Characterization of the micA gene encoding a small regulatory σE-dependent RNA in Salmonella enterica serovar Typhimurium.

The role of MicA (repressing small regulatory non-coding RNAs of two Salmonella porins) was determined in virulence of Salmonella enterica serovar Typhimurium. Transcriptional analysis revealed that the expression of the micA gene is driven by a single σ(E)-dependent promoter, micAp. Its activity increased towards stationary phase; in exponential phase, the activity was induced by several stresses by a DegS-dependent mechanism. Although phenotypic analysis revealed no significant differences between wild-type and the micA mutant strains, in vivo studies showed that this mutant is more virulent in the mouse model.

Identification and characterization of an indigoidine-like gene for a blue pigment biosynthesis in Streptomyces aureofaciens CCM 3239.

An incomplete oligoketide (PK; 'polyketide') gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.

Core promoters of the penicillin biosynthesis genes and quantitative RT-PCR analysis of these genes in high and low production strain of Penicillium chrysogenum.

The transcription start points of the penicillin biosynthesis genes from Penicillium chrysogenum were mapped using the primer extension method. For each of the three genes consensus sequences of the core promoter elements were identified, supporting the notion that the basal transcription of these genes is mediated separately. Interestingly, transcription start of the pcbC gene is located within the potential Inr element with no TATA box-like sequence being found at expected position. This is in contrast to pcbAB and penDE genes with proposed TATA boxes or even to Aspergillus nidulans ipnA (pcbC) gene indicating possible differences in basal transcription regulation. Using the quantitative RT-PCR analysis the expression of all three biosynthesis genes was monitored in both the high and low production strain of P. chrysogenum during a 3-d cultivation under production conditions. The differences were found between the strains in time regulation and transcript levels of the biosynthesis genes. Furthermore, we showed that the effect of higher gene dosage on productivity in the production strain is amplified by more efficient transcription of the biosynthesis genes with the RNA levels approximately 37- and 12-times higher, respectively, than in a low production strain.

Multiple regulatory genes in the salinomycin biosynthetic gene cluster of Streptomyces albus CCM 4719.

A DNA fragment containing part of the salinomycin biosynthetic gene cluster from industrial strain Streptomyces albus CCM 4719 was cloned. Sequence analysis of the 25.809-kbp fragment revealed the presence of 8 open reading frames (ORFs), including two large ORFs encoding three modular sets of oligoketide synthase, followed by three genes (salRI, salRII, salRIII) encoding transcriptional regulators. The first two regulators, SalRI and SalRII, belonged to the novel LAL family of large transcriptional regulators. SalRIII was highly similar to the NysRIV, AmphRIV, and FscRI transcriptional regulators from the oligoene macrolides nystatin, amphotericin, and R008/candicidin clusters, respectively.

Characterization of the alternative sigma factor sigmaG in Streptomyces coelicolor A3(2).

Using the previously established two-plasmid system for the identification of promoters recognized by a particular sigma factor, we identified two positive DNA fragments that were active only after induced sigG, encoding sigma factor sigmaG of Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping in the Escherichia coli two-plasmid system identified potential promoters, PG45 and PG54, whose sequences were similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. However, both putative sigmaG-dependent promoters were not active in S. coelicolor. Sequence analysis of the regions potentially governed by the promoters revealed a gene encoding a hypothetical protein SCO5555 and the rrnE gene encoding rRNA operon. To confirm that sigG encodes sigma factor, the sigmaG protein was overproduced in E. coli and purified. In an in vitro transcription assay, sigmaG, after complementation with S. coelicolor core RNA polymerase, was able to recognize both sigmaG-dependent promoters and initiate transcription.

Post-translational modification(s) and cell distribution of Streptomyces aureofaciens translation elongation factor Tu overproduced in Escherichia coli.

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.

Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress response sigma factor, sigmaF.

The previously established two-plasmid system in Escherichia coli for the identification of Mycobacterium tuberculosis promoters that are recognized by RNA polymerase containing the stress response sigma factor sigmaF was optimized. Expression of the M. tuberculosis sigmaF encoded by sigF gene was under the control of the isopropyl beta-D-thiogalactopyranoside (IPTG)-dependent Ptrc promoter. A low level of IPTG induced a nontoxic but sufficient level of sigmaF to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized the known sigmaF-dependent promoter, usfXp1, which was cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZalpha reporter gene. Primer extension analysis of the usfXp1 promoter in the E. coli two-plasmid system after IPTG-induced expression of M. tuberculosis sigF revealed a transcription start point that was identical as in M. tuberculosis. This new system has been shown to be useful for identification of M. tuberculosis sigmaF-dependent promoters.

Characterization of the Streptomyces coelicolor A3(2) wblE gene, encoding a homologue of the sporulation transcription factor.

The gene corresponding to the recently identified whiB-paralogous gene wblE in S. coelicolor was found after sequencing the downstream region of the stress-response sporulation-specific sigma-factor gene, sigH, in S. coelicolor A3(2). Sequence analysis has revealed an ORF exhibiting high similarity to sporulation transcription factors WhiB and WhiD. A stable null mutant of the wblE gene was obtained by integrative transformation, via double cross-over. Disruption of the S. coelicolor wblE gene appeared to have no obvious effect on growth, morphology, differentiation, and production of the pigmented antibiotics actinorhodin and undecylprodigiosin. Expression of the wblE gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared from S. coelicolor A3(2) and its isogenic sigF and sigH mutants grown to various developmental stages. A single promoter was identified upstream of the wblE coding region. The wblEp promoter was induced at the beginning of aerial mycelium formation and its activity decreased later in differentiation. No differences in expression of the wblEp promoter were detected in S. coelicolor A3(2) mutants in sigF and sigH genes for sporulation-specific sigma factors. Sequence of the wblEp promoter showed partial similarity to the consensus sequence of the extracytoplasmic sigma factors.

Response regulator ChiR regulates expression of chitinase gene, chiC, in Streptomyces coelicolor.

Transcription from the chiC promoter, directing expression of the chitinase gene, chiC, in Streptomyces coelicolor, was analyzed using xylE reporter gene and high-resolution S1-nuclease mapping. The transcription from the chiC promoter was induced by chitin, and this induction was dramatically reduced in the S. coelicolor chiR-disrupted strain. This indicated a dependence of chiC expression upon the chiR gene encoding a response regulator protein. To investigate this relationship, the S. coelicolor ChiR was overproduced using Escherichia coli T7 RNA polymerase expression system. However, gel mobility shift-assay with such a purified ChiR showed no binding in the chiC promoter region, which indicates a lack of specific phosphorylation of E. coli overproduced ChiR that is necessary for DNA-binding activity of response regulators.

Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli.

Bacterial accommodation to moderate concentrations of cadmium is accompanied by transient activation of general stress proteins as well as a sustained induction of other proteins of hitherto unknown functions. One of the latter proteins was previously identified as the product of the Escherichia coli yodA ORF. The yodA ORF encodes 216 aa residues (the YodA protein) and the increased synthesis of YodA during cadmium stress was found probably to be a result of transcriptional activation from one single promoter upstream of the structural yodA gene. Analysis of a transcriptional gene fusion, P(yodA)-lacZ, demonstrated that basal expression of yodA is low during exponential growth and expression is increased greater than 50-fold by addition of cadmium to growing cells. However, challenging cells with additional metals such as zinc, copper, cobalt and nickel did not increase the level of yodA expression. In addition, hydrogen peroxide also increased yodA expression whereas the superoxide-generating agent paraquat failed to do so. Surprisingly, cadmium-induced transcription of yodA is dependent on soxS and fur, but independent of oxyR. Moreover, a double relA spoT mutation abolished induction of yodA during cadmium exposure but ppGpp is not sufficient to induce yodA since expression of the gene is not elevated during stationary phase. After 45 min of cadmium exposure the YodA protein was primarily detected in the cytoplasmic fraction but was later (150 min) found in both the cytoplasmic and periplasmic compartments.

Some features of DNA-binding proteins involved in the regulation of the Streptomyces aureofaciens gap gene, encoding glyceraldehyde-3-phosphate dehydrogenase.

A gapR gene, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators, was previously identified upstream of the gap gene, coding for glyceraldehyde-3-phosphate dehydrogenase in Streptomyces aureofaciens. The GapR protein overproduced in Escherichia coli was shown to bind to the gap-P promoter region. Using the gel mobility shift assay with cell-free protein extracts from different developmental stages of S. aureofaciens, we identified several other proteins, in addition to GapR, that specifically bound to the S. aureofaciens gap-P promoter region. When cell-free extracts from S. aureofaciens cultivated in liquid medium with glucose were analyzed, only one complex corresponding to GapR was detected. A new protein interacting with the gap-P promoter was detected in stationary culture of S. aureofaciens grown in the presence of mannitol as carbon sources. The GapR protein was partially purified from S. aureofaciens cultivated in liquid medium containing glucose and used for binding studies. DNA footprinting analysis revealed an identical protected region as previously identified for the GapR protein overproduced from Escherichia coli. The direct role of the GapR protein in the regulation of gap expression in S. aureofaciens in vivo was confirmed but regulation of gap expression seems to be more complex, possibly involving other regulatory protein(s), depending on the developmental stage of S. aureofaciens.

The stress-response sigma factor sigma(H) controls the expression of ssgB, a homologue of the sporulation-specific cell division gene ssgA, in Streptomyces coelicolor A3(2).

By using a previously established method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, we identified a promoter in Streptomyces coelicolor A3(2) that is recognized by a heterologous RNA polymerase containing the late sporulation-specific sigma factor sigma(F). The promoter directed the expression of a gene named ssgB, which is related to the sporulation-specific cell division gene ssgA. These genes, together with three others, constitute a new family of paralogous genes specific for Streptomyces. S1-nuclease mapping using RNA prepared from an Escherichia coli strain that expresses ssgB under the control of sigma(F), and from S. coelicolor A3(2) at various developmental stages, identified identical transcription start points in both strains, corresponding to the promoter ssgBp. The promoter is developmentally regulated in S. coelicolor: it is induced at the time of aerial mycelium formation and is most active during sporulation. However, the level of the ssgB transcript was unaffected in a sigF mutant of S. coelicolor A3(2). Interestingly, the level of the transcript was substantially reduced in an S. coelicolor strain that was mutant for the sigH gene, which encodes a stress-response sigma factor (sigma(H)) that is essential for sporulation in S. coelicolor A3(2). This dependence of ssgB upon sigH was confirmed by an in vitro transcription assay, in which sigma(H), in the presence of the S. coelicolor core RNA polymerase, was able to recognize ssgBp. These results suggest that the S. coelicolor ssgB gene is under the control of the stress-response sigma(H). Transcription of ssgB was investigated in S. coelicolor A3(2) mutants with lesions in each of six known early whi genes required for sporulation septation. Expression of ssgB was deregulated in three of the mutants ( whiA, whiI, and whiJ). Based on these data, it is proposed that the ssgB gene product plays a role in the developmental process in S. coelicolor A3(2).

Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB, from Brevibacterium flavum.

Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomyces aureofaciens, we identified two sigma70-like genes in a library of Brevibacterium flavum. Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum. We designated them similarly sigA and sigB. Transcription of B. flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions. Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively. Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock. Expression of both genes is significantly reduced after heat shock. Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.

Phylogenetic analysis of the rplA genes encoding ribosomal protein L1.

Previously we have identified the rplA gene encoding ribosomal protein L1 in Streptomyces aureofaciens. Sequence comparison of ribosomal protein L1 among several bacterial genera revealed a high level of conservation. Based on this conservation, these proteins were used as a phylogenetic tool to compare evolutionary relationships among eubacteria and archaebacteria. This phylogenetic analysis of L1 ribosomal proteins including the S. aureofaciens rplA gene product revealed, except similar bacterial groupings, some new evolutionary relationships.

A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response sigma(E) in Escherichia coli.

We have previously established a two-plasmid system in Escherichia coli for identification of promoters recognized by RNA polymerase containing a heterologous sigma factor. Attempts to optimize this system for identification of promoters recognized by RNA polymerase containing E. coli extracytoplasmic stress response sigma(E) failed owing to high toxicity of the expressed rpoE. A new system for identification of sigma(E)-cognate promoters was established, and verified using the two known sigma(E)-dependent promoters, rpoEp2 and degPp. Expression of the sigma(E)-encoding rpoE gene was under the control of the AraC-dependent P(BAD) promoter. A low level of arabinose induced a non-toxic, however, sufficient level of sigma(E) to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized both known sigma(E)-dependent promoters, rpoEp2 and degPp, which were cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZ alpha reporter gene. This new system has proved to be useful for identification of E. coli sigma(E)-cognate promoters. Moreover, the system could be used for identification of ECF sigma-cognate promoters from other bacteria.

Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase of Streptomyces aureofaciens requires GapR, a member of the AraC/XylS family of transcriptional activators.

Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is developmentally regulated, and induced by glucose in Streptomyces aureofaciens. A gene, gapR, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators was identified upstream of gap. The gapR gene was constitutively expressed from a single promoter during the course of differentiation. By integrative transformation, via double crossover, a stable null mutant of the gapR gene was obtained. The mutation only slightly affected growth, and had no effect on differentiation of S. aureofaciens. However, transcription of the GAPDH-encoding gap gene was substantially reduced in the S. aureofaciens DeltagapR null mutant, irrespective of carbon source used. Though GAPDH activity was about 1.5-fold lower in the mutant, the substantial enzyme activity remained, suggesting the presence of a second GAPDH which is sufficient to ensure growth. The GapR protein, overproduced in Escherichia coli, was shown to bind upstream of the gap-P promoter region. The results indicate a direct role of GapR in regulation of gap expression in S. aureofaciens.

Identification of DNA-binding proteins involved in regulation of expression of the Streptomyces aureofaciens whiH gene encoding a sporulation transcription factor.

Using the gel mobility-shift assay with protein fractions from different developmental stages of solid-grown Streptomyces aureofaciens, we identified two different proteins specifically bound to the whiH promoter region. Only one protein (RwhA) was detected in young substrate mycelium cultivated in liquid medium. On comparing the mobility of the resulting complexes, one of the bound proteins present in substrate mycelium and in early stages of aerial mycelium seemed to be identical with the RwhA. The other detected protein with a higher mobility (RwhB) was present in all developmental stages except for mature spores. DNA footprinting analysis localized the binding site of RwhB to nucleotides -23 to +40 relative to the transcription start point of the PwhiH promoter. RwhA from young substrate mycelium protected the DNA fragment from -106 to -77 in coding strand and -126 to -82 in noncoding strand. WhiH has homology to a large family of metabolism-related repressors and seems to regulate negatively its own expression. These observations (and the results of transcription analysis of the whiH gene obtained earlier) suggest that two different proteins influence the expression of whiH gene in S. aureofaciens. The putative repressor-like RwhA protein protects expression of whiH in substrate mycelium either in liquid medium or during differentiation. The other detected protein, RwhB, which binds to the whiH promoter region during differentiation, may represent two forms of WhiH, one with a repressor role at the beginning of differentiation and second with the role of activator at the time of sporulation.

Streptomyces aureofaciens sporulation-specific sigma factor sigma(rpoZ) directs expression of a gene encoding protein similar to hydrolases involved in degradation of the lignin-related biphenyl compounds.

A previously established method for the identification of promoters recognized by a heterologous RNA polymerase holoenzyme containing a particular sigma factor was used to identify promoters dependent upon a sporulation specific sigma factor, sigma(RpoZ), of Streptomyces aureofaciens. Three new positive DNA fragments were identified, and these putative rpoZ-dependent promoters, P(ren24), P(ren57), and P(ren71), contained sequences similar to the consensus sequence of flagellar and chemotaxis promoters. However, only P(ren71) was active in S. aureofaciens. The promoter was induced at the time of aerial mycelium formation, and was inactive in an S. aureofaciens strain with an rpoZ-disrupted gene. The results suggest that the P(ren71) promoter is recognized by an RNA polymerase holoenzyme containing sigma(RpoZ) in S. aureofaciens. Sequence analysis of the region directed by P(ren71) revealed a gene, ren71, encoding a protein of 358 amino acids with an Mr 37,770. The deduced protein product showed end-to-end sequence similarity to the meta-cleavage compound hydrolase of Sphingomonas paucimobilis.