Biomedical subjects

Michael Hecker

Publications and source records attributed to Michael Hecker.

80 records · Page 5Linked to original sources

PubMed2002

ClpXP protease regulates the signal peptide cleavage of secretory preproteins in Bacillus subtilis with a mechanism distinct from that of the Ecs ABC transporter.

Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ alpha-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation.

ATP-Binding Cassette Transporters↗

PubMed2002

Heat shock proteome of Agrobacterium tumefaciens: evidence for new control systems.

The regulation of Agrobacterium tumefaciens heat shock genes involves a transcriptional activator (RpoH) and repressor elements (HrcA-CIRCE). Using proteome analysis and mutants in these control elements, we show that the heat shock induction of 32 (out of 56) heat shock proteins is independent of RpoH and HrcA. These results indicate the existence of additional regulatory factors in the A. tumefaciens heat shock response.

Agrobacterium tumefaciens↗

PubMed2002

Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis.

The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to DL-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

Bacillus subtilis↗

PubMed2002

Influence of the leuX-encoded tRNA5(Leu) on the regulation of gene expression in pathogenic Escherichia coli.

The leuX gene encoding the minor tRNA5(Leu) is important for the expression of several virulence factors of pathogenic Escherichia coli strains. The differential usage of minor codons to control the expression of specialized genes has been proposed to be a general mechanism of bacteria to regulate gene expression at the posttranscriptional level. The minor codon usage theory foots on the biased codon usage of bacterial genes and the selective availability of tRNA isoacceptors. We aimed at the further investigation of the regulatory role of the tRNA5(Leu) for gene expression in pathogenic E. coli. For this purpose, the molecular mechanism underlying the tRNA5(Leu)-dependent regulation of different virulence-associated genes of pathogenic E. coli as well as the regulation of leuX transcription under various growth conditions were investigated in detail. The global impact of the presence or absence of the leuX-encoded tRNA on gene expression of the uropathogenic E. coli strain 536 was studied by proteome analysis. The obtained results argue for a general importance of the tRNA5(Leu) for gene expression of E. coli and the involvement of this tRNA in global regulatory networks.

Animals↗

PubMed1999

Identification and transcriptional analysis of new members of the sigmaB regulon in Bacillus subtilis.

Bacillus subtilis responds to various stimuli (heat, ethanol and salt stress, energy starvation) with the induction of general stress proteins (GSPs). Most of them belong to the stress and stationary-phase regulon controlled by the alternative sigma factor sigmaB. The majority of sigmaB-dependent proteins are thought to provide a precautionary general stress resistance in stressed or starved cells. In this report, the identification and transcriptional analysis of nine new members of the sigmaB regulon are described. The biochemical function was not determined for any of the proteins encoded by the nine new sigmaB-dependent stress genes, however, similarities to proteins in the databases allowed a distinction between proteins with putative (i-iv) and unknown (v) function. The putative functions of BmrU, YcdF, YdaD, YdaP, YhdN and YocK underline the suggested protective role of sigmaB-dependent GSPs and also elucidate new areas where sigmaB might play an important role. (i) The finding that the bmrUR operon is under sigmaB control indicates that the elimination of multidrug compounds might be a new function in multiple stress resistance. (ii) YcdF and YdaD resemble NAD(P)-dependent dehydrogenases. Both proteins could be involved in the generation of NAD(P)H and therefore in the maintenance of the intracellular redox balance under stress. (iii) The ydaP gene might belong to the increasing number of sigmaB-dependent genes whose orthologues are under the control of sigmas in Escherichia coli, indicating that both regulons may fulfil similar functions. (iv) YhdN shows weak similarities to potassium ion channel proteins and YocK shows resemblance to the DnaK suppressor protein DksA. (v) Three new sigmaB-dependent genes (ydaE, ydaG and yfkM) encoding proteins with still unknown functions were also described. Further analyses of corresponding mutants might allow a first prediction of their function within the framework of the general stress regulon.

Bacillus subtilis↗

PubMed1997

Identification of vegetative proteins for a two-dimensional protein index of Bacillus subtilis.

Twenty-three of the most prominent spots which are visible on two-dimensional (2-D) protein gels of Bacillus subtilis crude extracts were selected as marker spots for the construction of a 2-D protein index. N-terminal sequencing of the corresponding proteins resulted in the identification of enzymes involved in glycolysis, TCA cycle, pentose phosphate cycle, amino acid metabolism, nucleotide biosynthesis and translation. Using computer analysis of the 2-D protein gels, most of these metabolic enzymes were found to be synthesized at a reduced rate after different stresses and glucose starvation. Such an approach permits a rapid and global evaluation of the regulation of different branches of metabolism in response to various physiological conditions.

Amino Acid Sequence↗

PubMed1997

Specific and general stress proteins in Bacillus subtilis--a two-deimensional protein electrophoresis study.

A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose of phosphate starvation. The data provided evidence that the induction of a least 42 general stress proteins absolutely required the alternative sigma factor sigmaB. However, at least seven stress proteins, among them ClpC, ClpP, Sod, AhpC and AhpF, remained stress-inducible in a sigB mutant. Such a detailed analysis also premitted the description of subgroups of general stress proteins which are subject to additional regulatory circuits, indicating a very thorough fine-tuning of this complex response. The relative synthesis rate of the general stress proteins constituted up to 40% of the total protein synthesis of stressed cells and thereby emphasizes the importance of the stress regulon. Besides the induction of these general or rather unspecific stress proteins, the induction of stress-specific proteins is shown and discussed.

Bacillus subtilis↗

PubMed1997

The Bacillus subtilis clpC operon encodes DNA repair and competence proteins.

ClpC of Bacillus subtilis, controlling competence gene expression and survival under stress conditions, is encoded by the fourth gene of a six-gene operon. The product of orf1 contains a potential helix-turn-helix motif, but shows no significant similarities with known protein sequences. The second and third genes encode proteins with similarities to zinc-finger proteins (orf2) and arginine kinases (orf3), respectively. The product of orf5 contains a zinc-finger motif and an ATP-binding domain, and is highly similar to the product of the Escherichia coli sms gene. A strain bearing a disruption of orf5 showed increased sensitivity to the alkylating agent methyl methanesulfonate. Furthermore, this mutant strain displayed decreased capacity for genetic recombination as measured by transformation experiments. The last open reading frame, orf6, encodes a protein with limited similarity in its C-terminal part to the B. subtilis comEA gene product and to the UvrC DNA repair excinuclease. Inactivation of orf5 resulted in strongly diminished transformation with all types of DNA. Mutations affecting either orf5 or orf6 resulted in strains with decreased resistance to UV-irradiation in the stationary phase, indicating that these proteins play a role in the development of a non-specific stationary-phase resistance to UV-irradiation. Moreover, these results suggest an involvement of both proteins in transformation and presumably in DNA repair.

Amino Acid Sequence↗