The response of Salmonella typhimurium to various forms of environmental stress was examined by using O'Farrell two-dimensional gel electrophoresis. Polypeptides (a total of 110) which quantitatively increased during various starvations, anaerobiosis, or heat shock were identified and cataloged in reference to a standard polypeptide map. Although significant overlap was noted during comparison of proteins induced by different starvations, only a few proteins produced during heat shock or anaerobiosis were also identified as starvation inducible.

Three new loci were discovered, each of which participates in the regulation of anaerobic gene expression. The regulatory gene earA negatively regulates the expression of the anaerobiosis-inducible gene aniG as well as that of at least three other genes, as determined by two-dimensional polyacrylamide gel electrophoresis. The earA locus maps at 86 min. The expression of aniG was also shown to be controlled by changes in external pH under aerobic and anaerobic conditions. Maximal expression was observed under anaerobic conditions at an external pH of 6.0. Significant transcriptional activity was also observed under aerobic conditions at pH 6.0. This was in contrast to hyd, whose expression was dependent upon anaerobiosis and varied with external pH. The pH dependence disappeared under fully aerobic conditions. Mutations in earA had no effect upon hyd expression. The two other regulators identified were oxrF, which controls aniH, and oxrG, which, in concert with oxrA and oxrB, controls aniC and anil. The oxrG locus was mapped to 88 min and appears to code for a positive regulator. Various oxr mutants were subjected to two-dimensional polyacrylamide electrophoretic analysis of anaerobiosis-inducible proteins. Several pathways of anaerobic control were observed by means of these techniques.There have been several reports identifying genes in Salmonella typhimurium and Escherichia coli whose expressions are controlled by the presence or absence of molecular oxygen (1,10,35,37). Anaerobiosis-inducible genes with unidentified products have been referred to as ani (1) and oxd (35). These genes were discovered by generating random lacZ operon fusions in the chromosome with Mu d(Ap lac) phage. Identified anaerobiosis-inducible genes include those involved with nitrate (nar; 4, 14, 34) and fumarate (frd; 21, 22) reductases, hydrogen sulfide production (phs; 1, 11), cobalamine biosynthesis (cob; 13), hydrogenase (hyd; 1, 20, 39), some peptidases and peptide permeases (tppB, pepT; 15, 18, 19, 35), anaerobic L-a-glycerophosphate dehydrogenase (glpAB; 23), NAD biosynthesis (nadAB; 17), degradative threonine dehydratase (28), formate hydrogen lyase (fhl; 31), a tertiary amine oxidase (torA; 24, 30), menaquinone synthesis (men; 24), and dimethyl sulfoxide reductase (23). Several genes have been implicated in S. typhimurium as participating in anaerobic transcriptional control. These include oxrA, a positive regulator equivalent to fnr in E. coli (7,25,35); oxrB, another positive regulator (35); oxrC (pgi), not a transcriptional regulator but involved with the generation of an inducing signal (19); and tppR, a positive regulator of tppB (18).The most characterized anaerobic regulatory locus is fnr of E. coli. Mutations in fnr prevent the anaerobic induction of several respiratory enzymes, such as the nitrate and fumarate reductases, hydrogenase 2, dimethyl sulfoxide reductase, and glycerol-3-phosphate dehydrogenase (3,7,23,25,34 (1,11,18,19,30,35). The molecular mechanism(s) controlling the expressions of these genes remains obs...

Using the technique of Mu dl(Ap lac)-directed lacZ operon fusions, several oxygen-regulated genetic loci were identified in SalmoneUa typhimurum. Thirteen anaerobically inducible and six aerobically inducible operon fusions were identified. Based on control by the oxrA and oxrB regulatory loci, the ani-lacZ fusions were grouped into three classes: class I loci were regulated by both oxr loci, class H genes were regulated by oxrA only, and class m loci were not affected by either regulatory locus. Several of the ani-lacZ fusions required growth in complex medium before they exhibited the inducible phenotype. While the expression of some of these loci was repressed when organisms were grown in nitrate, others were stimulated by nitrate. Fusions into the hyd and phs loci were identified among the Isolated ani-lacZ fusions. Six oxygen-inducible (oxi) operon fusions were also identified. Two of the oxi loci mapped near oxygen-regulatory loci: oxiC near oxrA and oxiE near oxyR. However, neither fusion appeared to occur within the regulatory locus. The data presented serve to further define the aerobic and anaerobic stimulons of S. typhimurium but indicate additional regulatory circuits above those already defined.

The recently described aniG locus exhibits a series of unique regulatory features. The gene is exogenously coinduced by acid and D-mannose, its expression is maximal under anaerobiosis, and the system is regulated in an unusual manner by cyclic AMP. The external acid regulatory locus, earA, is a repressor protein that mediates the acid and mannose control of aniG. The earA locus was cloned and found to code for a 33K protein associated with membrane- and soluble fractions. A second locus, earB, was located immediately upstream from earA. The earB locus or its product interferes with the repression of aniG by EarA. Mutations in crp and cya were found to prevent transcription of aniG but only in an earA+ background. Analysis of an earA-cat fusion established that crp does not affect earA expression. While the physiological role of aniG/earA is unclear, this system serves as a model for external pH-regulated gene expression. The present data indicate that it is used to sense the presence of mannose in an acidic extracellular environment. This is particularly intriguing in that the system is not involved in the utilization of mannose as a carbon source.

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