Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations.

Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s).

CcpA-mediated catabolite activation of the Bacillus subtilis ilv-leu operon and its negation by either CodY- or TnrA-mediated negative regulation.

The Bacillus subtilis ilv-leu operon functions in the biosynthesis of branched-chain amino acids. It undergoes catabolite activation involving a promoter-proximal cre which is mediated by the complex of CcpA and P-Ser-HPr. This activation of ilv-leu expression is negatively regulated through CodY binding to a high-affinity site in the promoter region under amino acid-rich growth conditions, and it is negatively regulated through TnrA binding to the TnrA box under nitrogen-limited growth conditions. The CcpA-mediated catabolite activation of ilv-leu required a helix face-dependent interaction of the complex of CcpA and P-Ser-HPr with RNA polymerase and needed a 19-nucleotide region upstream of cre for full activation. DNase I footprinting indicated that CodY binding to the high-affinity site competitively prevented the binding of the complex of CcpA and P-Ser-HPr to cre. This CodY binding not only negated catabolite activation but also likely inhibited transcription initiation from the ilv-leu promoter. The footprinting also indicated that TnrA and the complex of CcpA and P-Ser-HPr simultaneously bound to the TnrA box and the cre site, respectively, which are 112 nucleotides apart; TnrA binding to its box was likely to induce DNA bending. This implied that interaction of TnrA bound to its box with the complex of CcpA and P-Ser-HPr bound to cre might negate catabolite activation, but TnrA bound to its box did not inhibit transcription initiation from the ilv-leu promoter. Moreover, this negation of catabolite activation by TnrA required a 26-nucleotide region downstream of the TnrA box.

The mthA mutation conferring low-level resistance to streptomycin enhances antibiotic production in Bacillus subtilis by increasing the S-adenosylmethionine pool size.

Certain Str(r) mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel(+) but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 µg/ml, an antibiotic not produced (<1 µg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues.

Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

Expression of kinA and kinB of Bacillus subtilis, necessary for sporulation initiation, is under positive stringent transcription control.

Bacillus subtilis cells were exposed to decoyinine to trigger stringent transcription control through inhibition of GMP synthase; amino acid starvation results in the same control through inhibition of GMP kinase by 5'-diphosphate 3'-diphosphate guanosine. The positive and negative transcription control of the stringent genes involves adenine and guanine at the transcription initiation sites, whereby they sense an increase and a decrease in the in vivo ATP and GTP pools, respectively. Decoyinine also induces sporulation in minimum medium. DNA microarray analysis revealed that decoyinine induced two major sensor kinase genes, kinA and kinB, involved in the phosphorelay leading to spore formation. lacZ fusion experiments involving the core promoter regions of kinA and kinB, whose transcription initiation bases are adenines, indicated that decoyinine induced their expression. This induction was independent of CodY and AbrB. When the adenines were replaced with guanines or cytosines, the induction by decoyinine decreased. The in situ replacement of the adenines with guanines actually affected this decoyinine-induced sporulation as well as massive sporulation in nutrient medium. These results imply that operation of the positive stringent transcription control of kinA and kinB, which is mediated by an increase in the ATP pool, is likely a prerequisite for the phosphorelay to transfer the phosphoryl group to Spo0A to initiate sporulation.

Catabolite repression of the Bacillus subtilis FadR regulon, which is involved in fatty acid catabolism.

The Bacillus subtilis fadR regulon involved in fatty acid degradation comprises five operons, lcfA-fadR-fadB-etfB-etfA, lcfB, fadN-fadA-fadE, fadH-fadG, and fadF-acdA-rpoE. Since the lcfA-fadRB-etfBA, lcfB, and fadNAE operons, whose gene products directly participate in the ß-oxidation cycle, had been found to be probably catabolite repressed upon genome-wide transcript analysis, we performed Northern blotting, which indicated that they are clearly under CcpA-dependent catabolite repression. So, we searched for catabolite-responsive elements (cre's) to which the complex of CcpA and P-Ser-HPr binds to exert catabolite repression by means of a web-based cis-element search in the B. subtilis genome using known cre sequences, which revealed the respective candidate cre sequences in the lcfA, lcfB, and fadN genes. DNA footprinting indicated that the complex actually interacted with these cre's in vitro. Deletion analysis of each cre using the lacZ fusions with the respective promoter regions of the three operons with and without it, indicated that these cre's are involved in the CcpA-dependent catabolite repression of the operons in vivo.

Heavy involvement of stringent transcription control depending on the adenine or guanine species of the transcription initiation site in glucose and pyruvate metabolism in Bacillus subtilis.

In Bacillus subtilis cells, the GTP level decreases and the ATP level increases upon a stringent response. This reciprocal change in the concentrations of the substrates of RNA polymerase affects the rate of transcription initiation of certain stringent genes depending on the purine species at their transcription initiation sites. DNA microarray analysis suggested that not only the rrn and ilv-leu genes encoding rRNAs and the enzymes for synthesis of branched-chain amino acids, respectively, but also many genes, including genes involved in glucose and pyruvate metabolism, might be subject to this kind of stringent transcription control. Actually, the ptsGHI and pdhABCD operons encoding the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system and the pyruvate dehydrogenase complex were found to be negatively regulated, like rrn, whereas the pycA gene encoding pyruvate carboxylase and the alsSD operon for synthesis of acetoin from pyruvate were positively regulated, like ilv-leu. Replacement of the guanine at position 1 and/or position 2 of ptsGHI and at position 1 of pdhABCD (transcription initiation base at position 1) by adenine changed the negative stringent control of these operons in the positive direction. The initiation bases for transcription of pdhABCD and pycA were newly determined. Then the promoter sequences of these stringent operons were aligned, and the results suggested that the presence of a guanine(s) and the presence of an adenine(s) at position 1 and/or position 2 might be indispensable for negative and positive stringent control, respectively. Such stringent transcription control that affects the transcription initiation rate through reciprocal changes in the GTP and ATP levels likely occurs for numerous genes of B. subtilis.

Regulation of the Bacillus subtilis divergent yetL and yetM genes by a transcriptional repressor, YetL, in response to flavonoids.

DNA microarray analysis revealed that transcription of the Bacillus subtilis yetM gene encoding a putative flavin adenine dinucleotide-dependent monooxygenase was triggered by certain flavonoids during culture and was derepressed by disruption of the yetL gene in the opposite orientation situated immediately upstream of yetM, which encodes a putative MarR family transcriptional regulator. In vitro analyses, including DNase I footprinting and gel retardation analysis, indicated that YetL binds specifically to corresponding single sites in the divergent yetL and yetM promoter regions, with higher affinity to the yetM region; the former region overlaps the Shine-Dalgarno sequence of yetL, and the latter region contains a perfect 18-bp palindromic sequence (TAGTTAGGCGCCTAACTA). In vitro gel retardation and in vivo lacZ fusion analyses indicated that some flavonoids (kaempferol, apigenin, and luteolin) effectively inhibit YetL binding to the yetM cis sequence, but quercetin, galangin, and chrysin do not inhibit this binding, implying that the 4-hydroxyl group on the B-ring of the flavone structure is indispensable for this inhibition and that the coexistence of the 3-hydroxyl groups on the B- and C-rings does not allow antagonism of YetL.

Molecular mechanisms underlying the positive stringent response of the Bacillus subtilis ilv-leu operon, involved in the biosynthesis of branched-chain amino acids.

Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5' end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

Dual regulation of the Bacillus subtilis regulon comprising the lmrAB and yxaGH operons and yxaF gene by two transcriptional repressors, LmrA and YxaF, in response to flavonoids.

Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter beta-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.

Elaborate transcription regulation of the Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids through global regulators of CcpA, CodY and TnrA.

The Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis-elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases -96 to -82; +1 is the ilv-leu transcription initiation base) by means of DNase I-footprinting in vitro, and deletion and base-substitution analyses of cre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv-leu operon in overall metabolic regulation in this organism is discussed.

Negative transcriptional regulation of the ilv-leu operon for biosynthesis of branched-chain amino acids through the Bacillus subtilis global regulator TnrA.

The Bacillus subtilis ilv-leu operon is involved in the synthesis of branched-chain amino acids (valine, isoleucine, and leucine). The two- to threefold repression of expression of the ilv-leu operon during logarithmic-phase growth under nitrogen-limited conditions, which was originally detected by a DNA microarray analysis to compare the transcriptomes from the wild-type and tnrA mutant strains, was confirmed by lacZ fusion and Northern experiments. A genome-wide TnrA box search revealed a candidate box approximately 200 bp upstream of the transcription initiation base of the ilv-leu operon, the TnrA binding to which was verified by gel retardation and DNase I footprinting analyses. Deletion and base substitution of the TnrA box sequence affected the ilv-leu promoter activity in vivo, implying that TnrA bound to the box might be able to inhibit the promoter activity, possibly through DNA bending. The negative control of the expression of the ilv-leu operon by TnrA, which is considered to represent rather fine-tuning (two- to threefold), is a novel regulatory link between nitrogen and amino acid metabolism.

The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate.

A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km 102 and 10 s(-1) mM(-1), respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.

Mannitol-1-phosphate dehydrogenase (MtlD) is required for mannitol and glucitol assimilation in Bacillus subtilis: possible cooperation of mtl and gut operons.

We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons. The B. subtilis mtl operon consists of mtlA (encoding enzyme IICBA(mt1)) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome. The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-beta-D-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD. Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.

Organization and expression of the Bacillus subtilis sigY operon.

We investigated the organization and expression of the Bacillus subtilis sigY operon, the first gene of which codes for sigmaY, a member of the extracytoplasmic function (ECF) family of sigma factors. The sigY operon, comprising six genes (sigY, yxlC, D, E, F, and G), was induced upon nitrogen starvation; it was continuously transcribed from the 31st base upstream of sigY to a neighboring convergent gene, yxlH, resulting in a 4.2-kb mRNA. The expression of the sigY operon was also positively autoregulated through sigmaY, suggesting that its transcription is likely to be directed by sigmaY. Deletion analysis of the sigY promoter, which was localized by primer extension, revealed the promoter region of sigY with the "-10" and "-35" sequences of CGTC and TGAACG, respectively. The latter sequence was distinct from those recognized by sigmaW, sigmaX, and sigmaM. The sigmaY-directed transcription of sigY was under negative regulation involving YxlD. sigY disruption affected sporulation induced by nitrogen starvation, but sigY induction upon nitrogen starvation was not associated with the sporulation process. The organization and function of the sigY operon are significantly conserved in several microorganisms living in adverse living environments.