 of 130 (24.5%) seawater samples. Especially high levels were detected during summer to early autumn. All the seawater and bivalves contained less than 2 and 5% of the tdh and trh genes of the isolates, respectively, and seawater isolates possessed two fewer genes than the bivalve isolates. Of 23 antimicrobials tested, three agents (ofloxacin, norfloxacin, and trimethoprim/sulfamethoxazole) effectively treated V. parahaemolyticus illness due to the sensitivity of the isolates. The isolates were highly resistant to ampicillin, however, excluding it as a treatment option. More than half of the isolates exhibited resistance to at least three antimicrobials. These findings indicate the importance of an integrated monitoring and surveillance program noting the occurrence, virulence, and antimicrobial resistance patterns of V. parahaemolyticus in various aquatic sources for preventing human health risks from seafood consumption.

Due to the rapid elimination of bacteria through normal behaviour of filter feeding and excretion, the decontamination of hazardous contaminating bacteria from shellfish is performed by depuration. This process, under conditions that maximize shellfish filtering activity, is a useful method to eliminate microorganisms from bivalves. The microbiota composition in bivalves reflects that of the environment of harvesting waters, so quite different bacteriomes would be expected in shellfish collected in different locations. Bacterial accumulation within molluscan shellfish occurs primarily in the hepatopancreas. In order to assess the effect of the depuration process on these different bacteriomes, in this work we used 16S RNA pyrosequencing and metagenome prediction to assess the impact of 15 h of depuration on the whole hepatopancreas bacteriome of mussels collected in three different locations.

Anchialine lakes are a globally rare and unique ecosystem consisting of saline lakes surrounded by land and isolated from the surrounding marine environment. These lakes host a unique flora and fauna including numerous endemic species. Relatively few studies have, however, studied the prokaryote communities present in these lakes and compared them with the surrounding 'open water' marine environment. In the present study, we used a 16S rRNA gene barcoded pyrosequencing approach to examine prokaryote (Bacteria and Archaea) composition in three distinct biotopes (sediment, water and the mussel Brachidontes sp.) inhabiting four habitats, namely, three marine lakes and the surrounding marine environment of Berau, Indonesia. Biotope and habitat proved significant predictors of variation in bacterial and archaeal composition and higher taxon abundance. Most bacterial sequences belonged to OTUs assigned to the Proteobacteria. Compared to sediment and water, mussels had relatively high abundances of the classes Mollicutes and Epsilonproteobacteria. Most archaeal sequences, in turn, belonged to OTUs assigned to the Crenarchaeota with the relative abundance of crenarchaeotes highest in mussel samples. For both Bacteria and Archaea, the main variation in composition was between water samples on the one hand and sediment and mussel samples on the other. Sediment and mussels also shared much more OTUs than either shared with water. Abundant bacterial OTUs in mussels were related to organisms previously obtained from corals, oysters and the deepsea mussel Bathymodiolus manusensis. Abundant archaeal OTUs in mussels, in contrast, were closely related to organisms previously obtained from sediment.

Levels of Escherichia coli and male-specific bacteriophages (MSBs) were determined in the filter feeders obtained from retail markets, commercial farms, and wild beds in Korea. The accumulation and elimination of E. coli and MSBs were compared between ascidians and bivalves (oysters and mussels) during relaying and depuration. E. coli concentrations in ascidians from retail markets ranged between < 20 and 460 most probable number/100 g while MSBs were not detected. E. coli levels in bivalves from commercial farms and wild beds were not significantly different but bacterial levels in ascidians were consistently lower. Ascidians exhibited much lower ability than bivalves to accumulate E. coli and MSBs during relaying in a polluted coastal area. This study also shows that an equilibrium was developed between levels of microbes in water and ascidians and shellfish during relaying. E. coli and MSBs in ascidians decreased quickly during depuration in a clean seawater tank. However, after 1 day, E. coli in bivalves decreased by only 1.1-1.6 logs, and the elimination of MSBs was negligible. Therefore, depuration is an effective means to reduce the health risk of contaminated ascidians.

This study comprises the first systematic survey of the occurrence of Norovirus in Mediterranean mussels from harvesting areas in Montenegro coast of Adriatic Sea. Mussels may accumulate contaminants of public health concern, including pathogenic bacteria and viruses. Microbiological monitoring of harvesting areas is based on count of Escherichia coli in bivalve molluscs in the European Union. It is assumed that E. coli does not reflect contamination with enteric viruses. A structured field study was undertaken at six locations in Bay of Kotor, Montenegro, in order to investigate plausible influence of environmental factors on the variability of E. coli and norovirus (NoV). From July 2015 to July 2016, a total of 72 samples of mussels were collected in coastal harvesting areas of the Montenegro. The samples were screened for NoV of genogroups GI and GII using reverse transcription-qPCR (RT-qPCR). There were 43% NoV positive samples with higher presence of genogroup GII (74.2%). With regard to influence of environmental conditions on Norovirus presence, we have proved seasonal pattern of virus occurrence i.e., the largest number of positive samples was noticed during winter, while other physico-chemical factors were not of great significance. It was found that count of E. coli did not correlate with Norovirus prevalence. From the aspect of food safety, an upgrade of monitoring plans could lead to obtaining safer products.

According to the hologenome theory, the microbiota contributes to the fitness of the holobiont having an important role in its adaptation, survival, development, health, and evolution. Environmental stress also affects the microbiota and its capability to assist the holobiont in coping with stress factors. Here, we analyzed the diversity of cultivable bacteria associated with Manila clam tissues (mantle, gills, hemolymph) in two non-contaminated sites (Portugal and France) and one metal-contaminated site (Portugal). A total of 240 isolates were obtained. Representative isolates (n = 198) of the overall diversity were identified by 16S rDNA sequencing and subjected to functional characterization. Isolates affiliated with Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Proteobacteria (mostly Pseudoalteromonadaceae and Vibrionaceae) were dominant in non-contaminated sites while Actinobacteria (mostly Microbacteriaceae) dominated in the metal-contaminated site. The main factor affecting the microbiota composition was contamination. No significant differences were observed between clam tissues and geographic regions. Several isolates tested positive for antibacterial activity, biofilm formation, protease, and siderophore production. The results show that the Manila clam harbors a diverse microbiota that may contribute to clam protection and overall fitness, as well as to its adaptation to stressful environments. In addition, the Manila clam microbiota is revealed as a promising source of novel probiotics with potential application in aquaculture.

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.

Bacteria living in marine sediment play crucial roles in the benthic-pelagic interface coupling process. However, the complexity of the marine environment and the abundance of interfering materials hamper metaproteomic research of the marine mudflat environment. In this study, a modified sequential protein extraction method was used for marine mudflat sediment metaproteomic investigation. For marine sediment samples in cultured clam mudflat, more than 1000 protein spots were visualized in a two-dimensional gel electrophoresis map and 78 % of 194 randomly selected spots were successfully identified by mass spectrometry. We further applied this method to compare long-term clam aquaculture and natural mudflat sediment and identified 53 altered proteins from different microbe resources, which belonged to different functional categories or metabolic pathways. We found that proteins involved in stress/defense response process, ATP regeneration and protein folding more inclined to increase abundance while arginine biosynthesis and signal transduction process related proteins preferred to decrease in clam cultured mudflat sediment. Meanwhile, proteins were abundant in pathogens of bivalves, such as Vibrio and Photobacterium, and decreased in Acinetobacter, after about 8 months clam cultured. Furthermore, the terminal restriction fragment length polymorphism assay was performed to compare microbial community composition between sediments mentioned above. Results showed that the top three enrich genera in natural sediment were Cytophaga, Butyrivibrio and Spirochaeta, while Cytophaga, Spirochaeta and Azoarcus were found enrichment in long-term mudflat aquaculture sediment.

In the present study, we sampled bacterial communities associated with mussels inhabiting two distinct coastal marine ecosystems in Kalimantan, Indonesia, namely, marine lakes and coastal mangroves. We used 16S rRNA gene pyrosequencing and predicted metagenomic analysis to compare microbial composition and function. Marine lakes are small landlocked bodies of seawater isolated to varying degrees from the open sea environment. They contain numerous endemic taxa and represent natural laboratories of speciation. Our primary goals were to (1) use BLAST search to identify closely related organisms to dominant bacterial OTUs in our mussel dataset and (2) to compare bacterial communities and enrichment in the predicted bacterial metagenome among lakes. Our sequencing effort yielded 3553 OTUs belonging to 44 phyla, 99 classes and 121 orders. Mussels in the largest marine lake (Kakaban) and the coastal mangrove habitat were dominated by bacteria belonging to the phylum Proteobacteria whereas smaller lakes, located on the island of Maratua, were dominated by bacteria belonging to the phyla Firmicutes and Tenericutes. The single most abundant OTU overall was assigned to the genus Mycoplasma. There were several significant differences among locations with respect to metabolic pathways. These included enrichment of xenobiotic biodegradation pathways in the largest marine lake and coastal mangrove. These locations were also the most enriched with respect to nitrogen metabolism. The presence of genes related to isoquinoline alkaloids, polyketides, hydrolases, mono and dioxygenases in the predicted analysis of functional pathways is an indication that the bacterial communities of Brachidontes mussels may be potentially important sources of new marine medicines and enzymes of industrial interest. Future work should focus on measuring how mussel microbial communities influence nutrient dynamics within the marine lake environment and isolating microbes with potential biotechnological applications.

Vibrio tapetis CECT4600 is a pathogenic Gram-negative bacterium causing the brown ring disease in the Manila clam Ruditapes philippinarum. This vibriosis is induced by bacterial attachment on the periostracal lamina, yielding a decalcification of the bivalve shell. As in many bacterial species, pathogenesis is likely related to biofilm formation. The proteinaceous exoproducts of the marine bacterium Pseudoalteromonas sp. 3J6 inhibit the formation of biofilm by most of the tested marine bacteria without affecting their planktonic growth. In the present work, we examined the sensitivity of V. tapetis to Pseudoalteromonas sp. 3J6 and its exoproducts. In V. tapetis CECT4600-GFP-Pseudoalteromonas sp. 3J6 co-cultures, the latter outcompeted V. tapetis whatever the growth mode (planktonic or biofilm), which could result from a slower growth of V. tapetis. Biofilms containing only V. tapetis were grown in vitro on a glass substratum under dynamic conditions. When the glass was coated with a culture supernatant of Pseudoalteromonas sp. 3J6 (SN(3J6)) prior to inoculating V. tapetis CECT4600-GFP, the bacterial attachment was about fivefold lower than in control experiment without SN3J6 and the biofilm formation was delayed by about 24 h: A full biofilm was obtained at 48 versus 24 h for the control. Moreover, a preformed V. tapetis biofilm (grown on SN(3J6)-free glass substratum) could be disrupted by incubating it with SN3J6. This data suggest that Pseudoalteromonas sp. 3J6 is a good candidate to set up an anti-V. tapetis strategy usable in aquaculture to grow V. tapetis-free Manila clam spats.

A group of four strains isolated from clams (Venerupis decussata and Venerupis philippinarum) in Galicia (NW Spain) were subjected to a polyphasic characterization, based on the phenotypic characteristics, the analysis of chemotaxonomic features, the sequencing of the 16S rRNA and five housekeeping (atpA, pyrH, recA, rpoA and rpoD) genes, as well as DNA-DNA hybridization (DDH). The analysis of the phenotypic and chemotaxonomic characteristics and the results of a phylogenetic study, based on the 16S rRNA gene sequence analysis and multilocus sequence analysis, clearly indicated that these strains belong to the genus Vibrio and were allocated between the Splendidus and Anguillarum clades showing a close relationship with the type strains of Vibrio tapetis (98.8 %), Vibrio pomeroyi (98.0 %) and Vibrio crassostreae (97.9 %). DNA-DNA hybridization results confirmed that these isolates constitute a new species. The name Vibrio cortegadensis sp. nov. is proposed with C 16.17(T) (=CECT 7227(T)=LMG 27474(T)) as the type strain.

A large number of bacteria, including agents responsible for diseases, characterise sewage-polluted seawaters. Apart from standards for bathing waters and bivalve aquaculture waters, there are no general microbiological standards applicable to seawaters to help decide if bacterial pollution is within acceptable ranges. This study represents an attempt towards the issue of comparing the susceptibility of different marine invertebrates subjected to polluted seawater with a high microbial contamination. We explored the survival rates and the microbiological accumulation of mollusc bivalves, echinoderms and crustaceans species exposed to sewage-polluted seawaters. Microbiological analyses were performed on the polluted seawater and on the homogenates of exposed and unexposed specimens. Culturable bacteria (22 °C and 37 °C) and microbial pollution indicators (total coliforms, Escherichia coli and intestinal enterococci) were measured. When exposed to the sewage-polluted seawater, the examined invertebrates showed different survival rates. In the filter feeders, bacterial densities at 22 °C and 37 °C rose after 96 h of exposure to sewage. The highest concentrations of total coliforms and intestinal enterococci were found in exposed bivalve Mytilus galloprovincialis. The concentrations of bacteria growing at 37 °C were lower in the exposed deposit feeders compared to the polluted seawater. Some yeasts were absent in several exposed species although these yeasts were present in the polluted seawater. Our data suggest that the examined filter feeders, given their capability to survive and accumulate bacteria, may counteract the effects of sewage and restore seawater quality.

The aim of this study was to investigate the prevalence and genetic basis of multidrug resistance in Chryseobacterium indologenes from seawater and marine invertebrates used for human consumption, in Kaštela Bay, Adriatic Sea, Croatia. Out of 16 samples of seawater, Mediterranean mussel (Mytilus galloprovincialis Lam.), Rayed Mediterranean limpets (Patella caerulea L.) and Purple sea urchins (Paracentrotus lividus Lam.) collected, 15 were positive for C. indologenes. In total, 41 isolates were randomly selected and tested for antibiotic susceptibility by disc-diffusion and broth microdilution methods. PCR was used to detect alleles encoding extended-spectrum (ESBLs) and metallo-β-lactamases (MBLs). The clonality of β-lactamase-producing strains was evaluated by random amplified polymorphic DNA (RAPD) analysis. All C. indologenes isolates showed multiple resistance to at least 9 out of 16 antibiotics tested. Lowest resistance rates were found for piperacillin (9.7 %) and ciprofloxacin (24.4 %), whereas only piperacillin/tazobactam and trimethoprim/sulfamethoxazole showed 100 % activity. More than half of isolates carried bla (IND)-type gene, including 2 isolates carrying bla (IND-2) and 21 carrying bla (IND-7), that was identified as a major MBL genotype in isolates from Adriatic Sea. RAPD typing of IND-producing isolates revealed 6 major groups with no predominant clone in population. The presence of multidrug resistant and IND-producing C. indologenes in marine environment, including marine fauna, pose a risk for transmitting this opportunistic pathogen to humans through recreation or consummation of seafood. In addition, the antibiotic susceptibility test results have practical relevance for empirical treatment of C. indologenes infections.

We characterised the anti-Vibrio parahaemolyticus (anti-V. parahaemolyticus) marine bacteria DIT09, DIT44 and DIT46 isolated from the intertidal mussel Perumytilus purpuratus. The 16S rRNA gene sequences identify a Pseudoalteromonas sp. that form a clade with P. prydzensis and P. mariniglutinosa. The strains produced bacteriostatic anti-V. parahaemolyticus agents during the exponential growth phase, which were also active against V. cholerae and V. anguillarum, but not on other Gram positive and Gram negative bacteria. Bacteriostatic agents could be permeated by analytic ultra-filtration with 3.5 kDa cut-off, partially precipitated with 70 and 90 % ammonium sulphate, but not extracted with ethyl acetate. Reverse-phase HPLC revealed the production of a set of 5-6 active compounds by each strain (elution from 20 to 40 % acetonitrile), with similar but non identical HPLC patterns. Additionally, V. parahaemolyticus was able to progressively overcome the inhibition of antibiotics in trypticase soy agar with Fe(III) 0.5 up to 2 mM, suggesting the involvement of a set of novel siderophore or active molecules targeted at different Fe-siderophore uptake systems. The overall findings suggest that Pseudoalteromonas sp. DIT strains produce a putatively novel class of bacteriostatic and probably amphiphilic anti-Vibrio agents, indicating the need for further studies with chemical purification followed by their structural and functional characterization. Finally, the crude cell-free extracts, as well as the strains incubated at 10(3) and 10(5) c.f.u./mL, did not cause mortality in Artemia franciscana nauplii, suggesting that these bacteria are serious candidates for further probiotic evaluations with shellfish and fish cultures.

A novel Gram-positive, aerobic, motile, hemolytic, endospore-forming and rod-shaped bacterium TW25(T) was isolated from a dead ark clam during a mass mortality event on the South coast of Korea. The strain grew optimally at 30°C, at pH 8-9, and with 1% (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain TW25(T) was associated with the genus Ornithinibacillus and that it was most closely related to the type strain of Ornithinibacillus californiensis (98.5% similarity). The dominant cellular fatty acids were iso-C15:0, anteiso-C15:0 and C16:0. The peptidoglycan amino acid type was A4β, containing L: -ornithine and D: -aspartic acid. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, four unidentified phospholipids, two unidentified aminolipids and two unidentified lipids. The major respiratory quinone was menaquinone-7 (MK-7). The G + C content of genomic DNA was 36.7 mol%. DNA-DNA hybridization experiments with related strains revealed lower than 11 ± 3% relatedness. Based on this polyphasic taxonomic study, strain TW25(T) represents a novel species in the genus Ornithinibacillus, for which the name Ornithinibacillus scapharcae sp. nov. is proposed. The type strain is TW25(T) (=KACC 15116(T) = JCM 17314(T)).

Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from additional isolates of urease-positive thermophilic Campylobacter (UPTC) were performed. These isolates were obtained from the natural environment in Northern Ireland (n = 9 from mussels) and in England (n = 1 from sea water). All isolates carried the shorter flaA gene, [open reading frames (ORFs), 1,461 to 1,503 base pairs], without any internal termination codons, and did not carry any flaA pseudogenes. The UPTC isolates were well discriminated by the neighbor joining (NJ) phylogenetic tree constructed based on the putative flaA genes ORFs nucleotide sequence information. In addition, the NJ tree constructed based on the flaA-short variable region sequence information discriminated the Campylobacter lari isolates with a similar degree of discrimination power.

Two autochthonous clams, Venerupis aurea laeta and Cerastoderma edule/glaucum, collected for a period of 1 year, from May 2007 to January 2008, were studied in the Cape Peloro lagoon. On the samples, it was evaluated the presence of pathogenic microorganisms and proteins, lipids and yield edible part were determined. It was evaluated the concentration of mineral elements (Cd, Cu, Mn, Se, Pb, Zn) and organic contaminants (∑DDT and PCBs). Salmonella is never present in any sample. Total and faecal coliform values have always been under law limits for alive bivalves mussels production and sale. Zn, Cu, Cd and Pb levels were found in clams where they are slightly lower too. Both species do not show significant changes for Mn and Se. DDT and PCBs traces were discovered in Ganzirri Lake' samples, but in all observed cases, they were below law limits. As a result of all these observations, it is clear that, in recent years, the bivalve's use of Ganzirri Lake does not denote toxicological risk to human health.

Small heat shock proteins (sHSPs) encompass a widespread and diverse class of proteins with molecular chaperone activity. In the present study, two sHSP isoforms (VpsHSP-1 and VpsHSP-2) were cloned from Venerupis philippinarum haemocytes by Rapid Amplification of cDNA Ends (RACE) approaches. The expression profiles of these two genes under Vibrio anguillarum challenge and cadmium exposure were investigated by quantitative real-time reverse transcriptase polymerase chain reaction. The bacterial challenge could significantly up-regulate the mRNA expression of both VpsHSP-1 and VpsHSP-2, with the increase of VpsHSP-2 expression occurred earlier than that of VpsHSP-1. During the cadmium exposure experiment, the expression level of both VpsHSP-1 and VpsHSP-2 decreased significantly with larger amplitude in VpsHSP-2. As time progressed, the expression levels of both genes were up-regulated with more increment in the low-chemical exposure groups. The differences in the response to pathogen stimulation and cadmium exposure indicated that there were functional diversity between the two structurally different molecules, VpsHSP-1 and VpsHSP-2, and they probably played distinct roles in mediating the environmental stress and immune responses in calm.

To understand sulfur oxidation in thioauto-trophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox)(soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.

To understand reductive genome evolution (RGE), we comparatively analyzed the recently reported small genomes of two chemoautotrophic, intracellular symbionts of deep-sea clams, Calyptogena okutanii and C. magnifica. Both genomes lack most genes for DNA recombination and repair such as recA and mutY. Their genome architectures were highly conserved except one inversion. Many deletions from small (<100 bp) to large (1-11 kbp) sizes were detected and the deletion numbers decreased exponentially with size. Densities of deletions and short-repeats, as well as A+T content were higher in non-coding regions than in coding regions. Because Calyptogena symbiont genomes lack recA, we propose that deletions and the single inversion occurred by RecA-independent recombination (RIR) at short-repeats with simultaneous consumption of repeats, and that short-repeats were regenerated by accelerated mutations with enhanced A+T bias due to the absence of mutY. We further propose that extant Calyptogena symbiont genomes are in an actively reducing stage of RGE consisting of small and large deletions, and the deletions are caused by short-repeat dependent RIR along with regeneration of short-repeats. In future, the RGE rate will slowdown when the gene repertoires approach the minimum gene set necessary for intracellular symbiotic life.

Endosymbioses between chemosynthetic bacteria and marine invertebrates are remarkable biological adaptations to life in sulfide-rich environments. In these mutualistic associations, sulfur-oxidizing chemoautotrophic bacteria living directly within host cells both aid in the detoxification of toxic sulfide and fix carbon to support the metabolic needs of the host. Though best described for deep-sea vents and cold seeps, these symbioses are ubiquitous in shallow-water reducing environments. Indeed, considerable insight into sulfur-oxidizing endosymbioses in general comes from detailed studies of shallow-water protobranch clams in the genus Solemya. This review highlights the impressive body of work characterizing bacterial symbiosis in Solemya species, all of which are presumed to harbor endosymbionts. In particular, studies of the coastal Atlantic species Solemya velum and its larger Pacific congener Solemya reidi are the foundation for our understanding of the metabolism and physiology of marine bivalve symbioses, which are now known to occur in five families. Solemya velum, in particular, is an excellent model organism for symbiosis research. This clam can be collected easily from coastal eelgrass beds and maintained in laboratory aquaria for extended periods. In addition, the genome of the S. velum symbiont is currently being sequenced. The integration of genomic data with additional experimental analyses will help reveal the molecular basis of the symbiont-host interaction in Solemya, thereby complementing the wide array of research programs aimed at better understanding the diverse relationships between bacterial and eukaryotic cells.