Mechanistic insight into 3-methylmercaptopropionate metabolism and kinetical regulation of demethylation pathway in marine dimethylsulfoniopropionate-catabolizing bacteria.

The vast majority of oceanic dimethylsulfoniopropionate (DMSP) is thought to be catabolized by bacteria via the DMSP demethylation pathway. This pathway contains four enzymes termed DmdA, DmdB, DmdC and DmdD/AcuH, which together catabolize DMSP to acetylaldehyde and methanethiol as carbon and sulfur sources respectively. While molecular mechanisms for DmdA and DmdD have been proposed, little is known of the catalytic mechanisms of DmdB and DmdC, which are central to this pathway. Here, we undertake physiological, structural and biochemical analyses to elucidate the catalytic mechanisms of DmdB and DmdC. DmdB, a 3-methylmercaptopropionate (MMPA)-coenzyme A (CoA) ligase, undergoes two sequential conformational changes to catalyze the ligation of MMPA and CoA. DmdC, a MMPA-CoA dehydrogenase, catalyzes the dehydrogenation of MMPA-CoA to generate MTA-CoA with Glu435 as the catalytic base. Sequence alignment suggests that the proposed catalytic mechanisms of DmdB and DmdC are likely widely adopted by bacteria using the DMSP demethylation pathway. Analysis of the substrate affinities of involved enzymes indicates that Roseobacters kinetically regulate the DMSP demethylation pathway to ensure DMSP functioning and catabolism in their cells. Altogether, this study sheds novel lights on the catalytic and regulative mechanisms of bacterial DMSP demethylation, leading to a better understanding of bacterial DMSP catabolism.

Functional Redundancy in the Hydroxycinnamate Catabolism Pathways of the Salt Marsh Bacterium Sagittula stellata E-37.

The hydroxycinnamates (HCAs) ferulate and p-coumarate are among the most abundant constituents of lignin, and their degradation by bacteria is an essential step in the remineralization of vascular plant material. Here, we investigate the catabolism of these two HCAs by the marine bacterium Sagittula stellata E-37, a member of the roseobacter lineage with lignolytic potential. Bacterial degradation of HCAs is often initiated by the activity of a hydroxycinnamoyl-coenzyme A (hydroxycinnamoyl-CoA) synthase. Genome analysis of S. stellata revealed the presence of two feruloyl-CoA (fcs) synthase homologs, an unusual occurrence among characterized HCA degraders. In order to elucidate the role of these homologs in HCA catabolism, fcs-1 and fcs-2 were disrupted using insertional mutagenesis, yielding both single and double fcs mutants. Growth on p-coumarate was abolished in the fcs double mutant, whereas maximum cell yield on ferulate was only 2% of that of the wild type. Interestingly, the single mutants demonstrated opposing phenotypes, where the fcs-1 mutant showed impaired growth (extended lag and ∼60% of wild-type rate) on p-coumarate, and the fcs-2 mutant showed impaired growth (extended lag and ∼20% of wild-type rate) on ferulate, pointing to distinct but overlapping roles of the encoded fcs homologs, with fcs-1 primarily dedicated to p-coumarate utilization and fcs-2 playing a dominant role in ferulate utilization. Finally, a tripartite ATP-independent periplasmic (TRAP) family transporter was found to be required for growth on both HCAs. These findings provide evidence for functional redundancy in the degradation of HCAs in S. stellata E-37 and offer important insight into the genetic complexity of aromatic compound degradation in bacteria.IMPORTANCE Hydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacterium Sagittula stellata We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude that S. stellata is uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors.

Chemical differentiation of three DMSP lyases from the marine Roseobacter group.

Dimethylsulfoniopropionate (DMSP) catabolism of marine bacteria plays an important role in marine and global ecology. The genome of Ruegeria pomeroyi DSS-3, a model organism from the Roseobacter group, harbours no less than three genes for different DMSP lyases (DddW, DddP and DddQ) that catalyse the degradation of DMSP to dimethyl sulfide (DMS) and acrylate. Despite their apparent similar function these enzymes show no significant overall sequence identity. In this work DddQ and DddW from R. pomeroyi and the DddP homolog from Phaeobacter inhibens DSM 17395 were functionally characterised and their substrate scope was tested using several synthetic DMSP analogues. Comparative kinetic assays revealed differences in the conversion of DMSP and its analogues in terms of selectivity and overall velocity, giving additional insights into the molecular mechanisms of DMSP lyases and into their putatively different biological functions.

Structure of the Membrane-intrinsic Nitric Oxide Reductase from Roseobacter denitrificans.

Membrane-intrinsic nitric oxide reductases (NORs) are key components of bacterial denitrification pathways with a close evolutionary relationship to the cytochrome oxidase (COX) complex found in aerobic respiratory chains. A key distinction between COX and NOR is the identity of the metal directly opposite heme b3 within the active site. In NOR, this metal is iron (FeB), whereas in COX, it is copper (CuB). The purified NOR of Roseobacter denitrificans contains copper and has modest oxidase activity, raising the possibility that a COX-like active site might have independently arisen within the context of a NOR-like protein scaffold. Here we present the crystal structure of the Roseobacter denitrificans NorBC complex and anomalous scattering experiments probing the identity of each metal center. Our results refute the hypothesis that copper occupies the active site and instead reveal a new metal center in the small subunit not seen in any other NOR or COX.

Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149.

Two distinct bacterial and eukaryotic serine racemases (SRs) have been identified based on phylogenetic and biochemical characteristics. Although some reports have suggested that marine heterotrophic bacteria have the potential to produce d-serine, the gene encoding bacterial SRs is not found in those bacterial genomes. In this study, using in-depth genomic analysis, we found that eukaryotic SR homologues were distributed widely in various bacterial genomes. Additionally, we selected a eukaryotic SR homologue from a marine heterotrophic bacterium, Roseobacter litoralis Och 149 (RiSR), and constructed an RiSR gene expression system in Escherichia coli for studying the properties of the enzyme. Among the tested amino acids, the recombinant RiSR exhibited both racemization and dehydration activities only towards serine, similar to many eukaryotic SRs. Mg2+ and MgATP enhanced both activities of RiSR, whereas EDTA abolished these enzymatic activities. The enzymatic properties and domain structure of RiSR were similar to those of eukaryotic SRs, particularly mammalian SRs. However, RiSR showed lower catalytic efficiency for L-serine dehydration (kcat/Km=0.094 min(-1) mM(-1)) than those of eukaryotic SRs reported to date (kcat/Km=0.6-21 min(-1) mM(-1)). In contrast, the catalytic efficiency for L-serine racemization of RiSR (kcat/Km=3.14 min(-1) mM(-1)) was 34-fold higher than that of l-serine dehydration. These data suggested that RiSR primarily catalysed serine racemization rather than dehydration.

Pyrosequencing revealed SAR116 clade as dominant dddP-containing bacteria in oligotrophic NW Pacific Ocean.

Dimethyl sulfide (DMS) is a climatically active gas released into the atmosphere from oceans. It is produced mainly by bacterial enzymatic cleavage of dimethylsulfoniopropionate (DMSP), and six DMSP lyases have been identified to date. To determine the biogeographical distribution of bacteria relevant to DMS production, we investigated the diversity of dddP­the most abundant DMS-producing gene­in the northwestern Pacific Ocean using newly developed primers and the pyrosequencing method. Consistent with previous studies, the major dddP-containing bacteria in coastal areas were those belonging to the Roseobacter clade. However, genotypes closely related to the SAR116 group were found to represent a large portion of dddP-containing bacteria in the surface waters of the oligotrophic ocean. The addition of DMSP to a culture of the SAR116 strain Candidatus Puniceispirillum marinum IMCC1322 resulted in the production of DMS and upregulated expression of the dddP gene. Considering the large area of oligotrophic water and the wide distribution of the SAR116 group in oceans worldwide, we propose that these bacteria may play an important role in oceanic DMS production and biogeochemical sulfur cycles, especially via bacteria-mediated DMSP degradation.

Iron-sulfur cluster-dependent catalysis of chlorophyllide a oxidoreductase from Roseobacter denitrificans.

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.

The structure of RdDddP from Roseobacter denitrificans reveals that DMSP lyases in the DddP-family are metalloenzymes.

Marine microbes degrade dimethylsulfoniopropionate (DMSP), which is produced in large quantities by marine algae and plants, with DMSP lyases into acrylate and the gas dimethyl sulfide (DMS). Approximately 10% of the DMS vents from the sea into the atmosphere and this emission returns sulfur, which arrives in the sea through rivers and runoff, back to terrestrial systems via clouds and rain. Despite their key role in this sulfur cycle DMSP lyases are poorly understood at the molecular level. Here we report the first X-ray crystal structure of the putative DMSP lyase RdDddP from Roseobacter denitrificans, which belongs to the abundant DddP family. This structure, determined to 2.15 Å resolution, shows that RdDddP is a homodimeric metalloprotein with a binuclear center of two metal ions located 2.7 Å apart in the active site of the enzyme. Consistent with the crystallographic data, inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TRXF) revealed the bound metal species to be primarily iron. A 3D structure guided analysis of environmental DddP lyase sequences elucidated the critical residues for metal binding are invariant, suggesting all proteins in the DddP family are metalloenzymes.

Superoxide production by a manganese-oxidizing bacterium facilitates iodide oxidation.

The release of radioactive iodine (i.e., iodine-129 and iodine-131) from nuclear reprocessing facilities is a potential threat to human health. The fate and transport of iodine are determined primarily by its redox status, but processes that affect iodine oxidation states in the environment are poorly characterized. Given the difficulty in removing electrons from iodide (I(-)), naturally occurring iodide oxidation processes require strong oxidants, such as Mn oxides or microbial enzymes. In this study, we examine iodide oxidation by a marine bacterium, Roseobacter sp. AzwK-3b, which promotes Mn(II) oxidation by catalyzing the production of extracellular superoxide (O2(-)). In the absence of Mn(2+), Roseobacter sp. AzwK-3b cultures oxidized ∼90% of the provided iodide (10 µM) within 6 days, whereas in the presence of Mn(II), iodide oxidation occurred only after Mn(IV) formation ceased. Iodide oxidation was not observed during incubations in spent medium or with whole cells under anaerobic conditions or following heat treatment (boiling). Furthermore, iodide oxidation was significantly inhibited in the presence of superoxide dismutase and diphenylene iodonium (a general inhibitor of NADH oxidoreductases). In contrast, the addition of exogenous NADH enhanced iodide oxidation. Taken together, the results indicate that iodide oxidation was mediated primarily by extracellular superoxide generated by Roseobacter sp. AzwK-3b and not by the Mn oxides formed by this organism. Considering that extracellular superoxide formation is a widespread phenomenon among marine and terrestrial bacteria, this could represent an important pathway for iodide oxidation in some environments.

Comparative proteomics of Mn(II)-oxidizing and non-oxidizing Roseobacter clade bacteria reveal an operative manganese transport system but minimal Mn(II)-induced expression of manganese oxidation and antioxidant enzymes.

Manganese (Mn) is an essential nutrient and precipitates as minerals with technological and environmental relevance. To gain a proteomic understanding of how bacteria respond to Mn(II) and its connection to oxidation, a comparative examination of the proteomic response of Mn(II)-oxidizing (Roseobacter sp. AzwK-3b) and non-oxidizing (Ruegeria sp. TM1040) alphaproteobacteria was conducted. Both bacteria show an operative Mn(II) transport system. In the absence of Mn(II), both bacteria have higher expression of proteins that were homologous to SitA and SitB, known proteins in the Mn(II) transport system of other alphaproteobacteria. Overall, each bacterium demonstrated a varied response to Mn(II). Ru. TM1040 had a greater number of proteins differentially expressed in response to Mn(II) and also had a group of proteins related to chemotaxis at higher concentrations of Mn(II), suggesting a potential stress response. While both bacteria are able to generate extracellular superoxide and Mn(II) is a known antioxidant, the presence of Mn(II) did not significantly alter the expression of proteins related to antioxidant activity. Heme peroxidases, previously connected to Mn(II) oxidation, were found in the soluble protein extract of R. AzwK-3b, but only minor differential expression was observed as a function of Mn(II), indicating that their expression was not induced by Mn(II).

Identification of the genes encoding nitric oxide reductase in the aerobic photosynthetic bacterium Roseobacter denitrificans OCh114.

A cytochrome bc-type complex of Roseobacter denitrificans OCh114 was thought to be a novel cytochrome c oxidase. To determine its function, we deleted the genes encoding the complex. The mutant grew normally by aerobic respiration, but failed to grow by denitrification and lacked nitric oxide reductase activity, indicating that the physiological function of the gene product is nitric oxide reduction.

Proteomic responses of Roseobacter litoralis OCh149 to starvation and light regimen.

Roseobacter litoralis OCh149 is a type strain of aerobic anoxygenic phototrophic bacteria in marine Roseobacter clade. Its full genome has been sequenced; however, proteomic research, which will give deeper insights into the environmental stimuli on gene expression networks, has yet to be performed. In the present study, a proteomic approach was employed to analyze the status of R. litoralis OCh149 in carbon starvation during the stationary phase and its responses to a dark/light regimen (12 h:12 h) in both exponential and stationary phases. LC-MS/MS-based analysis of highly abundant proteins under carbon starvation revealed that proteins involved in transport, the transcription/translation process and carbohydrate metabolism were the major functional categories, while poly-ß-hydroxyalkanoate (PHA), previously accumulated in cells, was remobilized after stress. Glucose, as the sole carbon source in the defined medium, was broken down by Entner-Doudoroff and reductive pentose phosphate (PP) pathways. Carbohydrate catabolism-related proteins were down-regulated under light regardless of the growth phase, probably due to inhibition of respiration by light. In contrast, responses of amino acid metabolisms to light regimen varied among different proteins during growth phases depending on cellular requirements for proliferation, growth or survival. Fluorescence induction and relaxation measurements suggested that functional absorption cross-sections of the photosynthetic complexes decreased during the dark period and always recovered to about the previous level during the light period. Although the photosynthetic genes in R. litoralis OCh149 are located on the plasmid, these data indicate the regulatory mechanism of photoheterotroph metabolism by both carbon and light availability.

Co-cultures of Pseudomonas aeruginosa and Roseobacter denitrificans reveal shifts in gene expression levels compared to solo cultures.

Consistent biosynthesis of desired secondary metabolites (SMs) from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed "co-culturing" conditions and monitoring of messages via quantitative real-time PCR (qPCR). Gene expression of model bacterial strains (Pseudomonas aeruginosa PAO1 and Roseobacter denitrificans Och114) was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expression in vitro, Two P. aeruginosa genes (PhzH coding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN) and the RhdA gene for thiosulfate: cyanide sulfurtransferase (Rhodanese)) and two R. denitrificans genes (BetaLact for metallo-beta-lactamase and the DMSP gene for dimethylpropiothetin dethiomethylase) were assessed for differential expression. Results showed that R. denitrificans DMSP and BetaLact gene expression became elevated in a mixed culture. In contrast, P. aeruginosa co-cultures with R. denitrificans or a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expression in vitro and bypass complex genetic engineering manipulations.

DddW, a third DMSP lyase in a model Roseobacter marine bacterium, Ruegeria pomeroyi DSS-3.

Ruegeria pomeroyi DSS-3 is a model Roseobacter marine bacterium, particularly regarding its catabolism of dimethylsulfoniopropionate (DMSP), an abundant anti-stress molecule made by marine phytoplankton. We found a novel gene, dddW, which encodes a DMSP lyase that cleaves DMSP into acrylate plus the environmentally important volatile dimethyl sulfide (DMS). Mutations in dddW reduced, but did not abolish DMS production. Transcription of dddW was greatly enhanced by pre-growth of cells with DMSP, via a LysR-type regulator. Close DddW homologs occur in only one other Roseobacter species, and there are no close homologs and only a few related sequences in metagenomes of marine bacteria. In addition to DddW, R. pomeroyi DSS-3 had been shown to have two other, different, DMSP lyases, DddP and DddQ, plus an enzyme that demethylates DMSP, emphasizing the importance of this substrate for this model bacterium.

Correlating carbon monoxide oxidation with cox genes in the abundant Marine Roseobacter Clade.

The Marine Roseobacter Clade (MRC) is a numerically and biogeochemically significant component of the bacterioplankton. Annotation of multiple MRC genomes has revealed that an abundance of carbon monoxide dehydrogenase (CODH) cox genes are present, subsequently implying a role for the MRC in marine CO cycling. The cox genes fall into two distinct forms based on sequence analysis of the coxL gene; forms I and II. The two forms are unevenly distributed across the MRC genomes. Most (18/29) of the MRC genomes contain only the putative form II coxL gene. Only 10 of the 29 MRC genomes analysed have both the putative form II and the definitive form I coxL. None have only the form I coxL. Genes previously shown to be required for post-translational maturation of the form I CODH enzyme are absent from the MRC genomes containing only form II. Subsequent analyses of a subset of nine MRC strains revealed that only MRC strains with both coxL forms are able to oxidise CO.

Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade.

The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC-MS, and several MeSH-derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]-3-(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH- and MeSeH-derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH-derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

Optimization of extracellular thermotolerant alkaline protease produced by marine Roseobacter sp. (MMD040).

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.

Bacteria of the Roseobacter clade show potential for secondary metabolite production.

Members of the Roseobacter clade are abundant and widespread in marine habitats and have very diverse metabolisms. Production of acylated homoserine lactones (AHL) and secondary metabolites, e.g., antibiotics has been described sporadically. This prompted us to screen 22 strains of this group for production of signaling molecules, antagonistic activity against bacteria of different phylogenetic groups, and the presence of genes encoding for nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), representing enzymes involved in the synthesis of various pharmaceutically important natural products. The screening approach for NRPS and PKS genes was based on polymerase chain reaction (PCR) with degenerate primers specific for conserved sequence motifs. Additionally, sequences from whole genome sequencing projects of organisms of the Roseobacter clade were considered. Obtained PCR products were cloned, sequenced, and compared with genes of known function. With the PCR approach genes showing similarity to known NRPS and PKS genes were found in seven and five strains, respectively, and three PKS and NRPS sequences from genome sequencing projects were obtained. Three strains exhibited antagonistic activity and also showed production of AHL. Overall production of AHL was found in 10 isolates. Phylogenetic analysis of the 16S rRNA gene sequences of the tested organisms showed that several of the AHL-positive strains clustered together. Three strains were positive for three or four categories tested, and were found to be closely related within the genus Phaeobacter. The presence of a highly similar hybrid PKS/NRPS gene locus of unknown function in sequenced genomes of the Roseobacter clade plus the significant similarity of gene fragments from the strains studied to these genes argues for the functional requirement of the encoded hybrid PKS/NRPS complex. Our screening results therefore suggest that the Roseobacter clade is indeed employing PKS/NRPS biochemistry and should thus be further studied as a potential and largely untapped source of secondary metabolites.

Characterization of marine bacteria and the activity of their enzyme systems involved in degradation of the algal storage glucan laminarin.

The algal storage glucan laminarin is one of the most abundant carbon sources for marine prokaryotes. Its degradation was investigated in bacteria isolated during and after a spring phytoplankton bloom in the coastal North Sea. On average, 13% of prokaryotes detected by epifluorescence counts were able to grow in Most Probable Number dilution series on laminarin as sole carbon source. Several bacterial strains were isolated from different dilutions, and phylogenetic characterization revealed that they belonged to different phylogenetic groups. The activity of the laminarin-degrading enzyme systems was further characterized in three strains of Vibrio sp. that were able to grow on laminarin as sole carbon source. At least two types of activity were detected upon degradation of laminarin: release of glucose, and release of glucans larger than glucose. The expression of laminarinase activity was dependent on the presence of the substrate, and was repressed by the presence of glucose. In addition, low levels of activity were expressed under starvation conditions. Laminarinase enzymes showed minimal activity on substrates with similar glucosidic bonds to those of laminarin, but different sizes and secondary and/or tertiary structures. The characteristics found in these enzyme systems may help to elucidate factors hampering rapid carbohydrate degradation by prokaryotes.

Coupled photochemical and enzymatic Mn(II) oxidation pathways of a planktonic Roseobacter-Like bacterium.

Bacteria belonging to the Roseobacter clade of the alpha-Proteobacteria occupy a wide range of environmental niches and are numerically abundant in coastal waters. Here we reveal that Roseobacter-like bacteria may play a previously unrecognized role in the oxidation and cycling of manganese (Mn) in coastal waters. A diverse array of Mn(II)-oxidizing Roseobacter-like species were isolated from Elkhorn Slough, a coastal estuary adjacent to Monterey Bay in California. One isolate (designated AzwK-3b), in particular, rapidly oxidizes Mn(II) to insoluble Mn(III, IV) oxides. Interestingly, AzwK-3b is 100% identical (at the 16S rRNA gene level) to a previously described Pfiesteria-associated Roseobacter-like bacterium, which is not able to oxidize Mn(II). The rates of manganese(II) oxidation by live cultures and cell-free filtrates are substantially higher when the preparations are incubated in the presence of light. The rates of oxidation by washed cell extracts, however, are light independent. Thus, AzwK-3b invokes two Mn(II) oxidation mechanisms when it is incubated in the presence of light, in contrast to the predominantly direct enzymatic oxidation in the dark. In the presence of light, production of photochemically active metabolites is coupled with initial direct enzymatic Mn(II) oxidation, resulting in higher Mn(II) oxidation rates. Thus, Roseobacter-like bacteria may not only play a previously unrecognized role in Mn(II) oxidation and cycling in coastal surface waters but also induce a novel photooxidation pathway that provides an alternative means of Mn(II) oxidation in the photic zone.