Bronislava Rezuchova

New members of the Escherichia coli σE regulon identified by a two-plasmid system

A previously established method, based on a two-plasmid system, was used to identify promoters recognized by RNA polymerase containing the extracytoplasmic stress response sigma factor sigmaE in Escherichia coli. In addition to previously identified rpoE-dependent promoters, 11 new promoters potentially directing the expression of 15 genes were identified that were active only after over-expression of rpoE. The promoters were confirmed and transcriptional start points of the promoters were determined by primer extension analysis and S1-nuclease mapping. All the promoters contained sequences similar to the consensus sequence of rpoE-dependent promoters. The new rpoE-dependent promoters governed expression of genes encoding proteins involved in primary metabolism (fusA, tufA, recR), phospholipid and lipopolysaccharide biosynthesis (psd, lpxP), signal transduction (sixA), proposed inner or outer membrane proteins (bacA, sbmA, smpA, yeaY), and proteins with unknown function (ybaB, yaiW, yiiS, yiiT, yfeY).

Transcriptional analysis of the rpoE gene encoding extracytoplasmic stress response sigma factor σE in Salmonella enterica serovar Typhimurium

The rpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium), which encodes the extracytoplasmic stress response sigma factor sigmaE, is critically important for the virulence of S. Typhimurium. We analysed expression of rpoE by wild-type and mutant bacteria grown in different conditions by S1-nuclease mapping using RNA, and using in vivo reporter gene fusions. Three promoters, rpoEp1, rpoEp2 and rpoEp3, were located upstream of the S. Typhimurium rpoE gene. The promoters were differentially expressed during growth and under several stress conditions including cold shock. Expression from the rpoEp3 promoter was absent in an S. Typhimurium rpoE mutant, demonstrating its dependence upon sigmaE. The level of mRNA corresponding to rpoEp3 was also higher in a cpxR mutant, indicating a negative regulation of the promoter by the Cpx system. Using this rpoE-dependent promoter, we optimised a two-plasmid system for identification of promoters recognised by S. Typhimurium sigmaE. The rpoEp3 promoter was active in the Escherichia coli two-plasmid system and has an identical transcription start point as in S. Typhimurium but only after induction of S. Typhimurium rpoE expression.

Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on sigma(E) (RpoE). S. Typhimurium htrA also possesses a sigma(E)-dependent promoter; however, we found that the absence of sigma(E) had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was sigma(E) dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor sigma(70). These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 degrees C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

Cloning and characterization of a polyketide synthase gene cluster involved in biosynthesis of a proposed angucycline-like polyketide auricin in Streptomyces aureofaciens CCM 3239.

A new polyketide gene cluster, aur1, was identified in Streptomyces aureofaciens CCM3239 by using genes for the spore-pigment polyketide synthase of the Streptomyces coelicolor whiE operon as a probe. Sequence analysis of three overlapping DNA fragments (encompassing 15,100 bp) revealed 15 open reading frames, the majority of which showed high similarity to the previously characterized type II polyketide synthase genes. The highest similarity was to three Streptomyces polyketide gene clusters involved in biosynthesis of angucycline antibiotics, jadomycin, urdamycin and landomycin. The proposed S. aureofaciens ketosynthase (Aur1D) was phylogenetically more related to all known ketosynthases for polyketide antibiotics in Streptomyces than to spore-pigment ketosynthases. Interestingly, the aur1 gene cluster contained a gene encoding a proposed malonyl-CoA:ACP transacylase that has not been identified in any of the previously characterized type II polyketide synthase cluster. Transcriptional analysis of aur1 revealed a single promoter upstream the first open reading frame (the aur1A gene) that was active in all stages of differentiation with increased activity at the time of aerial mycelium formation. The aur1 gene cluster was disrupted by a homologous recombination, replacing the three genes (aur1B,C,D) including ketosynthase, with antibiotic resistance marker gene in S. aureofaciens chromosome. Disruption did not affect growth and differentiation; disrupted strain produced spores with wild-type gray-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild-type and aur1-disrupted strains revealed an antibacterial compound that was missing in the mutant. The results indicated a role of the S. aureofaciens aur1 gene cluster in biosynthesis of a polyketide secondary metabolite (which we named auricin), and not in the spore pigment biosynthesis.

A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response σE 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.

Small outer-membrane lipoprotein, SmpA, is regulated by sigmaE and has a role in cell envelope integrity and virulence of Salmonella enterica serovar Typhimurium.

SmpA is a small outer-membrane lipoprotein that is a component of the essential YaeT outer-membrane protein assembly complex. In Salmonella enterica serovar Typhimurium (S. Typhimurium), expression of the smpA gene was shown to be directed by two promoters, smpAp1 and smpAp2. The more distal promoter, smpAp1, is dependent upon the extracytoplasmic stress response sigma factor sigma(E). An smpA null mutant was constructed in S. Typhimurium SL1344 and was shown to be more sensitive than its wild-type parent to growth at high temperature and in the presence of sodium cholate, SDS plus EDTA, and the hydrophobic antibiotic rifampicin. The lack of SmpA in S. Typhimurium elicits a sigma(E)-dependent stress response. These findings are indicative of altered outer-membrane integrity in the smpA mutant, probably due to a defect in outer-membrane protein biogenesis. SmpA was not important for entry or survival within murine macrophages; however, the S. Typhimurium smpA mutant was attenuated in mice by both the oral and parenteral routes of infection, and SmpA appeared to be most important for the growth of S. Typhimurium at systemic sites.

Differential expression of two sporulation specific σ factors of Streptomyces aureofaciens correlates with the developmental stage

In previous experiments, the Streptomyces aureofaciens (Sa) rpoZ, and sigF genes have been shown to encode putative sigma factors essential in differentiation. In an attempt to investigate expression of these genes during differentiation, we have performed S1 nuclease mapping using RNA prepared from Sa in various developmental stages. Two putative promoters were identified upstream of the rpoZ coding region. The promoters significantly differed in their strength, and were active in distinct developmental stages; the weaker, rpoZ-P1, was active only in substrate mycelium, and the stronger, rpoZ-P2, was induced at the beginning of aerial mycelium formation. Transcriptional analysis of sigF revealed two apparent transcription start points, both being detectable only in the late phase of aerial mycelium formation. No sigF transcription was detected in an rpoZ-disrupted Sa strain. Promoter-bearing DNA fragments from rpoZ and sigF were inserted into several promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis. The results implicate temporally different expression of rpoZ and sigF during the differentiation of Sa, and direct or indirect dependence of sigF expression on the rpoZ-encoded putative sigma factor, thus indicating a cascade of sigma factors in Streptomyces development.

Disruption of a glycogen-branching enzyme gene, glgB, specifically affects the sporulation-associated phase of glycogen accumulation in Streptomyces aureofaciens

In the course of Streptomyces differentiation, glycogen is accumulated in two discrete phases: in substrate hyphae that undergo aerial mycelium formation (phase I), and during septation of aerial hyphae (phase II). We have disrupted a previously identified gene, glgB, encoding a putative glycogen-branching enzyme in Streptomyces aureofaciens. Disruption of the gene had no profound effect on sporulation. However, the amount of glycogen-like polysaccharides, compared to wild-type (WT) S. aureofaciens, decreased in the late stage of differentiation of the glgB-disrupted strain. Absorption spectra of polysaccharides extracted from the WT and glgB-disrupted strains have shown the presence of glycogen in both strains in the first stage of differentiation (aerial mycelium formation), and unbranched glucan was detected in the glgB-disrupted strain in the late stage of differentiation. The results were confirmed by electron microscopy after silver proteinate staining of glycogen granules. Two distinct glycogen-branching enzymes, which had temporally different expression during differentiation, were detected in WT S. aureofaciens. The absence of this enzyme activity in the late stage of differentiation in the glgB mutant suggests that the product of the glgB gene is responsible for phase II glycogen accumulation.

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.

Strict control of auricin production in Streptomyces aureofaciens CCM 3239 involves a feedback mechanism

The polyketide gene cluster aur1 is responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is regulated in a complex manner involving several regulators, including a key pathway-specific positive regulator Aur1P that belongs to the family of ‘atypical’ response regulators. Production of auricin is induced after entry into stationary phase. However, auricin was produced in only a short time interval of several hours. We found that the decrease of auricin production was due to a strict regulation of auricin biosynthetic genes at the transcriptional level by a feedback mechanism; auricin and/or its intermediate(s) inhibited binding of Aur1P to its cognate biosynthetic promoter aur1Ap and consequently stopped its activation. In addition, we also determined that synthesised auricin is unstable during growth of S. aureofaciens CCM3239 in the production medium even though purified auricin is stable for days in various organic solvents. The critical parameter affecting its stability was pH. Auricin is stable at acid pH and unstable at neutral and alkaline pH. The drop in auricin concentration was due to an increase of pH shortly after induction of auricin production during cultivation of S. aureofaciens CCM3239.