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.

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.

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.

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.

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.

Identification of a region critically involved in the interaction of phlorizin with the rabbit sodium-D-glucose cotransporter SGLT1.

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604-610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of alpha-methyl-D-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K(i) for phlorizin increased at least by a factor of 5 compared to the wild-type K(i) of 4.6 +/- 0.7 micromol/l; most striking changes were observed for Y604K (K(i) = 75.3 +/- 19.0 micromol/l) and C608K (K(i) = 83.6 +/- 13.9 micromol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (Km = 0.8 +/- 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside.

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.

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.

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.

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.

Identification of DNA-binding proteins involved in regulation of expression of the Streptomyces aureofaciens sigF gene, which encodes sporulation sigma factor sigma(F).

Expression of the sigF gene encoding a sporulation-specific sigma factor, sigma(F), in Streptomyces aureofaciens is restricted only to sporulation. Gel mobility-shift assays using protein fractions from different developmental stages of S. aureofaciens revealed two different putative proteins specifically bound to the sigF promoter region: a protein (designated RsfA) present in young substrate mycelium, and a protein (designated RsfB) present in the course of sporulation. Based on the characteristic profiles of their appearance during differentiation, RsfA might be a repressor and RsfB an activator of sigF expression. The location of a specific binding site of the repressor-like protein (RsfA) was determined by gel mobility-shift assays of promoter deletion fragments and by DNase I footprinting analysis. The binding site mapped from nucleotides -87 to -25 relative to the transcription start point of the sigF promoter, and overlapped the -35 promoter region. Given the dependence of sigF expression upon whiH, the putative sporulation transcription factor WhiH was overproduced in Escherichia coli and used in the mobility-shift assays with the sigF promoter. However, no specific binding was detected, indicating an indirect dependence of sigF upon whiH.

Cloning of a two-component regulatory system probably involved in the regulation of chitinase in Streptomyces coelicolor A3(2).

Using the method for the identification of promoters recognized by the sporulation specific sigma factor (sigma F), we identified a positive 950 bp Sau3AI DNA fragment in Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, PF35, in the E. coli two-plasmid system similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor (sigma B). However, the putative sigF-dependent promoter, PF35, was inactive in S. coelicolor in the course of differentiation, and it was located divergently in the promoter region directing expression of the chiC gene encoding chitinase. Sequence analysis of the region potentially governed by PF35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component system chiS, chiR, regulating chitinase activity in Streptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the PF35 promoter. Disruption of the S. coelicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, the chiR disruption did not affect the overall chitinase activity.

Probing transmembrane topology of the high-affinity Sodium/Glucose cotransporter (SGLT1) with histidine-tagged mutants.

To reexamine the existing predictions about the general membrane topology of the high-affinity Na+/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 x Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na+/glucose cotransport activity at 0.1 mM D-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541-aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition.

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2).

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.

The Streptomyces aureofaciens homologue of the sporulation gene whiH is dependent on rpoZ-encoded sigma factor.

Using the method for the identification of promoters recognized by a sporulation specific sigma factor RpoZ, we identified a promoter in Streptomyces aureofaciens, directing expression of a gene having high sequence similarity (83% amino acid identity) to sporulation transcription factor WhiH of Streptomyces coelicolor. High-resolution S1-nuclease mapping using RNA prepared from S. aureofaciens from various developmental stages showed high similarity of PwhiH promoter to the consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in S. aureofaciens strain with rpoZ-disrupted gene. The results suggest that the PwhiH promoter is recognized by sigma factor RpoZ in S. aureofaciens.

Cloning of the putative aldehyde dehydrogenase, aldA, gene from Streptomyces aureofaciens.

A Streptomyces aureofaciens gene, gap, encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was previously identified. Hybridization studies suggested the presence of a second gap gene in S. aureofaciens. To clone the gene, S. aureofaciens subgenomic library was screened with an oligonucleotide probe encoding a peptide motif conserved in all GAPDH. 3352 bp positive BamHI fragment was identified, the length of which correlated with the hybridization signal. The nucleotide sequence of the fragment was determined, and analysis of the sequence revealed the presence of three open reading frames (ORF). However, none of the genes coded for GAPDH. All three genes formed an operon, consisting of gene orf251, with a high homology to a conserved gene present only in archaeabacteria, and the aldA and adhA genes homologous to various eukaryotic and prokaryotic aldehyde- and alcohol-dehydrogenases, with maximum homology to the phenylacetaldehyde dehydrogenases and arylalcohol dehydrogenases, respectively.

The gene downstream of Streptomyces aureofaciens whiB encodes a large protein with proposed transmembrane localization, and is induced by glucose.

The sequence analysis of the region downstream of the Streptomyces aureofaciens whiB sporulation gene revealed a long open reading frame (1219 amino acids; Mr 128 209) encoding protein with potential transmembrane structure. By integrative transformation, via double cross-over, a stable null mutant of the gene, orf1219, was prepared. This mutation appeared to have no obvious effect on vegetative growth and differentiation. In vitro and in vivo transcriptional analysis of the downstream gene revealed a single apparent promoter induced by glucose.

The Streptomyces aureofaciens homologue of the whiB gene is essential for sporulation; its expression correlates with the developmental stage.

In previous experiments, a Streptomyces aureofaciens gene highly similar to the sporulation-specific whiB gene of Streptomyces coelicolor was identified. By integrative transformation, via double cross-over, a stable null mutant of the whiB-homologous gene of S. aureofaciens was obtained. The disruption blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores, producing white aerial hyphae without septation. Expression of the whiB gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared from S. aureofaciens in various developmental stages. Two putative promoters were identified upstream of the whiB coding region. The stronger promoter, whiB-P2, was induced at the beginning of aerial mycelium formation, and the weaker promoter, whiB-P1, was expressed fairly constantly during differentiation. No differences in the expression of the whiB promoters were detected in an rpoZ-disrupted S. aureofaciens strain. The promoter bearing DNA fragment was inserted into the promoter-probe vector pARC1 to produce an expression pattern consistent with the results of direct RNA analysis.