Biomedical subjects

Jan Kormanec

Publications and source records attributed to Jan Kormanec.

27 records · Page 2Linked to original sources

PubMed2004

Role of the two-component regulator CpxAR in the virulence of Salmonella enterica serotype Typhimurium.

The CpxAR (Cpx) two-component regulator controls the expression of genes in response to a variety of environmental cues. The Cpx regulator has been implicated in the virulence of several gram-negative pathogens, although a role for Cpx in vivo has not been demonstrated directly. Here we investigate whether positive or negative control of gene expression by Cpx is important for the pathogenesis of Salmonella enterica serotype Typhimurium. The Cpx signal pathway in serotype Typhimurium was disrupted by insertional inactivation of the cpxA and cpxR genes. We also constitutively activated the Cpx pathway by making an internal in-frame deletion in cpxA (a cpxA* mutation). Activation of the Cpx pathway inhibited induction of the envelope stress response pathway controlled by the alternative sigma factor sigma(E) (encoded by rpoE). Conversely, the Cpx pathway was highly up-regulated (>40-fold) in a serotype Typhimurium rpoE mutant. The cpxA* mutation, but not the cpxA or the cpxR mutation, significantly reduced the capacity of serotype Typhimurium to adhere to and invade eucaryotic cells, although intracellular replication was not affected. The cpxA and cpxA* mutations significantly impaired the ability of serotype Typhimurium to grow in vivo in mice. To our knowledge, this is the first demonstration that the Cpx system is important for a bacterial pathogen in vivo.

Animals↗

PubMed2004

Microarray-based analysis of the Staphylococcus aureus sigmaB regulon.

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by sigmaB activity. While sigmaB was found to positively control 198 genes by a factor of > or =2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of sigmaB. Gene products that were found to be influenced by sigmaB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by sigmaB were preceded by a nucleotide sequence that resembled the sigmaB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by sigmaB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the sigmaB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

Bacterial Proteins↗

PubMed2003

Transcriptional analysis of the rpoE gene encoding extracytoplasmic stress response sigma factor sigmaE 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.

Artificial Gene Fusion↗

PubMed2003

The ssgB gene, encoding a member of the regulon of stress-response sigma factor sigmaH, is essential for aerial mycelium septation in Streptomyces coelicolor A3(2).

The Streptomyces coelicolor A3(2) gene ssgB belongs to the regulon of stress-response sigma factor sigmaH. By integrative transformation via double cross-over, a stable null mutant of ssgB was obtained. This mutation had no obvious effect on vegetative growth, but critically affected aerial mycelium septation. The S. coelicolor ssgB mutant produced aerial hyphae without any signs of septation into spore compartments. The mutation was complemented in trans by wild-type ssgB including the sigmaH-dependent ssgBp promoter. The results proved that ssgB belongs a developmental branch of the sigmaH regulon.

Bacterial Proteins↗

PubMed2003

New members of the Escherichia coli sigmaE 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).

Base Sequence↗

PubMed2002

The Brevibacterium flavum sigma factor SigB has a role in the environmental stress response.

We have previously cloned a gene encoding a SigB, a principal-like sigma factor in Brevibacterium flavum, which was induced by several stress conditions. To clarify the in vivo function of this sigma factor, the sigB gene was disrupted by a homologous recombination, replacing the internal essential coding region in B. flavum chromosome by a kanamycin resistance marker gene. This mutation dramatically decreased vegetative growth rates of B. flavum. Studies of the effect of the sigB mutation on growth and viability of the cells under conditions of stress showed that the sigB mutant had increased susceptibility to acid, salt, alcohol, heat and cold stress. The plasmid-born wild-type sigB gene complemented the mutation. Based on the results, we propose that SigB has a role in vegetative growth and in response to various environmental stresses.

Bacterial Proteins↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

Stress-response sigma factor sigma(H) directs expression of the gltB gene encoding glutamate synthase in Streptomyces coelicolor A3(2).

Using the previously established Escherichia coli two-plasmid system, we identified a promoter recognized by the Streptomyces coelicolor stress-response sigma factor sigma(H). The promoter directed expression of the gltB gene, encoding a protein with considerable homology with large subunit of glutamate synthases. S1-nuclease mapping using RNA prepared from S. coelicolor identified an identical transcription start point corresponding to the promoter. The level of the transcript from this promoter was substantially reduced in a S. coelicolor sigH mutant. In addition to this sigH-dependent gltBp2 promoter, expression of the S. coelicolor gltB gene was directed by two other promoters, gltBp1 and gltBp3, independent upon sigH. S. coelicolor core RNA polymerase, after complementation with sigma(H), was able to recognize the gltBp2 promoter in vitro. These results suggested that the S. coelicolor gltB gene is under the control of stress-response sigma(H).

Bacterial Proteins↗

PubMed2002

Activity of the Streptomyces coelicolor stress-response sigma factor sigmaH is regulated by an anti-sigma factor.

The alternative sigma factor sigmaH has been shown to play an important role in stress response and morphological differentiation in Streptomyces coelicolor. Its gene, sigH, is located in an operon with the gene encoding proposed anti-sigma factor UshX, and one of the promoters directing expression of the operon is dependent upon sigH. To clarify the function of S. coelicolor UshX, both the sigmaH and UshX proteins were overproduced in Escherichia coli and purified. In an in vitro transcription assay, sigmaH, after complementation with S. coelicolor core RNA polymerase, was able to recognize the sigH-dependent promoter, sigH-P2. This transcription was inhibited by UshX, if it was incubated with sigmaH prior to the addition of the core RNA polymerase. When sigmaH and UshX were incubated and electrophoresed through non-denaturing polyacrylamide gels, they formed a specific complex. These results showed that UshX is a specific anti-sigma factor for sigmaH, and the S. coelicolor sigH operon is directly autoregulated.

Bacterial Proteins↗