Elizabeth A. Karr

The alternative oxidase (AOX) gene in Vibrio fischeri is controlled by NsrR and upregulated in response to nitric oxide

Alternative oxidase (AOX) is a respiratory oxidase found in certain eukaryotes and bacteria; however, its role in bacterial physiology is unclear. Exploiting the genetic tractability of the bacterium Vibrio fischeri, we explore the regulation of aox expression and AOX function. Using quantitative PCR and reporter assays, we demonstrate that aox expression is induced in the presence of nitric oxide (NO), and that the NO‐responsive regulatory protein NsrR mediates the response. We have identified key amino acid residues important for NsrR function and experimentally confirmed a bioinformatically predicted NsrR binding site upstream of aox. Microrespirometry demonstrated that oxygen consumption by V. fischeri CydAB quinol oxidase is inhibited by NO treatment, whereas oxygen consumption by AOX is less sensitive to NO. NADH oxidation assays using inverted membrane vesicles confirmed that NO directly inhibits CydAB, and that AOX is resistant to NO inhibition. These results indicate a role for V. fischeri AOX in aerobic respiration during NO stress.

The Methanogen-Specific Transcription Factor MsvR Regulates the fpaA-rlp-rub Oxidative Stress Operon Adjacent to msvR in Methanothermobacter thermautotrophicus

Methanogens represent some of the most oxygen-sensitive organisms in laboratory culture. Recent studies indicate that they have developed mechanisms to deal with brief oxygen exposure. MsvR is a transcriptional regulator that has a domain architecture unique to a select group of methanogens. Here, runoff in vitro transcription assays were used to demonstrate that MsvR regulates transcription of the divergently transcribed fpaA-rlp-rub operon in Methanothermobacter thermautotrophicus in addition to transcription from its own promoter. The protein products of the fpaA-rlp-rub operon have previously been implicated in oxidative stress responses in M. thermautotrophicus. Additionally, electrophoretic mobility shift assays (EMSAs) and DNase I footprinting were used to confirm a binding site inferred by bioinformatic analysis. Sequence mutations within these binding sites did not significantly alter EMSA shifting patterns on longer templates but did on shorter 50-bp fragments encompassing only the region containing the binding sites. Footprinting confirmed that the regions protected for the longer mutant templates are at different positions within the intergenic region compared to those seen in the intact intergenic region. Oxidized and reduced preparations of MsvR demonstrated different EMSA binding patterns and regions of protection on the intergenic sequence, suggesting that MsvR may play a role in detecting the redox state of the cell.

TrpY regulation of trpB2 transcription in Methanothermobacter thermautotrophicus

TrpY binds specifically to TRP box sequences upstream of trpB2, but the repression of trpB2 transcription requires additional TrpY assembly that is stimulated by but not dependent on the presence of tryptophan. Inhibitory complex formation is prevented by insertions within the regulatory region and by a G149R substitution in TrpY, even though TrpY(G149R) retains both TRP box DNA- and tryptophan-binding abilities.

Transcription regulation in the third domain.

The ability of organisms to sense and respond to their environment is essential to their survival. This is no different for members of the third domain of life, the Archaea. Archaea are found in diverse and often extreme habitats. However, their ability to sense and respond to their environment at the level of gene expression has been understudied when compared to bacteria and eukaryotes. Over the last decade, the field has expanded, and a variety of unique and interesting regulatory schemes have been unraveled. In this review, the current state of knowledge of archaeal transcription regulation is explored.

Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues

BackgroundMethanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. The mechanisms by which they sense and respond to oxidizing conditions are unknown. MsvR is a transcription regulatory protein unique to the methanoarchaea. Initially identified and characterized in the methanogen Methanothermobacter thermautotrophicus (Mth), MthMsvR displays differential DNA binding under either oxidizing or reducing conditions. Since MthMsvR regulates a potential oxidative stress operon in M. thermautotrophicus, it was hypothesized that the MsvR family of proteins were redox-sensitive transcription regulators.ResultsAn MsvR homologue from the methanogen Methanosarcina acetivorans, MaMsvR, was overexpressed and purified. The two MsvR proteins bound the same DNA sequence motif found upstream of all known MsvR encoding genes, but unlike MthMsvR, MaMsvR did not bind the promoters of select genes involved in the oxidative stress response. Unlike MthMsvR that bound DNA under both non-reducing and reducing conditions, MaMsvR bound DNA only under reducing conditions. MaMsvR appeared as a dimer in gel filtration chromatography analysis and site-directed mutagenesis suggested that conserved cysteine residues within the V4R domain were involved in conformational rearrangements that impact DNA binding.ConclusionsResults presented herein suggest that homodimeric MaMsvR acts as a transcriptional repressor by binding Ma PmsvR under non-reducing conditions. Changing redox conditions promote conformational changes that abrogate binding to Ma PmsvR which likely leads to de-repression.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

ABSTRACT Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA. Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

The Methanosarcina acetivorans thioredoxin system activates DNA binding of the redox-sensitive transcriptional regulator MsvR

The production of biogas (methane) by an anaerobic digestion is an important facet to renewable energy, but is subject to instability due to the sensitivity of strictly anaerobic methanogenic archaea (methanogens) to environmental perturbations, such as oxygen. An understanding of the oxidant-sensing mechanisms used by methanogens may lead to the development of more oxidant tolerant (i.e., stable) methanogen strains. MsvR is a redox-sensitive transcriptional regulator that is found exclusively in methanogens. We show here that oxidation of MsvR from Methanosarcina acetivorans (MaMsvR) with hydrogen peroxide oxidizes cysteine thiols, which inactivates MaMsvR binding to its own promoter (PmsvR). Incubation of oxidized MaMsvR with the M. acetivorans thioredoxin system (NADPH, MaTrxR, and MaTrx7) results in reduction of the cysteines back to thiols and activation of PmsvR binding. These data confirm that cysteines are critical for the thiol-disulfide regulation of PmsvR binding by MaMsvR and support a role for the M. acetivorans thioredoxin system in the in vivo activation of MaMsvR. The results support the feasibility of using MaMsvR and PmsvR, along with the Methanosarcina genetic system, to design methanogen strains with oxidant-regulated gene expression systems, which may aid in stabilizing anaerobic digestion.

Examination of metal corrosion by Desulfomicrobium thermophilum, Archaeoglobus fulgidus, and Methanothermobacter thermautotrophicus

Abstract. Metal corrosion resulting from microbial processes constitutes a serious expense in many industrial applications. Oil pipelines are plagued by these events leading not only to economic losses but also severe environmental consequences. Therefore, our understanding of the microorganisms that contribute to metal corrosion is vital. Here, the roles of a methanogen and either a sulfate-reducing bacterium or archaeon in corrosion are addressed. The methanogen Methanothermobacter thermautotrophicus was used to assess the effect of methanogenesis in corrosion. The sulfate-reducing bacterium Desulfomicrobium thermophilum and the sulfate-reducing archaeon Archaeoglobus fulgidus were used to measure the effect of sulfate reduction on corrosion. To assess the combined effect of sulfate-reduction and methanogenesis on corrosion, M. thermautotrophicus was grown in combination with A. fulgidus or D. thermophilum. This allowed investigation of corrosion impact at different temperatures and salinities. All organisms contributed to corrosion, measured by metal weight loss over time, either independently or in conjunction with a partner organism. Corrosion resulting from strictly biotic processes was dominant at lower temperature and salinities. At higher salinities and temperatures, approximately half of the corrosion measured was because of abiotic reactions as indicated by experimental controls. This work demonstrates the role of these microbial metabolisms in metal corrosion in anaerobic environments.

Spontaneous trpY Mutants and Mutational Analysis of the TrpY Archaeal Transcription Regulator

Over 90% of Methanothermobacter thermautotrophicus mutants isolated as spontaneously resistant to 5-methyl tryptophan had mutations in trpY. Most were single-base-pair substitutions that identified separate DNA- and tryptophan-binding regions in TrpY. In vivo and in vitro studies revealed that DNA binding was sufficient for TrpY repression of trpY transcription but that TrpY must bind DNA and tryptophan to assemble a complex that represses trpEGCFBAD.

Crystal structure and DNA binding activity of a PadR family transcription regulator from hypervirulent Clostridium difficile R20291.

BackgroundClostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. We examined the structure and function of a gene product with the locus tag CDR20291_0991 (cdPadR1) as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent C. difficile strain R20291. cdPadR1 is genomically positioned near genes that are involved in stress response and virulence. In addition, it was previously reported that cdPadR1 and a homologue from the historical C. difficile strain 630 (CD630_1154) were differentially expressed when exposed to stressors, including antibiotics.ResultsThe crystal structure of cdPadR1 was determined to 1.9 Å resolution, which revealed that it belongs to the PadR-s2 subfamily of PadR transcriptional regulators. cdPadR1 binds its own promoter and other promoter regions from within the C. difficile R20291 genome. DNA binding experiments demonstrated that cdPadR1 binds a region comprised of inverted repeats and an AT-rich core with the predicted specific binding motif, GTACTAT(N2)ATTATA(N)AGTA, within its own promoter that is also present in 200 other regions in the C. difficile R20291 genome. Mutation of the highly conserved W in α4 of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other PadR-s2 proteins, resulted in alterations of cdPadR1 binding to the predicted binding motif, potentially due to loss of higher order oligomerization.ConclusionsOur results indicate that cdPadR1 binds a region within its own promoter consisting of the binding motif GTACTAT(N2)ATTATA(N)AGTA and seems to associate non-specifically with longer DNA fragments in vitro, which may facilitate promoter and motif searching. This suggests that cdPadR1 acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence.