Joy E. M. Watts

Metamorphosis of a Scleractinian Coral in Response to Microbial Biofilms

ABSTRACT Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.

Identification of a Bacterium That Specifically Catalyzes the Reductive Dechlorination of Polychlorinated Biphenyls with Doubly Flanked Chlorines

ABSTRACT A microorganism whose growth is linked to the dechlorination of polychlorinated biphenyls (PCBs) with doubly flanked chlorines was identified. Identification was made by reductive analysis of community 16S ribosomal DNA (rDNA) sequences from a culture enriched in the presence of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB), which was dechlorinated at the para position. Denaturing gradient gel electrophoresis (DGGE) analysis of total 16S rDNA extracted from the culture led to identification of three operational taxonomic units (OTUs 1, 2, and 3). OTU 1 was always detected when 2,3,4,5-CB or other congeners with doubly flanked chlorines were present and dechlorinated. Only OTUs 2 and 3 were detected in the absence of PCBs and when other PCBs (i.e., PCBs lacking doubly flanked chlorines) were not dechlorinated. Partial sequences of OTUs 2 and 3 exhibited 98.2% similarity to the sequence of “Desulfovibrio caledoniensis” (accession no. DCU53465 ). A sulfate-reducing vibrio isolated from the culture generated OTUs 2 and 3. This organism could not dechlorinate 2,3,4,5-CB. From these results we concluded that OTU 1 represents the dechlorinating bacterium growing in a coculture with a Desulfovibrio sp. The 16S rDNA sequence of OTU 1 is most similar to the 16S rDNA sequence of bacterium o-17 (89% similarity), an ortho-PCB-dechlorinating bacterium. The PCB dechlorinator, designated bacterium DF-1, reductively dechlorinates congeners with doubly flanked chlorines when it is supplied with formate or H2-CO2 (80:20).

Characterization of the microbial community and nitrogen transformation processes associated with moving bed bioreactors in a closed recirculated mariculture system

The microbial consortium of a moving bed bioreactor (MBB) connected to a marine recirculating aquaculture system was examined by denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA gene fragments. Both ammonia and nitrite oxidizers, Nitrosomonas cryotolerans and Nitrospira marina, respectively, were found associated with the marine system as well as a number of heterotrophic bacteria, including Pseudomonas sp. and Sphingomonas sp. In addition, two Planctomycetes sp. were detected in the system suggesting the capability for anaerobic ammonia oxidation (anammox). The potential for carrying out different nitrogen transformation processes—nitrification, denitrification and anammox—by the bead consortium in both low and high organic load MBBs was measured by short-term batch incubation. Beads with a high organic load exhibited a lower nitrification rate (25 mg NH3–N/m2/h) than low organic load beads (31.5 mg NH3–N/m2/h) as well as the ability to carry out denitrification and anammox processes. The potential of using MBBs to induce different nitrogen transformation processes was evaluated, and it was found that this type of bioreactor has the capability to serve as a platform for mediating desired anoxic processes such as denitrification and anammox.

Lignocellulose degradation mechanisms across the Tree of Life

Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.

Sequential Reductive Dechlorination of meta-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different Chloroflexi Phylotypes

ABSTRACT Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.

Anaerobic ammonium-oxidizing (Anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system

ABSTRACT Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.

Anaerobic ammonia-oxidizing bacteria and related activity in Baltimore inner harbor sediment.

ABSTRACT The discovery of bacteria capable of anaerobic ammonia oxidation (anammox) has generated interest in understanding the activity, diversity, and distribution of these bacteria in the environment. In this study anammox activity in sediment samples obtained from the Inner Harbor of Baltimore, Md., was detected by 15N tracer assays. Anammox-specific oligonucleotide primer sets were used to screen a Planctomycetales-specific 16S rRNA gene library generated from sediment DNA preparations, and four new anammox bacterial sequences were identified. Three of these sequences form a cohesive new branch of the anammox group, and the fourth sequence branches separately from this group. Denaturing gradient gel electrophoresis analysis of sediment incubated with anammox-specific media confirmed the presence of the four anammox-related 16S rRNA gene sequences. Evidence for the presence of anammox bacteria in Inner Harbor sediment was also obtained by using an anammox-specific probe in fluorescence in situ hybridization studies. To our knowledge, this is the first report of anammox activity and related bacterial 16S rRNA gene sequences from the Chesapeake Bay basin area, and the results suggest that this pathway plays an important role in the nitrogen cycle of this estuarine environment. Furthermore, the presence of these bacteria and their activity in sediment strengthen the contention that anammox-related Plactomycetales are globally distributed.

Detection and phylogenetic analysis of novel crenarchaeote and euryarchaeote 16S ribosomal RNA gene sequences from a Great Barrier Reef sponge

Abstract: The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was investigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluorescently labeled oligonucleotide probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first report of euryarchaeaotes associated with marine sponges.

Development of Two PCR-Based Techniques for Detecting Helical and Coccoid Forms of Helicobacter pylori

ABSTRACT The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

The Rising Tide of Antimicrobial Resistance in Aquaculture: Sources, Sinks and Solutions

As the human population increases there is an increasing reliance on aquaculture to supply a safe, reliable, and economic supply of food. Although food production is essential for a healthy population, an increasing threat to global human health is antimicrobial resistance. Extensive antibiotic resistant strains are now being detected; the spread of these strains could greatly reduce medical treatment options available and increase deaths from previously curable infections. Antibiotic resistance is widespread due in part to clinical overuse and misuse; however, the natural processes of horizontal gene transfer and mutation events that allow genetic exchange within microbial populations have been ongoing since ancient times. By their nature, aquaculture systems contain high numbers of diverse bacteria, which exist in combination with the current and past use of antibiotics, probiotics, prebiotics, and other treatment regimens—singularly or in combination. These systems have been designated as “genetic hotspots” for gene transfer. As our reliance on aquaculture grows, it is essential that we identify the sources and sinks of antimicrobial resistance, and monitor and analyse the transfer of antimicrobial resistance between the microbial community, the environment, and the farmed product, in order to better understand the implications to human and environmental health.