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Results for “Gene Expression Regulation, Bacterial”

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At least 19 recordsLinked to original sources
PubMed2002

Cloning, purification, and crystallization of a bacterial gene expression regulator--Hfq protein from Escherichia coli.

Thermostable RNA-binding protein Hfq (also denoted HF1) is a multifunctional expression regulator of many bacterial genes. The regulation takes place both at a translation level (directly) and transcription level (indirectly through the stimulation of bacterial RNA polymerase sigmaS-subunit translation). We have cloned and overexpressed the hfq gene from E. coli and developed a purification procedure for the protein. Using gel filtration and ultracentrifugation techniques it was shown that the obtained Hfq protein is highly homogeneous and well dissolved. It has been crystallized and can be used for structural investigations.

Amino Acid Sequence↗
PubMed2002

Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression.

Although proteins fulfil most of the requirements that biology has for structural and functional components such as enzymes and receptors, RNA can also serve in these capacities. For example, RNA has sufficient structural plasticity to form ribozyme and receptor elements that exhibit considerable enzymatic power and binding specificity. Moreover, these activities can be combined to create allosteric ribozymes that are modulated by effector molecules. It has also been proposed that certain messenger RNAs might use allosteric mechanisms to mediate regulatory responses depending on specific metabolites. We report here that mRNAs encoding enzymes involved in thiamine (vitamin B(1)) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the need for protein cofactors. The mRNA-effector complex adopts a distinct structure that sequesters the ribosome-binding site and leads to a reduction in gene expression. This metabolite-sensing regulatory system provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.

Allosteric Regulation↗
PubMed2002

Macrophage-induced genes of Legionella pneumophila: protection from reactive intermediates and solute imbalance during intracellular growth.

A promoter-probe strategy was devised to identify genes specifically expressed by Legionella pneumophila during growth within the macrophage. Random fragments from the L. pneumophila chromosome were inserted upstream of a promoterless phage T4 td gene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes the L. pneumophila ortholog of ahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis of ahpC gene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate of L. pneumophila in cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.

Base Sequence↗
PubMed1998

amlC, another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24.

The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.

Amino Acid Sequence↗
PubMed2001

Identification and analysis of Staphylococcus aureus components expressed by a model system of growth in serum.

A model system mimicking Staphylococcus aureus bacteremia was developed by growth in serum under microaerobic conditions. Eight genes induced by growth in serum were identified, including an antimicrobial peptide biosynthesis locus, amino acid biosynthetic loci, and genes encoding putative surface proteins. Nine independent insertions were found in the major lysine biosynthesis operon, which encodes eight genes, is repressed by lysine in vitro, and is expressed in vivo.

Aerobiosis↗
PubMed1998

Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in corynebacterium glutamicum.

The metA gene encoding homoserine acetyltransferase, the first enzyme of the methionine biosynthetic pathway, was isolated from a pMT1-based corynebacterium glutamicum gene library via complementation of an Escherichia coli metA mutant. A DNA-sequence analysis of the cloned DNA is identified an open-reading frame of 1,137 bp which encodes a protein with the molecular weight of 41,380 comprising 379 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. The internal fragment of the cloned DNA was successfully used to disrupt chromosomal metA, demonstrating the identity of the cloned gene. The C. glutamicum metA mutant lost the ability to grow on glucose minimal medium supplemented with homoserine. However, the mutant could grow on a minimal medium supplemented with cystathionine, demonstrating that C. glutamicum uses the cystathionine route to synthesize methionine. Introduction of a plasmid carrying cloned metA into C. glutamicum resulted in a 10-fold increase in enzyme activities and expression of a protein product of M(r) 41,000, which agrees with the sequence data and is similar in size to those of other homoserine acetyltransferases. Unlike E. coli whose metA product uses succinyl coenzyme A as a substrate, the cloned metA gene produced homoserine acetyltransferase which uses only acetyl coenzyme A as the acyl donor.

Acetyltransferases↗
PubMed1993

The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes.

Regulation of gene expression in bacteria, as in eukaryotic cells, is often achieved by variation of mRNA levels. Since the steady state levels of mRNA depend on both the rate of synthesis and the rate of decay, both mechanisms are important for gene regulation. After considerable effort undertaken over many years to understand the regulation of transcription, mRNA degradation has recently gained increasing attention as an important step in the regulation of some bacterial genes, and many investigations have addressed the mechanisms involved in mRNA decay. The puf mRNA of Rhodobacter capsulatus encoding pigment binding proteins has become a model system to study decay of a polycistronic mRNA and the role of mRNA degradation in gene expression. Individual segments of the polycistronic puf mRNA display extremely different half-lives. These differences in stability of mRNA segments are involved in the differential expression of puf encoded genes and consequently contribute to the stoichiometry of light-harvesting I and reaction centre complexes that results in optimal growth. In addition, control of mRNA stability is involved in the oxygen-dependent regulation of photosynthesis genes. High oxygen tension results in decreased stability of the reaction-centre specific puf mRNA segment, most likely by affecting the rate of endonucleolytic cleavage within the reaction centre coding region. The results obtained from studying puf mRNA degradation in R. capsulatus and Escherichia coli suggest that a specific distribution of decay promoting and decay impeding mRNA elements along the polycistronic mRNA is responsible for the different half-lives of individual puf segments.

Bacterial Proteins↗
PubMed2005

Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941.

The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.

Bacillus megaterium↗
PubMed2000

Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion.

Lipases from Pseudomonas bacteria are widely used for a variety of biotechnological applications. Overexpression in heterologous hosts like Escherichia coli failed to produce enzymatically active lipase prompting to study the molecular mechanisms underlying the regulation of lipase gene expression and secretion. The prototype lipase from P. aeruginosa is encoded in a bicistronic operon which is transcribed from two different promotors, one of which depends on the alternative sigma factor RpoN (sigma(54)). Recently, a two-component regulatory system was identified as an element controlling transcription of the lipase operon. P. aeruginosa lipase is secreted via a type II pathway. The cytoplasmic prelipase contains a 26 amino acid N-terminal signal sequence mediating secretion across the inner membrane via the Sec-machinery. In the periplasm, lipase folds into an enzymatically active conformation assisted by its specific intermolecular chaperone Lif and by unspecific accessory folding catalysts including Dsb-proteins which catalyze the formation of a disulfide bond. Enzymatically active and secretion-competent lipase is finally transported through a complex secretion machinery consisting of 12 different Xcp-proteins of which XcpQ forms a pore-like structure in the outer membrane allowing the release of lipase into the extracellular medium. Biotechnologically important lipases from Burkholderia glumae and P. alcaligenes also use such a type II secretion pathway whereas lipases from P. fluorescens and Serratia marcescens, which lack a typical signal sequence are secreted via a type I pathway. Future challenges to produce Pseudomonas lipases may include artificial up-regulation of lipase gene transcription and construction of more efficient expression strains in which both folding and secretion of lipase are optimized.

Gene Expression Regulation, Bacterial↗
PubMed2003

Identification of histidine kinases that act as sensors in the perception of salt stress in Synechocystis sp. PCC 6803.

In plants and microorganisms, salt stress regulates the expression of large numbers of genes. However, the machinery that senses salt stress remains to be characterized. In this study we identified sensory histidine kinases that are involved in the perception of salt stress in the cyanobacterium Synechocystis sp. strain PCC 6803. A library of strains with mutations in all 43 histidine kinases was screened by DNA microarray analysis of genomewide gene expression under salt stress. The results suggested that four histidine kinases, namely, Hik16, Hik33, Hik34, and Hik41, perceived and transduced salt signals. However, Hik33, Hik34, and Hik16 acting with Hik41 regulated the expression of different sets of genes. These histidine kinases regulated the expression of approximately 20% of the salt-inducible genes, whereas the induction of the remaining salt-inducible genes was unaffected by mutations in any of the histidine kinases, suggesting that additional sensory mechanisms might operate in the perception of salt stress. We also used DNA microarrays to investigate the effect of various salts on gene expression. Our results indicate that Hik33 responds to sodium salts and not to KCl, whereas the Hik16/Hik41 system responds only to NaCl.

Base Sequence↗
PubMed2002

Regulatory interactions between the Pho and sigma(B)-dependent general stress regulons of Bacillus subtilis.

When Bacillus subtilis is subjected to phosphate starvation, the Pho and sigma(B)-dependent general stress regulons are activated to elicit, respectively, specific and non-specific responses to this nutrient-limitation stress. A set of isogenic mutants, with a beta-galactosidase reporter gene transcriptionally fused to the inactivated target gene, was used to identify genes of unknown function that are induced or repressed under phosphate limitation. Nine phosphate-starvation-induced (psi) genes were identified: yhaX, yhbH, ykoL and yttP were regulated by the PhoP-PhoR two-component system responsible for controlling the expression of genes in the Pho regulon, while ywmG (renamed csbD), yheK, ykzA, ysnF and yvgO were dependent on the alternative sigma factor sigma(B), which controls the expression of the general stress genes. Genes yhaX and yhbH are unique members of the Pho regulon, since they are phosphate-starvation induced via PhoP-PhoR from a sporulation-specific sigma(E) promoter or a promoter that requires the product of a sigma(E)-dependent gene. Null mutations in key regulatory genes phoR and sigB showed that the Pho and sigma(B)-dependent general stress regulons of Bacillus subtilis interact to modulate the levels at which each are activated.

Bacillus subtilis↗
PubMed1989

Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase genes.

The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase (EC 1.13.11.3) were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5-kilobase-pair PstI fragment into the broad-host-range cloning vector pRO2317. The resultant clone was able to complement protocatechuate 3,4-dioxugenase mutations in P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host. Since the cloned genes exhibited normal induction patterns when present in P. cepacia DBO1, it was concluded that induction was subject to negative control. Regulatory studies with P. cepacia wild-type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate. Further studies of P. cepacia DBO1 showed that p-hydroxybenzoate hydroxylase (EC 1.14.13.2), the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hydroxybenzoate and beta-ketoadipate.

Chromosome Mapping↗
PubMed2000

ctsR of Lactococcus lactis encodes a negative regulator of clp gene expression.

Bacteria undergo a complex programme of differential gene expression in response to stress. In Bacillus subtilis, it was recently shown that CtsR, a negative transcriptional regulator, mediates stress-induced expression of components of the Clp protease complex. In this study, a gene was identified in the Gram-positive bacterium Lactococcus lactis that encodes a 17 kDa product with 38% identity to the CtsR protein of B. subtilis. By Northern analyses it was found that in a L. lactis strain carrying a large internal deletion of ctsR, including the region encoding a putative helix-turn-helix motif, the amounts of clpC, clpP, clpB and clpE mRNAs were increased 3-8-fold compared to those present in wild-type L. lactis MG1363. In another ctsR mutant strain in which only one-third of CtsR was deleted, leaving the putative DNA-binding domain and the C-terminal 29 amino acids intact, only minor derepression of clp gene expression was observed and, furthermore, all the clp genes were still induced by heat. These results indicate that the amino acids of CtsR involved in temperature sensing are located either close to the DNA-binding domain or in the C-terminal part of the protein. Thus, in L. lactis in addition to B. subtilis, CtsR is a key regulator of heat-shock-induced gene expression, suggesting that the presence of CtsR-homologous DNA-binding sites observed in many Gram-positive bacteria reflects functional heat-shock regulatory systems.

Adenosine Triphosphatases↗
PubMed1994

Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. carotovora in response to DNA-damaging agents.

In most soft-rotting Erwinia spp., including E. carotovora subsp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (Pnl) is activated by DNA-damaging agents such as mitomycin C (MC). Induction of Pnl production in Ecc71 requires a functional recA gene and the rdg locus. DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressors of lambdoid phages, specially phi 80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix-turn-helix motifs. By replacing the rdgB promoter with the IPTG-inducible tac promoter, we have determined that rdgB by itself can activate Pnl production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnlA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of Pnl production, are expressed differently in Ecc71.

Amino Acid Sequence↗
PubMed1990

Regulation of bacterial gene expression by metal-protein complexes.

Metal ions are essential cofactors in several transacting bacterial gene regulators. Upon binding of the metal, the receptor proteins act either as repressors of gene expression or, in other systems, as transcriptional activators. Other metal-dependent regulatory proteins may function, directly or indirectly, as sensors of the cellular oxygen status, and may even be mediators in light-responsive gene regulation.

Bacterial Proteins↗
PubMed2003

Bacteria-host communication: the language of hormones.

The interbacterial communication system known as quorum sensing (QS) utilizes hormone-like compounds referred to as autoinducers to regulate bacterial gene expression. Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is the agent responsible for outbreaks of bloody diarrhea in several countries. We previously proposed that EHEC uses a QS regulatory system to "sense" that it is within the intestine and activate genes essential for intestinal colonization. The QS system used by EHEC is the LuxS/autoinducer 2 (AI-2) system extensively involved in interspecies communication. The autoinducer AI-2 is a furanosyl borate diester whose synthesis depends on the enzyme LuxS. Here we show that an EHEC luxS mutant, unable to produce the bacterial autoinducer, still responds to a eukaryotic cell signal to activate expression of its virulence genes. We have identified this signal as the hormone epinephrine and show that beta- and alpha-adrenergic antagonists can block the bacterial response to this hormone. Furthermore, using purified and in vitro synthesized AI-2 we showed that AI-2 is not the autoinducer involved in the bacterial signaling. EHEC produces another, previously undescribed autoinducer (AI-3) whose synthesis depends on the presence of LuxS. These results imply a potential cross-communication between the luxS/AI-3 bacterial QS system and the epinephrine host signaling system. Given that eukaryotic cell-to-cell signaling typically occurs through hormones, and that bacterial cell-to-cell signaling occurs through QS, we speculate that QS might be a "language" by which bacteria and host cells communicate.

Bacterial Proteins↗