Structure-function studies of Escherichia coli RpoH (sigma32) by in vitro linker insertion mutagenesis

The sigma factor RpoH (sigma(32)) is the key regulator of the heat shock response in Escherichia coli. Many structural and functional properties of the sigma factor are poorly understood. To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background. Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity. Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2.     

Stress induced cross-protection against environmental challenges on prokaryotic and eukaryotic microbes

Prokaryotic and eukaryotic microbes thrive successfully in stressful environments such as high osmolarity, acidic or alkali, solar heat and u.v. radiation, nutrient starvation, oxidative stress, and several others. To live under these continuous stress conditions, these microbes must have mechanisms to protect their proteins, membranes, and nucleic acids, as well as other mechanisms that repair nucleic acids. The stress responses in bacteria are controlled by master regulators, which include alternative sigma factors, such as RpoS and RpoH. The sigma factor RpoS integrates multiple signals, such as the general stress response regulators and the sigma factor RpoH regulates the heat shock proteins. These response pathways extensively overlap and are induced to various extents by the same environmental stresses. In eukaryotes, two major pathways regulate the stress responses: stress proteins, termed heat shock proteins (HSP), which appear to be required only for growth during moderate stress, and stress response elements (STRE), which are induced by different stress conditions and these elements result in the acquisition of a tolerant state towards any stress condition. In this review, the mechanisms of stress resistance between prokaryotic and eukaryotic microbes will be described and compared.     

Phenotypes of Bacillus subtilis mutants altered in the precursor-specific region of sigma E.

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode sigma E directly. The sequence specifying the first amino acid of sigma E (GGC) was placed immediately downstream of the initiating codon (ATG) of P31. The resulting sigE allele (sigE delta 84) encodes a sigma E-like protein which differs from the "processed product" by a single Met residue at its amino terminus. B. subtilis strains which carried this allele were Spo- and contained no detectable sigma E. The sigE delta 84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele. A sigma E-like product was also not detected in two B. subtilis strains with missense mutations in the sequence encoding the processed region of P31. These results suggest that sigma E is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence. A mutant allele (sigE delta 48) which made an active sigma E-like protein in B. subtilis was isolated. This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site. The sigE delta 48 product was not processed, but it activated the sigma E -dependent spoIID promoter in vivo. The sigE delta 48 product therefore lost both an essential target for processing and a region which inhibited sigma sigma E activity. Cells which carried sig E delta 48 were Spo-. The basis of the sigE delta 48-dependent defect in sporulation is unknown, but the sigma E delta 48 activity appeared to persist beyond the time in development (4 h after onset sporulation) when wild-type sigma E activity declines. Thus, it may interfere with the proper regulation of late sporulation genes.     

Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.     

The C-terminal end of LpxC is required for degradation by the FtsH protease

Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane-bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co-purified with LpxC. However, degradation of LpxC was DnaK/J-independent in contrast to turnover of the heat shock sigma factor sigma32 (RpoH). The stability of LpxC in a bacterial one-hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C-terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half-life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C-terminus of LpxC contains a signal sequence necessary for FtsH-dependent degradation.     

Principal sigma subunit of the Caulobacter crescentus RNA polymerase.

We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.     

Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase.

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.