Catalases HPI and HPII in Escherichia coli are induced independently

Three strains of Escherichia coli differing only in the catalase locus mutated by transposon Tn10 were constructed. These strains produced only catalase HPI (katE::Tn10 and katF::Tn10 strains) or catalase HPII (katG::Tn10). HPI levels increased gradually about twofold during logarithmic growth but did not increase during growth into stationary phase in rich medium. HPII levels, which were initially threefold lower than HPI levels, did not change during logarithmic growth but did increase tenfold during growth into stationary phase. HPI levels increased in response to ascorbate or H2O2 being added to the medium but HPII levels did not. In minimal medium, any carbon source derived from the tricarboxylic acid cycle caused five- to tenfold higher HPII levels during logarithmic growth but had very little effect on HPI levels. Active electron transport did not affect either HPI or HPII levels.     

Transcriptional regulation of katE in Escherichia coli K-12

Escherichia coli produces two distinct species of catalase, hydroperoxidases I and II, which differ in kinetic properties and regulation. To further examine catalase regulation, a lacZ fusion was placed into one of the genes that is involved in catalase synthesis. Transductional mapping revealed the fusion to be either allelic with or very close to katE, a locus which together with katF controls the synthesis of the aerobically inducible hydroperoxidase (hydroperoxidase II). katE was expressed under anaerobic conditions at levels that were approximately one-fourth of those found in aerobically grown cells and was found to be induced to higher levels in early-stationary-phase cells relative to levels of exponentially growing cells under both anaerobic and aerobic conditions. katE was fully expressed in air and was not further induced when the growth medium was sparged with 100% oxygen. Expression of katE was unaffected by the addition of hydrogen peroxide or by the presence of additional lesions in oxyR or sodA, indicating that it is not part of the oxyR regulon. When katF::Tn10 was introduced into a katE::lacZ strain, beta-galactosidase synthesis was largely eliminated and was no longer inducible, suggesting that katF is a positive regulator of katE expression.     

Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli

A class of catalase-deficient mutants that was unlinked to katE was localized between mutS and cys at 59.0 min on the Escherichia coli genome. This locus was named katF. Transposon Tn10 insertions were isolated that mapped in both katE and katF loci. The catalase species present in katE+ and katF+ recombinants was found to be different from the main catalase activities, HPI and HPII, in several respects. It did not have an associated peroxidase activity; it was electrophoretically slower on native polyacrylamide gels; it eluted from DEAE-Sephadex A50 at a higher salt concentration; its Km for H2O2 was 30.9 mM as compared with 3.7 mM for HPI and HPII; its synthesis was not induced by ascorbate; and it did not cross react with HPI-HPII antisera. This new catalase was labeled HPIII.     

Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp.

Strains of Escherichia coli which lack detectable guanosine 3',5'-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth.     

Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli

Two genes, katE and katF, affecting the synthesis of catalase HPII in Escherichia coli, have been cloned. The multistep cloning protocol involved: screening for the tet gene in a transposon interrupting the genes, selecting DNA adjacent to the transposon, and using it to probe a library of wild-type DNA to select clones from which katE and katF were subcloned into pAT153. The clones were physically characterized and the presence of the genes confirmed by complementation of their respective mutations. The location of the transposon insertions in the two genes was determined by Southern blotting of genomic digests to further confirm the identity of the cloned genes. A 93-kDa protein, the same size as the subunit of HPII, was encoded by the katE plasmid, indicating that katE was the structural gene for HPII. A 44-kDa protein was encoded by the katF plasmid.     

Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene.

The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes as 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes. The promoter was identified following primer extension analysis and is similar to sigma 70-like promoters. Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.     

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

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.     

Expression analysis and characterization of the mutant of a growth-phase- and starvation-regulated monofunctional catalase gene from Xanthomonas campestris pv. phaseoli

Analysis of the Xanthomonas campestris pv. phaseoli (Xp) catalase profile using an activity gel revealed at least two distinct monofunctional catalase isozymes denoted Kat1 and Kat2. Kat1 was expressed throughout growth, whereas Kat2 was expressed only during the stationary phase of growth. The nucleotide sequence of a previously isolated monofunctional catalase gene, Xp katE, was determined. The deduced amino acid sequence of Xp KatE showed a high percentage identity to an atypical group of monofunctional catalases that includes the well-characterized E. coli katE. Expression of Xp katE was growth phase-dependent but was not inducible by oxidants. In addition, growth of Xp in a carbon-starvation medium induced expression of the gene. An Xp katE mutant was constructed, and analysis of its catalase enzyme pattern showed that Xp katE coded for the Kat2 isozyme. Xp katE mutant had resistance levels similar to the parental strain against peroxide and superoxide killing at both exponential and stationary phases of growth. Interestingly, the level of total catalase activity in the mutant was similar to that of the parental strain even in stationary phase. These results suggest the existence of a novel compensatory mechanism for the activity of Xp catalase isozymes.     

Proteomic analysis of survival of Rhodococcus jostii RHA1 during carbon starvation

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ～250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.     

Growth conditions and heat resistance of Citrobacter freundii

The influence of the growth medium and the growth temperature on the heat resistance of Citrobacter freundii has been established. Logarithmic growth phase cells grown on rich media have a higher heat resistance than cells of the same phase grown on minimal media. This finding was independent of type of carbon source in the growth medium, but the kind of carbon source has a definite influence on the heat resistance. Logarithmic phase cells grown at 37°C are much more heat stable than cells grown at 20 or 41°C. Stationary growth phase cells are much more heat resistant than logarithmic phase cells, whereas Mg²⁺-or glucose-starved cells are even slightly more heat stable than stationary phase cells.