Hans K. Carlson

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

H-NOX Regulation of c-di-GMP Metabolism and Biofilm Formation in Legionella pneumophila

Haem Nitric oxide/OXygen (H‐NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H‐NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H‐NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Δhnox1 strain displays a hyper‐biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF‐EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well‐studied diguanylate cyclase, vca0956, results in a hyper‐biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)‐NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Δhnox1 background results in reversion of the hyper‐biofilm phenotype back to wild‐type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c‐di‐GMP production by lpg1057 and biofilm formation in response to NO.

H-NOX–mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri

The bioluminescent bacterium Vibrio fischeri initiates a specific, persistent symbiosis in the light organ of the squid Euprymna scolopes. During the early stages of colonization, V. fischeri is exposed to host-derived nitric oxide (NO). Although NO can be both an antimicrobial component of innate immunity and a key signaling molecule in eukaryotes, potential roles in beneficial host–microbe associations have not been described. V. fischeri hnoX encodes a heme NO/oxygen-binding (H-NOX) protein, a member of a family of bacterial NO- and/or O2-binding proteins of unknown function. We hypothesized that H-NOX acts as a NO sensor that is involved in regulating symbiosis-related genes early in colonization. Whole-genome expression studies identified 20 genes that were repressed in an NO- and H-NOX–dependent fashion. Ten of these, including hemin-utilization genes, have a promoter with a putative ferric-uptake regulator (Fur) binding site. As predicted, in the presence of NO, wild-type V. fischeri grew more slowly on hemin than a hnoX deletion mutant. Host-colonization studies showed that the hnoX mutant was also 10-fold more efficient in initially colonizing the squid host than the wild type; similarly, in mixed inoculations, it outcompeted the wild-type strain by an average of 16-fold after 24 h. However, the presence of excess hemin or iron reversed this dominance. The advantage of the mutant in colonizing the iron-limited light-organ tissues is caused, at least in part, by its greater ability to acquire host-derived hemin. Our data suggest that V. fischeri normally senses a host-generated NO signal through H-NOXVf and modulates the expression of its iron uptake capacity during the early stages of the light-organ symbiosis.

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP

Significance Bacteria are hidden forces of nature. For example, Geobacter bacteria play important roles in geochemistry by reducing metals in the environment. Scientists also are exploring the application of these bacteria toward toxic metal remediation and as “living batteries” that can generate electricity from biowaste. However, there is limited understanding of the signaling pathways that regulate this extracellular metal-reducing activity. Here we have discovered that Geobacter sulfurreducens use riboswitch sensors for a signaling molecule called cAG to regulate this process, which is an unexpected finding because cAG was previously associated only with pathogenic bacteria. Furthermore, we have adapted the riboswitch to generate a fluorescent biosensor that can be used to visualize cAG signaling in live bacteria. Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3′-5′, 3′-5′ cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

Toward a mechanistic understanding of anaerobic nitrate-dependent iron oxidation: balancing electron uptake and detoxification

The anaerobic oxidation of Fe(II) by subsurface microorganisms is an important part of biogeochemical cycling in the environment, but the biochemical mechanisms used to couple iron oxidation to nitrate respiration are not well understood. Based on our own work and the evidence available in the literature, we propose a mechanistic model for anaerobic nitrate-dependent iron oxidation. We suggest that anaerobic iron-oxidizing microorganisms likely exist along a continuum including: (1) bacteria that inadvertently oxidize Fe(II) by abiotic or biotic reactions with enzymes or chemical intermediates in their metabolic pathways (e.g., denitrification) and suffer from toxicity or energetic penalty, (2) Fe(II) tolerant bacteria that gain little or no growth benefit from iron oxidation but can manage the toxic reactions, and (3) bacteria that efficiently accept electrons from Fe(II) to gain a growth advantage while preventing or mitigating the toxic reactions. Predictions of the proposed model are highlighted and experimental approaches are discussed.

Inhibition of microbial sulfate reduction in a flow-through column system by (per)chlorate treatment.

Microbial sulfate reduction is a primary cause of oil reservoir souring. Here we show that amendment with chlorate or perchlorate [collectively (per)chlorate] potentially resolves this issue. Triplicate packed columns inoculated with marine sediment were flushed with coastal water amended with yeast extract and one of nitrate, chlorate, or perchlorate. Results showed that although sulfide production was dramatically reduced by all treatments, effluent sulfide was observed in the nitrate (10 mM) treatment after an initial inhibition period. In contrast, no effluent sulfide was observed with (per)chlorate (10 mM). Microbial community analyses indicated temporal community shifts and phylogenetic clustering by treatment. Nitrate addition stimulated Xanthomonadaceae and Rhizobiaceae growth, supporting their role in nitrate metabolism. (Per)chlorate showed distinct effects on microbial community structure compared with nitrate and resulted in a general suppression of the community relative to the untreated control combined with a significant decrease in sulfate reducing species abundance indicating specific toxicity. Furthermore, chlorate stimulated Pseudomonadaceae and Pseudoalteromonadaceae, members of which are known chlorate respirers, suggesting that chlorate may also control sulfidogenesis by biocompetitive exclusion of sulfate-reduction. Perchlorate addition stimulated Desulfobulbaceae and Desulfomonadaceae, which contain sulfide oxidizing and elemental sulfur-reducing species respectively, suggesting that effluent sulfide concentrations may be controlled through sulfur redox cycling in addition to toxicity and biocompetitive exclusion. Sulfur isotope analyses further support sulfur cycling in the columns, even when sulfide is not detected. This study indicates that (per)chlorate show great promise as inhibitors of sulfidogenesis in natural communities and provides insight into which organisms and respiratory processes are involved.

Identification of a perchlorate reduction genomic island with novel regulatory and metabolic genes.

ABSTRACT A comparative analysis of the genomes of four dissimilatory (per)chlorate-reducing bacteria has revealed a genomic island associated with perchlorate reduction. In addition to the characterized metabolic genes for perchlorate reductase and chlorite dismutase, the island contains multiple conserved uncharacterized genes possibly involved in electron transport and regulation.

Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate.

We investigated perchlorate (ClO4−) and chlorate (ClO3−) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H2S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance. Using tagged-transposon pools we identified genes responsible for sensitivity and resistance in Desulfovibrio alaskensis G20. We found that mutants in Dde_2702 (Rex), a repressor of the central sulfate-reduction pathway were resistant to both (per)chlorate and nitrate. In general, Rex derepresses its regulon in response to increasing intracellular NADH:NAD+ ratios. In cells in which respiratory sulfate reduction is inhibited, NADH:NAD+ ratios should increase leading to derepression of the sulfate-reduction pathway. In support of this, in (per)chlorate or nitrate-stressed wild-type G20 we observed higher NADH:NAD+ ratios, increased transcripts and increased peptide counts for genes in the core Rex regulon. We conclude that one mode of (per)chlorate and nitrate toxicity is as direct inhibitors of the central sulfate-reduction pathway. Our results demonstrate that (per)chlorate are more potent inhibitors than nitrate in both pure cultures and communities, implying that they represent an attractive alternative for controlling sulfidogenesis in industrial ecosystems. Of these, perchlorate offers better application logistics because of its inhibitory potency, solubility, relative chemical stability, low affinity for mineral cations and high mobility in environmental systems.

Use of a Semisynthetic Epitope to Probe Histidine Kinase Activity and Regulation

Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this article, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPgammaS as a substrate to form a stable thiophosphohistidine even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine, and tyrosine residues is shown to recognize thiophosphohistidine and thiophosphoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators.

Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms.

Despite the environmental and economic cost of microbial sulfidogenesis in industrial operations, few compounds are known as selective inhibitors of respiratory sulfate reducing microorganisms (SRM), and no study has systematically and quantitatively evaluated the selectivity and potency of SRM inhibitors. Using general, high-throughput assays to quantitatively evaluate inhibitor potency and selectivity in a model sulfate-reducing microbial ecosystem as well as inhibitor specificity for the sulfate reduction pathway in a model SRM, we screened a panel of inorganic oxyanions. We identified several SRM selective inhibitors including selenate, selenite, tellurate, tellurite, nitrate, nitrite, perchlorate, chlorate, monofluorophosphate, vanadate, molydate, and tungstate. Monofluorophosphate (MFP) was not known previously as a selective SRM inhibitor, but has promising characteristics including low toxicity to eukaryotic organisms, high stability at circumneutral pH, utility as an abiotic corrosion inhibitor, and low cost. MFP remains a potent inhibitor of SRM growing by fermentation, and MFP is tolerated by nitrate and perchlorate reducing microorganisms. For SRM inhibition, MFP is synergistic with nitrite and chlorite, and could enhance the efficacy of nitrate or perchlorate treatments. Finally, MFP inhibition is multifaceted. Both inhibition of the central sulfate reduction pathway and release of cytoplasmic fluoride ion are implicated in the mechanism of MFP toxicity.