Marine Proteobacteria metabolize glycolate via the ß-hydroxyaspartate cycle.

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments1. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria2-4, the further fate of this C2 metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the ß-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago5. We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing-to our knowledge-the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton.

The human gut microbiome in early-onset type 1 diabetes from the TEDDY study.

Type 1 diabetes (T1D) is an autoimmune disease that targets pancreatic islet beta cells and incorporates genetic and environmental factors1, including complex genetic elements2, patient exposures3 and the gut microbiome4. Viral infections5 and broader gut dysbioses6 have been identified as potential causes or contributing factors; however, human studies have not yet identified microbial compositional or functional triggers that are predictive of islet autoimmunity or T1D. Here we analyse 10,913 metagenomes in stool samples from 783 mostly white, non-Hispanic children. The samples were collected monthly from three months of age until the clinical end point (islet autoimmunity or T1D) in the The Environmental Determinants of Diabetes in the Young (TEDDY) study, to characterize the natural history of the early gut microbiome in connection to islet autoimmunity, T1D diagnosis, and other common early life events such as antibiotic treatments and probiotics. The microbiomes of control children contained more genes that were related to fermentation and the biosynthesis of short-chain fatty acids, but these were not consistently associated with particular taxa across geographically diverse clinical centres, suggesting that microbial factors associated with T1D are taxonomically diffuse but functionally more coherent. When we investigated the broader establishment and development of the infant microbiome, both taxonomic and functional profiles were dynamic and highly individualized, and dominated in the first year of life by one of three largely exclusive Bifidobacterium species (B. bifidum, B. breve or B. longum) or by the phylum Proteobacteria. In particular, the strain-specific carriage of genes for the utilization of human milk oligosaccharide within a subset of B. longum was present specifically in breast-fed infants. These analyses of TEDDY gut metagenomes provide, to our knowledge, the largest and most detailed longitudinal functional profile of the developing gut microbiome in relation to islet autoimmunity, T1D and other early childhood events. Together with existing evidence from human cohorts7,8 and a T1D mouse model9, these data support the protective effects of short-chain fatty acids in early-onset human T1D.

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.

Pass-back chain extension expands multimodular assembly line biosynthesis.

Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally effect a linear sequence of catalytic cycles. Here, we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS-PKS assembly lines that defy many standard rules of assembly line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. We generate a series of precise domain-inactivating mutations in thalassospiramide assembly lines, and present evidence for an unprecedented biosynthetic model that invokes intermodule substrate activation and tailoring, module skipping and pass-back chain extension, whereby the ability to pass the growing chain back to a preceding module is flexible and substrate driven. Expanding bidirectional intermodule domain interactions could represent a viable mechanism for generating chemical diversity without increasing the size of biosynthetic assembly lines and challenges our understanding of the potential elasticity of multimodular megaenzymes.

Hup-Type Hydrogenases of Purple Bacteria: Homology Modeling and Computational Assessment of Biotechnological Potential.

Three-dimensional structures of six closely related hydrogenases from purple bacteria were modeled by combining the template-based and ab initio modeling approach. The results led to the conclusion that there should be a 4Fe3S cluster in the structure of these enzymes. Thus, these hydrogenases could draw interest for exploring their oxygen tolerance and practical applicability in hydrogen fuel cells. Analysis of the 4Fe3S cluster's microenvironment showed intragroup heterogeneity. A possible function of the C-terminal part of the small subunit in membrane binding is discussed. Comparison of the built models with existing hydrogenases of the same subgroup (membrane-bound oxygen-tolerant hydrogenases) was carried out. Analysis of intramolecular interactions in the large subunits showed statistically reliable differences in the number of hydrophobic interactions and ionic interactions. Molecular tunnels were mapped in the models and compared with structures from the PDB. Protein-protein docking showed that these enzymes could exchange electrons in an oligomeric state, which is important for oxygen-tolerant hydrogenases. Molecular docking with model electrode compounds showed mostly the same results as with hydrogenases from E. coli, H. marinus, R. eutropha, and S. enterica; some interesting results were shown in case of HupSL from Rba. sphaeroides and Rvi. gelatinosus.

Acetic acid bacteria encode two levansucrase types of different ecological relationship.

Acetic acid bacteria (AAB) are associated with plants and insects. Determinants for the targeting and occupation of these widely different environments are unknown. However, most of these natural habitats share plant-derived sucrose, which can be metabolized by some AAB via polyfructose building levansucrases (LS) known to be involved in biofilm formation. Here, we propose two LS types (T) encoded by AAB as determinants for habitat selection, which emerged from vertical (T1) and horizontal (T2) lines of evolution and differ in their genetic organization, structural features and secretion mechanism, as well as their occurrence in proteobacteria. T1-LS are secreted by plant-pathogenic α- and γ-proteobacteria, while T2-LS genes are common in diazotrophic, plant-growth-promoting α-, ß- and γ-proteobacteria. This knowledge may be exploited for a better understanding of microbial ecology, plant health and biofilm formation by sucrase-secreting proteobacteria in eukaryotic hosts.

Gut microbial beta-glucuronidase and glycerol/diol dehydratase activity contribute to dietary heterocyclic amine biotransformation.

BACKGROUND: Consuming red and processed meat has been associated with an increased risk of colorectal cancer (CRC), which is partly attributed to exposure to carcinogens such as heterocyclic amines (HCA) formed during cooking and preservation processes. The interaction of gut microbes and HCA can result in altered bioactivities and it has been shown previously that human gut microbiota can transform mutagenic HCA to a glycerol conjugate with reduced mutagenic potential. However, the major form of HCA in the colon are glucuronides (HCA-G) and it is not known whether these metabolites, via stepwise microbial hydrolysis and acrolein conjugation, are viable precursors for glycerol conjugated metabolites. We hypothesized that such a process could be concurrently catalyzed by bacterial beta-glucuronidase (B-GUS) and glycerol/diol dehydratase (GDH) activity. We therefore investigated how the HCA-G PhIP-N2-ß-D-glucuronide (PhIP-G), a representative liver metabolite of PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), which is the most abundant carcinogenic HCA in well-cooked meat, is transformed by enzymatic activity of human gut microbial representatives of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. RESULTS: We employed a combination of growth and enzymatic assays, and a bioanalysis approach combined with metagenomics. B-GUS of Faecalibacterium prausnitzii converted PhIP-G to PhIP and GDH of Flavonifractor plautii, Blautia obeum, Eubacterium hallii, and Lactobacillus reuteri converted PhIP to PhIP-M1 in the presence of glycerol. In addition, B-GUS- and GDH-positive bacteria cooperatively converted PhIP-G to PhIP-M1. A screen of genes encoding B-GUS and GDH was performed for fecal microbiome data from healthy individuals (n = 103) and from CRC patients (n = 53), which revealed a decrease in abundance of taxa with confirmed GDH and HCA transformation activity in CRC patients. CONCLUSIONS: This study for the first time demonstrates that gut microbes mediate the stepwise transformation of PhIP-G to PhIP-M1 via the intermediate production of PhIP. Findings from this study suggest that targeted manipulation with gut microbes bearing specific functions, or dietary glycerol supplementation might modify gut microbial activity to reduce HCA-induced CRC risk.

Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation.

Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans.

Horizontal gene transfer of three co-inherited methane monooxygenase systems gave rise to methanotrophy in the Proteobacteria.

The critical role that bacterial methanotrophs have in regulating the environmental concentrations of the potent greenhouse gas, methane, under aerobic conditions is dependent on monooxygenase enzymes which oxidise the substrate as both a carbon and energy source. Despite the importance of these organisms, the evolutionary origins of aerobic methane oxidation capability and its relationship to proteobacterial evolution is not well understood. Here we investigated the phylogenetic relationship of proteobacterial methanotrophs with related, non-methanotrophic bacteria using 16S rRNA and the evolution of two forms of methane monooxygenase: membrane bound (pMMO and pXMO) and cytoplasmic (sMMO). Through analysis we have concluded that extant proteobacterial methanotrophs evolved from up to five ancestral species, and that all three methane monooxygenase systems, pMMO, pXMO and sMMO, were likely present in the ancestral species (although pXMO and sMMO are not present in most of the present day methanotrophs). Here we propose that the three monooxygenase systems entered the ancestral species by horizontal gene transfer, with these likely to have pre-existing physiological and metabolic attributes that supported conversion to methanotrophy. Further, we suggest that prior to these enzyme systems developing methane oxidation capabilities, the membrane-bound and cytoplasmic monooxygenases were already both functionally and phylogenetically associated. These results not only suggest that sMMO and pXMO have a far greater role in methanotrophic evolution than previously understood but also implies that the co-inheritance of membrane bound and cytoplasmic monooxygenases have roles additional to that of supporting methanotrophy.

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase.

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase.

Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s-1 were determined and resulted in a catalytic efficiency (kcatKm-1) of 0.64 s-1 µM-1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcatKm-1 of 5.21 s-1 µM-1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg-1 and 0.46 U mg-1, as well as on benzyl methyl sulfide of 1.62 U mg-1 and 3.11 U mg-1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.

Epilithic Biofilms in Lake Baikal: Screening and Diversity of PKS and NRPS Genes in the Genomes of Heterotrophic Bacteria.

A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria , Firmicutes , Actinobacteria , and Bacteroidetes . To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus ) was 34% and NRPS genes were found in 78%. In Proteobacteria , PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria , respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus) was 34% and NRPS genes were found in 78%. In Proteobacteria, PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria, respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of ß-Aryl Ether Bonds in Lignin.

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via ß-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent ß-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because ß-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic ß-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

New Arsenate Reductase Gene (arrA) PCR Primers for Diversity Assessment and Quantification in Environmental Samples.

The extent of arsenic contamination in drinking water and its potential threat to human health have resulted in considerable research interest in the microbial species responsible for arsenic reduction. The arsenate reductase gene (arrA), an important component of the microbial arsenate reduction system, has been widely used as a biomarker to study arsenate-reducing microorganisms. A new primer pair was designed and evaluated for quantitative PCR (qPCR) and high-throughput sequencing of the arrA gene, because currently available PCR primers are not suitable for these applications. The primers were evaluated in silico and empirically tested for amplification of arrA genes in clones and for amplification and high-throughput sequencing of arrA genes from soil and groundwater samples. In silico, this primer pair matched (≥90% DNA identity) 86% of arrA gene sequences from GenBank. Empirical evaluation showed successful amplification of arrA gene clones of diverse phylogenetic groups, as well as amplification and high-throughput sequencing of independent soil and groundwater samples without preenrichment, suggesting that these primers are highly specific and can amplify a broad diversity of arrA genes. The arrA gene diversity from soil and groundwater samples from the Cache Valley Basin (CVB) in Utah was greater than anticipated. We observed a significant correlation between arrA gene abundance, quantified through qPCR, and reduced arsenic (AsIII) concentrations in the groundwater samples. Furthermore, we demonstrated that these primers can be useful for studying the diversity of arsenate-reducing microbial communities and the ways in which their relative abundance in groundwater may be associated with different groundwater quality parameters. IMPORTANCE: Arsenic is a major drinking water contaminant that threatens the health of millions of people worldwide. The extent of arsenic contamination and its potential threat to human health have resulted in considerable interest in the study of microbial species responsible for the reduction of arsenic, i.e., the conversion of AsV to AsIII In this study, we developed a new primer pair to evaluate the diversity and abundance of arsenate-reducing microorganisms in soil and groundwater samples from the CVB in Utah. We observed significant arrA gene diversity in the CVB soil and groundwater samples, and arrA gene abundance was significantly correlated with the reduced arsenic (AsIII) concentrations in the groundwater samples. We think that these primers are useful for studying the ecology of arsenate-reducing microorganisms in different environments.

Comparison of bacterial community characteristics between complete and shortcut denitrification systems for quinoline degradation.

For quinoline-denitrifying degradation, very few researches focused on shortcut denitrification process and its bacterial community characteristics. In this study, complete and shortcut denitrification systems were constructed simultaneously for quinoline degradation. By calculation, specific quinoline removal rates were 0.905 and 1.123 g/(gVSS d), respectively, in the complete and shortcut systems, and the latter was 1.24 times of the former. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing, and quantitative PCR (qPCR) techniques based on 16S rRNA were jointly applied to compare microbial community structures of two systems. Many denitrifying bacteria phyla, classes, and genera were detected in the two systems. Phylum Proteobacteria, Class Gammaproteobacteria, and Genus Alicycliphilus denitrificans were the dominant contributors for quinoline-denitrifying degradation. In the shortcut denitrification system, main and specific strains playing crucial roles were more; the species richness and the total abundance of functional genes (narG, nirS, nirK, and nosZ) were higher compared with the complete denitrification system. It could be supposed that inorganic-nitrogen reductase activity of bacterial community was stronger in the shortcut denitrification system, which was the intrinsic reason to result in higher denitrification rate.

Distinct distribution patterns of proteobacterial nirK- and nirS-type denitrifiers in the Yellow River estuary, China.

Denitrification is considered to be the critical process in removing reactive nitrogen in estuarine ecosystems. In the present study, the abundance, diversity, and community structure of nirK- and nirS-type denitrifiers were compared in sediments from the Yellow River estuary. Quantitative polymerase chain reaction showed that the 2 types of denitrifiers exhibited different distribution patterns among the samples, indicating their distinct habitat preference. Phylogenetic analysis revealed that most of the sequences from clusters I, III, IV, and V for nirK-type denitrifiers were dominant and were distributed at sites where dissolved oxygen (DO) was lower, and the sequences in the other clusters were dominant at sites with higher DO. However, there was no spatially heterogeneous distribution for the nirS-type denitrifier community. Canonical correlation analysis and correlation analysis demonstrated that the community structure of nirK was more responsive to environmental factors than was that of nirS. Inversely, the abundance and α-diversity targeting nirS gene could be more easily influenced by environmental parameters. These findings can extend our current knowledge about the distribution patterns of denitrifying bacteria and provide a basic theoretical reference for the dynamics of denitrifying communities in estuarine ecosystem of China.

Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.

Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes.

Analysis of the metatranscriptome of microbial communities of an alkaline hot sulfur spring revealed different gene encoding pathway enzymes associated with energy metabolism.

Alkaline sulfur hot springs notable for their specialized and complex ecosystem powered by geothermal energy are abundantly rich in different chemotrophic and phototrophic thermophilic microorganisms. Survival and adaptation of these organisms in the extreme environment is specifically related to energy metabolism. To gain a better understanding of survival mechanism of the organisms in these ecosystems, we determined the different gene encoding enzymes associated with anaerobic pathways of energy metabolism by applying the metatranscriptomics approach. The analysis of the microbial population of hot sulfur spring revealed the presence of both aerobic and anaerobic organisms indicating dual mode of lifestyle of the community members. Proteobacteria (28.1 %) was the most dominant community. A total of 988 reads were associated with energy metabolism, out of which 33.7 % of the reads were assigned to nitrogen, sulfur, and methane metabolism based on KEGG classification. The major lineages of hot spring communities were linked with the anaerobic pathways. Different gene encoding enzymes (hao, nir, nar, cysH, cysI, acs) showed the involvement of microbial members in nitrification, denitrification, dissimilatory sulfate reduction, and methane generation. This study enhances our understanding of important gene encoding enzymes involved in energy metabolism, required for the survival and adaptation of microbial communities in the hot spring.

Unicellular cyanobacteria Synechocystis accommodate heterotrophic bacteria with varied enzymatic and metal resistance properties.

The interactions between heterotrophic bacteria and primary producers have a profound impact on the functioning of marine ecosystem. We characterized the enzymatic and metal resistance properties of fourteen heterotrophic bacteria isolated from a unicellular cyanobacterium Synechocystis sp. that came from a heavy metal contaminated region of Cochin estuary, southwest coast of India. Based on 16S rRNA gene sequence similarities, the heterotrophic bacteria were grouped into three phyla: namely Actinobacteria, Firmicute, and Proteobacteria. Overall Proteobacteria showed a higher level of enzyme expression while Actinobacteria and Firmicutes showed higher tolerance to heavy metals. Among Proteobacteria, an isolate of Marinobacter hydrocarbonoclasticus (MMRF-584) showed highest activities of ß-glucosidase (1.58 ± 0.2 µMml(-1) min(-1) ) and laminarinase (1170.17 ± 95.4 µgml(-1) min(-1) ), while other two isolates of M. hydrocarbonoclasticus, MMRF-578 and 581, showed highest phosphatase (44.71 ± 0.2 µMml(-1) min(-1) ) and aminopeptidase (33.22 ± 0 µMml(-1) min(-1) ) activities respectively. Among Firmicutes, the Virgibacillus sp. MMRF-571 showed exceptional resistance against the toxic heavy metals Cd (180 mM), Pb (150 mM), and Hg (0.5 mM). Bacillus cereus, MMRF-575, showed resistance to the highest concentrations of Co (250 mM), Cd (150 mM), Pb (180 mM), Hg (0.5 mM), Ni (280 mM), and Zn (250 mM) tested. Our results show that heterotrophic bacteria with varied enzymatic and metal resistance properties are associated with Synechocystis sp. Further studies to delineate the role of these heterotrophic bacteria in protecting primary producers from toxic effects of heavy metals and their potential application in bioremediation will be appreciated.

Construction and Characterization of a Cellulolytic Consortium Enriched from the Hindgut of Holotrichia parallela Larvae.

Degradation of rice straw by cooperative microbial activities is at present the most attractive alternative to fuels and provides a basis for biomass conversion. The use of microbial consortia in the biodegradation of lignocelluloses could reduce problems such as incomplete synergistic enzymes, end-product inhibition, and so on. In this study, a cellulolytic microbial consortium was enriched from the hindgut of Holotrichia parallela larvae via continuous subcultivation (20 subcultures in total) under static conditions. The degradation ratio for rice straw was about 83.1% after three days of cultivation, indicating its strong cellulolytic activity. The diversity analysis results showed that the bacterial diversity and richness decreased during the consortium enrichment process, and the consortium enrichment process could lead to a significant enrichment of phyla Proteobacteria and Spirochaetes, classes Clostridia, Epsilonproteobacteria, and Betaproteobacteria, and genera Arcobacter, Treponema, Comamonas, and Clostridium. Some of these are well known as typical cellulolytic and hemicellulolytic microorganisms. Our results revealed that the microbial consortium identified herein is a potential candidate for use in the degradation of waste lignocellulosic biomass and further highlights the hindgut of the larvae as a reservoir of extensive and specific cellulolytic and hemicellulolytic microbes.