Transcriptional Control of hgcAB by an ArsR-Like Regulator in Pseudodesulfovibrio mercurii ND132.

The hgcAB gene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg methylator Pseudodesulfovibrio mercurii ND132, we evaluated transcriptional control of hgcAB by a putative ArsR encoded upstream and cotranscribed with hgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosylhomocysteine (SAH)-responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR proteins in P. mercurii. Using quantitative PCR (qPCR) and RNA sequencing (RNA-seq) transcriptome analyses, we confirmed this ArsR regulates hgcAB transcription and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link between hgcAB activity and arsenic transformations, with significant upregulation of other ArsR-regulated arsenic resistance operons alongside hgcAB. Interestingly, wild-type ND132 was less sensitive to As(V) (but not As(III)) than an hgcAB knockout strain, supporting the idea that hgcAB may be linked to arsenic resistance. Arsenic significantly impacted rates of Hg methylation by ND132; however, responses varied with culture conditions. Differences in growth and metabolic activity did not account for arsenic impacts on methylation. While arsenic significantly increased hgcAB expression, hgcAB gene and transcript abundance was not a good predictor of Hg methylation rates. Taken together, these results support the idea that Hg and As cycling are linked in P. mercurii ND132. Our results may hold clues to the evolution of hgcAB and the controls on Hg methylation in nature. IMPORTANCE This work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that control hgcAB expression. We show that hgcAB expression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosylhomocysteine in our model organism, Pseudodesulfovibrio mercurii ND132. Exposure to arsenic also significantly impacted Pseudodesulfovibrio mercurii ND132 mercury methylation rates. However, expression of hgcAB was not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls on hgcAB expression, which is needed to better predict environmental methylmercury production.

Sulfur Metabolites Play Key System-Level Roles in Modulating Denitrification.

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. It is now recognized that intermediates of nitrogen and sulfur cycling (e.g., hydrogen sulfide, nitrite, etc.) can also directly impact NRB and SRB activities in freshwater, wastewater, and sediment and therefore may play important roles in competitive interactions. Here, through comparative transcriptomic and metabolomic analyses, we have uncovered mechanisms of hydrogen sulfide- and cysteine-mediated inhibition of nitrate respiratory growth for the NRB Intrasporangium calvum C5. Specifically, the systems analysis predicted that cysteine and hydrogen sulfide inhibit growth of I. calvum C5 by disrupting distinct steps across multiple pathways, including branched-chain amino acid (BCAA) biosynthesis, utilization of specific carbon sources, and cofactor metabolism. We have validated these predictions by demonstrating that complementation with BCAAs and specific carbon sources relieves the growth inhibitory effects of cysteine and hydrogen sulfide. We discuss how these mechanistic insights give new context to the interplay and stratification of NRB and SRB in diverse environments.IMPORTANCE Nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) colonize diverse anoxic environments, including soil subsurface, groundwater, and wastewater. NRB and SRB compete for resources, and their interplay has major implications on the global cycling of nitrogen and sulfur species, with undesirable outcomes in some contexts. For instance, the removal of reactive nitrogen species by NRB is desirable for wastewater treatment, but in agricultural soils, NRB can drive the conversion of nitrates from fertilizers into nitrous oxide, a potent greenhouse gas. Similarly, the hydrogen sulfide produced by SRB can help sequester and immobilize toxic heavy metals but is undesirable in oil wells where competition between SRB and NRB has been exploited to suppress hydrogen sulfide production. By characterizing how reduced sulfur compounds inhibit growth and activity of NRB, we have gained systems-level and mechanistic insight into the interplay of these two important groups of organisms and drivers of their stratification in diverse environments.

Robust Mercury Methylation across Diverse Methanogenic Archaea.

Methylmercury (MeHg) production was compared among nine cultured methanogenic archaea that contain hgcAB, a gene pair that codes for mercury (Hg) methylation. The methanogens tested produced MeHg at inherently different rates, even when normalized to growth rate and Hg availability. Eight of the nine tested were capable of MeHg production greater than that of spent- and uninoculated-medium controls during batch culture growth. Methanococcoides methylutens, an hgcAB+ strain with a fused gene pair, was unable to produce more MeHg than controls. Maximal conversion of Hg to MeHg through a full batch culture growth cycle for each species (except M. methylutens) ranged from 2 to >50% of the added Hg(II) or between 0.2 and 17 pmol of MeHg/mg of protein. Three of the species produced >10% MeHg. The ability to produce MeHg was confirmed in several hgcAB+ methanogens that had not previously been tested (Methanocella paludicola SANAE, Methanocorpusculum bavaricum, Methanofollis liminatans GKZPZ, and Methanosphaerula palustris E1-9c). Maximal methylation was observed at low sulfide concentrations (<100 µM) and in the presence of 0.5 to 5 mM cysteine. For M. hollandica, the addition of up to 5 mM cysteine enhanced MeHg production and cell growth in a concentration-dependent manner. As observed for bacterial Hg methylators, sulfide inhibited MeHg production. An initial evaluation of sulfide and thiol impacts on bioavailability showed methanogens responding to Hg complexation in the same way as do Deltaproteobacteria The mercury methylation rates of several methanogens rival those of the better-studied Hg-methylating sulfate- and iron-reducing DeltaproteobacteriaIMPORTANCEArchaea, specifically methanogenic organisms, play a role in mercury methylation in nature, but their global importance to MeHg production and the subsequent risk to ecosystems are not known. Methanogenesis has been linked to Hg methylation in several natural habitats where methylmercury production incurs risk to people and ecosystems, including rice paddies and permafrost. In this study, we confirm that most methanogens carrying the hgcAB gene pair are capable of Hg methylation. We found that methylation rates vary inherently among hgcAB+ methanogens but that several species are capable of MeHg production at rates that rival those of the better-know Hg-methylating sulfate- and iron-reducing bacteria. Methanogens may need to be considered equally with sulfate and iron reducers in evaluations of MeHg production in nature.

Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem.

Microbial methylation and demethylation are two competing processes controlling the net production and bioaccumulation of neurotoxic methylmercury (MeHg) in natural ecosystems. Although mercury (Hg) methylation by anaerobic microorganisms and demethylation by aerobic Hg-resistant bacteria have both been extensively studied, little attention has been given to MeHg degradation by anaerobic bacteria, particularly the iron-reducing bacterium Geobacter bemidjiensis Bem. Here we report, for the first time, that the strain G. bemidjiensis Bem can mediate a suite of Hg transformations, including Hg(II) reduction, Hg(0) oxidation, MeHg production and degradation under anoxic conditions. Results suggest that G. bemidjiensis utilizes a reductive demethylation pathway to degrade MeHg, with elemental Hg(0) as the major reaction product, possibly due to the presence of genes encoding homologues of an organomercurial lyase (MerB) and a mercuric reductase (MerA). In addition, the cells can strongly sorb Hg(II) and MeHg, reduce or oxidize Hg, resulting in both time and concentration-dependent Hg species transformations. Moderate concentrations (10-500 µM) of Hg-binding ligands such as cysteine enhance Hg(II) methylation but inhibit MeHg degradation. These findings indicate a cycle of Hg methylation and demethylation among anaerobic bacteria, thereby influencing net MeHg production in anoxic water and sediments.

Site-directed mutagenesis of HgcA and HgcB reveals amino acid residues important for mercury methylation.

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative "cap helix" region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1-V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.

Global detection and characterization of hypothetical proteins in Shewanella oneidensis MR-1 using LC-MS based proteomics.

The availability of whole genome sequences has enabled the application of powerful tools for assaying global expression patterns in environmentally relevant bacteria such as Shewanella oneidensis MR-1. A large number of genes in prokaryote genomes, including MR-1, have been annotated as hypothetical, indicating that no similar protein has yet been identified in other organisms. Using high-sensitivity MS coupled with accurate mass and time (AMT) tag methodology, 1078 tryptic peptides were collectively detected in MR-1 cultures, 671 of which were unique to their parent protein. Using only these unique tryptic peptides and a minimum of two peptides per protein, we identified, with high confidence, the expression of 258 hypothetical proteins. These proteins ranged from 3.5 to 139 kDa, with 47 being 100 amino acid residues or less. Using a combination of information including detection in cells grown under specific culture conditions, presence within a specific cell fraction, and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some hypothetical proteins. Further, by applying this approach a number of proteins were found not only to be expressed, but only expressed under certain culturing conditions, thereby suggesting function while at the same time isolating several proteins to distinct locales of the cell. These results demonstrate the utility of the AMT tag methodology for comprehensive profiling of the microbial proteome while confirming the expression of a large number of hypothetical genes.