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The YvrI alternative sigma factor is essential for acid stress induction of oxalate decarboxylase in Bacillus subtilis.

ABSTRACT: YvrI is a recently identified alternative sigma factor in Bacillus subtilis that requires the coactivator YvrHa to activate transcription. Previously, a strain engineered to overproduce YvrI was found to overproduce oxalate decarboxylase (OxdC), and further analysis identified three YvrI-activated promoters preceding the yvrI-yvrHa, yvrJ, and oxdC-yvrL operons. Independently, proteome analyses identified OxdC as a highly abundant, cell wall-associated protein that accumulated under acidic growth conditions. We show here that the accumulation of OxdC in the cell wall proteome under acidic growth conditions is absolutely dependent on YvrI and is correlated with enhanced transcription of both the yvrI-yvrHa and the oxdC-yvrL operons. Conversely, OxdC accumulates to a high level even under nonacidic growth conditions in cells lacking YvrL, a negative regulator of YvrI/YvrHa-dependent transcription. These results indicate that YvrI and its associated coregulators YvrHa and YvrL are required for the regulation of OxdC expression by acid stress. The high-level accumulation of OxdC depends, in part, on a strong oxdC promoter. A regulatory sequence with similarity to an upstream promoter element (UP) was identified upstream of the oxdC promoter and is required for high-level promoter activity. Conservation of the YvrI/YvrHa/YvrL regulatory system among related species allowed us to deduce an expanded consensus sequence for the compositionally unusual promoters recognized by this new sigma factor.

SUBMITTER: MacLellan SR

PROVIDER: S-EPMC2632099 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A previously unidentified sigma factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis.

Project description:We have investigated the function of a cell envelope stress-inducible gene, yvrI, which encodes a 22.5 kDa protein that includes a predicted sigma(70) region 4 domain, but lacks an apparent region 2 domain. YvrI interacts with RNA polymerase and overexpression of YvrI results in induction of OxdC, an oxalate decarboxylase maximally expressed under low-pH conditions. We have used microarray-based analyses to define the YvrI regulon. YvrI is required for the transcription of three operons (oxdC-yvrL, yvrJ and yvrI-yvrHa) each of which is preceded by a highly similar promoter sequence. Activation of these promoters requires both YvrI and the product of the second gene in the yvrI-yvrHa operon, YvrHa. YvrI and YvrHa together allow recognition of the oxdC promoter, stimulate DNA melting and activate transcription by core RNA polymerase. Together, these results suggest that YvrI is a previously unrecognized sigma factor in Bacillus subtilis and that the 9.5 kDa YvrHa protein acts as a required co-activator of transcription. A yvrL deletion results in the upregulation of YvrI activity suggesting that YvrL is a negative regulator of YvrI-dependent transcription, possibly functioning as an anti-sigma factor.

| S-EPMC2688449 | biostudies-literature

Nitric oxide reversibly inhibits Bacillus subtilis oxalate decarboxylase.

Project description:Membrane inlet mass spectrometry (MIMS) has been employed to assay the catalytic activity of oxalate decarboxylase (OxDC), allowing us to demonstrate that nitric oxide (NO) reversibly inhibits the enzyme under dioxygen-depleted conditions. X-band EPR measurements do not provide any direct evidence for the interaction of NO with either of the Mn(II) centers in OxDC raising the possibility that there is a separate dioxygen-binding pocket in the enzyme.

| S-EPMC3057162 | biostudies-literature

Biochemical properties and oxalate-degrading activity of oxalate decarboxylase from bacillus subtilis at neutral pH.

Project description:Oxalate decarboxylase (OxDC) from Bacillus subtilis is a Mn-dependent hexameric enzyme that converts oxalate to carbon dioxide and formate. OxDC has greatly attracted the interest of the scientific community, mainly due to its biotechnological and medical applications in particular for the treatment of hyperoxaluria, a group of pathologic conditions caused by oxalate accumulation. The enzyme has an acidic optimum pH, but most of its applications involve processes occurring at neutral pH. Nevertheless, a detailed biochemical characterization of the enzyme at neutral pH is lacking. Here, we compared the structural-functional properties at acidic and neutral pH of wild-type OxDC and of a mutant form, called OxDC-DSSN, bearing four amino acid substitutions in the lid (Ser161-to-Asp, Glu162-to-Ser, Asn163-toSer, and Ser164-to-Asn) that improve the oxalate oxidase activity and almost abolish the decarboxylase activity. We found that both enzymatic forms do not undergo major structural changes as a function of pH, although OxDC-DSSN displays an increased tendency to aggregation, which is counteracted by the presence of an active-site ligand. Notably, OxDC and OxDC-DSSN at pH 7.2 retain 7 and 15% activity, respectively, which is sufficient to degrade oxalate in a cellular model of primary hyperoxaluria type I, a rare inherited disease caused by excessive endogenous oxalate production. The significance of the data in the light of the possible use of OxDC as biological drug is discussed. © 2019 IUBMB Life, 1-11, 2019.

| S-EPMC6850040 | biostudies-literature

Formation of Hexacoordinate Mn(III) in Bacillus subtilis Oxalate Decarboxylase Requires Catalytic Turnover.

Project description:Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C-C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).

| S-EPMC8183094 | biostudies-literature

Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4.

Project description:This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin-trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion, both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping are similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein.

| S-EPMC4355160 | biostudies-literature

Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis.

Project description:Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.

| S-EPMC2041844 | biostudies-literature

Immobilization of Bacillus subtilis oxalate decarboxylase on a Zn-IMAC resin.

Project description:Oxalate decarboxylase, a bicupin enzyme coordinating two essential manganese ions per subunit, catalyzes the decomposition of oxalate into carbon dioxide and formate in the presence of oxygen. Current efforts to elucidate its catalytic mechanism are focused on EPR studies of the Mn. We report on a new immobilization strategy linking the enzyme's N-terminal His6-tag to a Zn-loaded immobilized metal affinity resin. Activity is lowered somewhat due to the expected crowding effect. High-field EPR spectra of free and immobilized enzyme show that the resin affects the coordination environment of the active site Mn ions only minimally. The immobilized preparation was used to study the effect of varying pH on the same sample. Repeated freeze-thaw cycles lead to break down of the resin beads and some enzyme loss from the sample. However, the EPR signal increases due to higher packing efficiency on the sample column.

| S-EPMC5668902 | biostudies-other

Translational activation by an alternative sigma factor in Bacillus subtilis

Project description:Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, B, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, A, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription fromB-dependent promoters liberates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between B- and A-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.

2021-03-06 | GSE168393 | GEO

Phenolic acid-mediated regulation of the padC gene, encoding the phenolic acid decarboxylase of Bacillus subtilis.

Project description:In Bacillus subtilis, several phenolic acids specifically induce expression of padC, encoding a phenolic acid decarboxylase that converts these antimicrobial compounds into vinyl derivatives. padC forms an operon with a putative coding sequence of unknown function, yveFG, and this coding sequence does not appear to be involved in the phenolic acid stress response (PASR). To identify putative regulators involved in the PASR, random transposon mutagenesis, combined with two different screens, was performed. PadR, a negative transcriptional regulator of padC expression, was identified. padR is not located in the vicinity of padC, and the expression of padR is low and appears constitutive. This is in contrast with what occurs in other gram-positive bacteria, in which padR is autoregulated and induced by phenolic acids. Further screening of the transposon library failed to identify genes other than padR involved in the PASR. Modest inactivation of padR by phenolic acids was obtained in recombinant Escherichia coli expressing padC and padR, and this translates into induction of decarboxylase activity. Gel shift promoter binding assays performed with and without MgCl(2), and with and without phenolic acids, demonstrated that phenolic acids were able to abolish the binding of PadR to the yveFG-padC promoter in the absence of MgCl(2). Altogether, our results indicate that (i) PadR is inactivated directly by phenolic acids in vitro, (ii) inhibition of PadR in response to phenolic acids may occur without the need for a sensor-like effector in B. subtilis, and (iii) phenolic acids are able to modulate PadR activity in E. coli in the absence of any additional effector.

| S-EPMC2347383 | biostudies-literature

Gene cloning, transcriptional analysis, purification, and characterization of phenolic acid decarboxylase from Bacillus subtilis.

Project description:Bacillus subtilis displays a substrate-inducible decarboxylating activity with the following three phenolic acids: ferulic, p-coumaric, and caffeic acids. Based on DNA sequence homologies between the Bacillus pumilus ferulate decarboxylase gene (fdc) (A. Zago, G. Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484-4486, 1995) and the Lactobacillus plantarum p-coumarate decarboxylase gene (pdc) (J.-F. Cavin, L. Barthelmebs, and C. Diviès, Appl. Environ. Microbiol. 63:1939-1944, 1997), a DNA probe of about 300 nucleotides for the L. plantarum pdc gene was used to screen a B. subtilis genomic library in order to clone the corresponding gene in this bacterium. One clone was detected with this heterologous probe, and this clone exhibited phenolic acid decarboxylase (PAD) activity. The corresponding 5-kb insertion was partially sequenced and was found to contain a 528-bp open reading frame coding for a 161-amino-acid protein exhibiting 71 and 84% identity with the pdc- and fdc-encoded enzymes, respectively. The PAD gene (pad) is transcriptionally regulated by p-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. The pad gene was overexpressed constitutively in Escherichia coli, and the stable purified enzyme was characterized. The difference in substrate specificity between this PAD and other PADs seems to be related to a few differences in the amino acid sequence. Therefore, this novel enzyme should facilitate identification of regions involved in catalysis and substrate specificity.

| S-EPMC106171 | biostudies-literature

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