Influence of impaired chaperone or secretion function on SecB production in Escherichia coli.

The efficient export of proteins through the cytoplasmic membrane of Escherichia coli requires chaperones to maintain protein precursors in a translocation-competent conformation. In addition to SecB, the major chaperone facilitating export of particular precursors, heat shock-induced chaperones DnaK-DnaJ and GroEL-GroES are also involved in this process. By use of secB'-lacZ gene fusions and immunoprecipitation experiments, SecB production was studied in E. coli strains containing conditional lethal mutations in chaperone or sec genes. While the loss of heat shock chaperones resulted in an increased production of SecB, mutations in sec genes showed only minor effects on SecB synthesis. Neither the plasmid-mediated overexpression of precursors of exoproteins nor the overexpression of secB altered the synthesis of SecB. These results suggest that under conditions where chaperones become depleted, E. coli responds by raising the expression of secB. These data confirm the supposed synergy of different chaperones involved in protein export.     

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.     

Renaturation of denatured lambda repressor requires heat shock proteins

The temperature-sensitive bacteriophage lambda cI857 repressor protein rapidly renatures after thermal inactivation. E. coli mutants in the heat shock protein genes dnaK, dnaJ, and grpE do not efficiently reactivate heat-denatured repressor. Our results suggest that protein refolding is promoted by heat shock proteins and that such a process is the basis of the homeostatic role played by these proteins in the heat shock response.     

A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress.

Escherichia coli treated with nontoxic levels of the superoxide-generating redox-cycling agents menadione and paraquat showed dramatic changes in protein composition as monitored by two-dimensional gel analysis. The distribution of proteins synthesized after treatment with these agents overlapped significantly with that seen after hydrogen peroxide treatment, and it included all the proteins in the oxyR regulon. The redox-cycling agents also elicited the synthesis of at least 33 other proteins that were not seen with hydrogen peroxide, including three heat shock proteins, the Mn-containing superoxide dismutase, the DNA repair protein endonuclease IV, and glucose-6-phosphate dehydrogenase. At least some of these redox-inducible proteins appear to be part of a specific response to intracellular superoxide. E. coli is thus equipped with a network of inducible responses against oxidative damage, controlled in multiple regulatory pathways.     

Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates.

BACKGROUND: Mycobacterium tuberculosis is a significant human pathogen capable of replicating in mononuclear phagocytic cells. Exposure to reactive oxygen and nitrogen intermediates is likely to represent an important aspect of the life cycle of this organism. The response of M. tuberculosis to these agents may be of significance for its survival in the host. MATERIALS AND METHODS: Patterns of de novo proteins synthesized in M. tuberculosis H37Rv exposed to compounds that generate reactive oxygen and nitrogen intermediates were studied by metabolic labeling and two-dimensional electrophoresis. RESULTS: Menadione, a redox cycling compound which increases intracellular superoxide levels, caused enhanced synthesis of seven polypeptides, six of which appeared to be heat shock proteins. Chemical release of nitric oxide induced eight polypeptides of which only one could be identified as a heat shock protein. Nitric oxide also exhibited a mild inhibitory action on general protein synthesis in the concentration range tested. Hydrogen peroxide did not cause differential gene expression and exerted a generalized inhibition in a dose-dependent manner. Cumene hydroperoxide caused mostly inhibition but induction of two heat shock proteins was detectable. CONCLUSIONS: The presented findings indicate major differences between M. tuberculosis and the paradigms of oxidative stress response in enteric bacteria, and are consistent with the multiple lesions found in oxyR of this organism. The effect of hydrogen peroxide, which in Escherichia coli induces eight polypeptides known to be controlled by the central regulator oxyR, appears to be absent in M. tuberculosis. Superoxide and nitric oxide responses, which in E. coli overlap and are controlled by the same regulatory system soxRS, represent discrete and independent phenomena in M. tuberculosis.     

Bacterial growth inhibition by overproduction of protein

Multicopy plasmids that have been engineered to produce large quantitites of a single gratuitous (non-functional, non-toxic) protein are often problematic. When fully induced, these engineered constructions produce very sick bacteria. The reasons for this may be found in the physiology of wild-type laboratory strains that have been selected to grow at maximum rates with optimal quantities of their proteins. Such bacteria apparently experience the accumulation of gratuitous proteins as an internal shift down and they respond to this with a starvation response. Unlike the shift down associated with a change of growth media, the production of large quantities of gratuitous protein is not associated with a new pre-programmed steady-state of balanced growth. Consequently, the starvation response continues until the bacteria commit suicide by, among other things, destroying their ribosomes.     

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.     

Expression of genes for guanyl-specific ribonucleases in Bacillus intermedius and Bacillus pumilus is regulated by the PhoP-PhoR two-component signal transduction system of PHO regulon of Bacillus subtilis]

Promoters of the genes of guanyl-specific ribonucleases of Bacillus intermedius (binase) and Bacillus pumilus (RNase Bp) were found to contain sequences homologous to those recognizable by the regulatory protein PhoP in the promoters of the PHO regulon of B. subtilis, as well as regions partially homologous to the binding sites of another regulatory protein, PhoB, in the promoters of the PHO regulon of Escherichia coli. The role of the two-component regulatory systems PhoP-PhoR and PhoB-PhoR in the regulation of expression of the genes of binase and RNase Bp in recombinant strains of B. subtilis and E. coli was studied by using mutant strains. It was established that the expression of these genes in recombinant B. subtilis cells is stringently controlled by the PhoP-PhoR two-component regulatory system, whereas the expression of these genes in E. coli cells is not controlled by the regulatory proteins PhoB or PhoR. Presumably, regulatory systems of the response to phosphate starvation, analogous to the PHO regulon of B. subtilis, also function in other representatives of the genus Bacillus.     

Global versus local regulatory roles for Lrp-related proteins: Haemophilus influenzae as a case study

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp(+) and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.