The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::lacZ fusions using the lambda placMu system. One operon fusion (csi2::lacZ) has been studied in detail. csi2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::lacZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::lacZ or csi2::Tn10 did not produce glycogen, did not develop thermotolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::lacZ defines a regulatory gene of central importanc e for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' ('sigma S') as appropriate designations.

Identification of a central regulator of stationary‐phase gene expression in Escherichia coli

As the decade progresses this prediction is rapidly being realized, and microbial enzymes are becoming increasingly important in such diverse fields as medicine, brewing, and timber preservation. The genus Bacillus has played a major role in this development as evidenced by the distribution of the papers read at the Fifth International Fermentation Symposium, 1976 (64). Of 23 papers in the session devoted to "Microbial Enzymes of Industrial Interest" no less than ten were concerned with enzymes from bacilli. Reasons for the predominance of these bacteria in this area of study are several. First, they comprise a group of chemoorganotrophs that can be easily maintained and cultivated and yet are markedly heterogeneous in character. Psychrophiles, mesophiles, and thermophiles, in addition to alkalophilic, neutrophilic, and acidophilic species are well represented. Furthermore, virtually all 48 species of the genus listed in Bergey's Manual ofDeterminative Bacteriology (92) secrete a variety of soluble extracellular enzymes, which reflects the diversity of the parental habitats. Amylases that can liquefy starch under pressure at 11000 (194) and proteases that are stable and active at pH 12.0 (6) are extreme examples of enzyme adaption. This article will attempt to review the recent literature concerned with the characterization and properties of the exoenzymes synthesized by the bacilli and the control and mechanisms of their synthesis. It is restricted to this genus because the commercial importance of extracellular enzymes and academic interest in the process of sporulation have prompted a considerable amount of research into this general area. Nevertheless, in the final section I have attempted to equate our present knowledge of exoenzyme synthesis in procaryotes other than

Extracellular enzyme synthesis in the genus Bacillus.

The second vegetative sigma factor sigma S (encoded by the rpoS gene) is the master regulator in a complex regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. Using a combination of gene-fusion technology and quantitative immunoblot, pulse-labeling, and immunoprecipitation analyses, we demonstrate here that rpoS/sigma S expression is not only transcriptionally controlled, but is also extensively regulated at the levels of translation and protein stability. rpoS transcription is inversely correlated with growth rate and is negatively controlled by cAMP-CRP. In complex medium rpoS transcription is stimulated during entry into stationary phase, whereas in minimal media, it is not significantly induced. rpoS translation is stimulated during transition into stationary phase as well as by an increase in medium osmolarity. A model involving mRNA secondary structure is suggested for this novel type of post-transcriptional growth phase-dependent and osmotic regulation. Furthermore, sigma S is a highly unstable protein in exponentially growing cells (with a half-life of 1.4 min), that is stabilized at the onset of starvation. When cells are grown in minimal glucose medium, translational induction and sigma S stabilization occur in a temporal order with the former being stimulated already in late exponential phase and the latter taking place at the onset of starvation. Although sigma S does not control its own transcription, it is apparently indirectly involved in a negative feedback control that operates on the post-transcriptional level. Our analysis also indicates that at least five different signals [cAMP, a growth rate-related signal (ppGpp?), a cell density signal, an osmotic signal, and a starvation signal] are involved in the control of all these processes that regulate rpoS/sigma S expression.

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The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability.

Cyclic 3',5'-nucleotide phosphodiesterase. Evidence for and properties of a protein activator.

The concentration of cyclic adenosine 3′,5′-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of β-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.

Cyclic adenosine 3',5'-monophosphate in Escherichia coli.

The cyclic 3′,5′-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier.

H.V. Rickenberg

Papers

Cyclic adenosine 3',5'-monophosphate in Escherichia coli.

Cyclic 3',5'-adenosine monophosphate phosphodiesterase of Escherichia coli.

The enzymic degradation of 3′, 5′ cyclic AMP in strains of E., coli sensitive and resistant to catabolite repression

Occurrence of the adenylate cyclase “G Protein” in membranes of Dictyosteliumdiscoideum

Catabolite repression in Escherichiacoli: The role of glucose 6-phosphate