Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli

https://mmbr.asm.org/content/mmbr/55/3/371.full.pdf

Control site location and transcriptional regulation in Escherichia coli.

Because of their ability to stimulate gene expression from distant sites and regardless of their orientation, transcriptional enhancer elements are a puzzling class of regulatory DNA sequences (42). Although enhancers are a common feature of eukaryotic and viral genes, the mechanism by which they communicate with promoters and stimulate gene expression is still not very well understood. However, our understanding of the mechanism of gene activation at a distance has been boosted by the study of naturally occurring enhancer-like elements (ELE) in prokaryotes. The ELE are palindromic or nearly palindromic nucleotide sequences normally found between 100 and 200 bp upstream of a certain class of promoters, and, like the eukaryotic enhancers, they still stimulate transcription when moved thousands of nucleotides upstream or downstream from the transcription start and regardless of their orientation (7, 47). The extensive genetic characterization of many bacterial ELE and the development of defined in vitro transcription systems has greatly facilitated the study of activation by enhancers, and now some of the molecular details of distal activation are well established (34). It has been demonstrated that the ELE are binding sites for regulatory proteins named bacterial enhancer-binding proteins (EBPs). The best-studied EBPs are NtrC and NifA, which stimulate the expression of genes required for nitrogen assimilation and nitrogen fixation, respectively, in a number of organisms (37). These proteins activate genes transcribed by the RNA polymerase holoenzyme containing the alternative sigma factor &F4 (EO(J4). The u54 system is widely represented in the alpha and gamma proteobacteria (previously called purple bacteria), and it has also been reported to be present in Bacillus subtilis (13, 14). Among other physiological processes controlled by C54 are nitrogen assimilation and fixation, dicarboxylic acid transport, hydrogen oxidation, alginate utilization, degradation of aromatic compounds, pilus formation and formate utilization (for reviews, see references 35 and 56). A common feature of all these processes is that they are not absolutely required for cell survival, and each is activated by a different EBP interacting with a distinctive ELE nucleotide sequence. In addition to its ability to be activated at a distance, the Er54 system has several other unique characteristics that are not common among bacterial holoenzymes. E&'4 recognizes promoter DNA with unusually close boxes, centered at

The sigma 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains.

Promoters recognized by the RNA-polymerase with the alternative sigma factor sigma(54) (Esigma54) are unique in having conserved positions around -24 and -12 nucleotides upstream from the transcriptional start site, instead of the typical -35 and -10 boxes. Here we compile 186 -24/-12 promoter sequences reported in the literature and generate an updated and extended consensus sequence. The use of the extended consensus increases the probability of identifying genuine -24/-12 promoters. The effect of several reported mutations at the -24/-12 elements on RNA-polymerase binding and promoter strength is discussed in the light of the updated consensus.

Compilation and analysis of sigma(54)-dependent promoter sequences.

Summary
In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli.

The fdhF gene, encoding the selenopolypeptide of formate dehydrogenase (FDHH), has a -12/-24 nif-type consensus promoter. A cis-acting DNA element, which is required for the regulation of the promoter by formate under anaerobic conditions, has been identified. This regulatory sequence of about 25 bp in length is located 110 bp upstream of the transcription start site. By analysing a variety of mutant constructs in this region (5' deletions, internal deletions and point mutations) we were able to identify a hexanucleotide sequence -GTCACG-, which is important for the formate regulation of the fdhF promoter. The data also indicate that this element has many of the properties characteristic of eukaryotic enhancers.

Characterization of a cis regulatory DNA element necessary for formate induction of the formate dehydrogenase gene (fdhF) of Escherichia coli.

Summary

Hybrid 5′ regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF′-′lacZ or nif′-′lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF 5’flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the nfrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed In front of the nifD spacer plus promoter from B. Japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH′-′lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.

A. Birkmann

Papers

Characterization of a cis regulatory DNA element necessary for formate induction of the formate dehydrogenase gene (fdhF) of Escherichia coli.

Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes.