Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli

In Escherichia coli the beta-lactam mecillinam specifically inhibits penicillin-binding protein 2 (PBP2), a peptidoglycan transpeptidase essential for maintaining rod shape. We have previously shown that PBP2 inactivation results in a cell division block and that an increased concentration of the nucleotide ppGpp, effector of the RelA-dependent stringent response, confers mecillinam resistance and allows cells to divide as spheres in the absence of PBP2 activity. In this study we have characterized an insertion mutation which confers mecillinam resistance in wild-type and DeltarelA strains but not in DeltarelADeltaspoT strains, devoid of ppGpp. The mutant has an insertion in the fes gene, coding for enterochelin esterase. This cytoplasmic enzyme hydrolyses enterochelin-Fe(3+) complexes, making the scavenged iron available to the cells. We show that inactivation of the fes gene causes iron limitation on rich medium plates and a parallel SpoT-dependent increase of the ppGpp pool, as judged by the induction of the iron-regulated fiu::lacZ fusion and the repression of the stringently controlled P1(rrnB)::lacZ fusion respectively. We further show, by direct ppGpp assays, that iron starvation in liquid medium produces a SpoT-dependent increase of the ppGpp pool, strongly suggesting a role for iron in the balance of the two activities of SpoT, synthesis and hydrolysis of (p)ppGpp. Finally, we present evidence that ppGpp exerts direct or indirect positive control on iron uptake, suggesting a simple homeostatic regulatory circuit: iron limitation leads to an increased ppGpp pool, which increases the expression of iron uptake genes, thereby alleviating the limitation.     

Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80.

Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.     

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate ⁵⁵Fe³⁺ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of ⁵⁵Fe³⁺ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.     

Inhibition of lacZ Gene Translation Initiation in trp-lac Fusion Strains

Different levels of beta-galactosidase are found in various trp-lac fusion strains. These levels of beta-galactosidase fall within a 60-fold range. The amount of thiogalactoside transacetylase activity detected in these same strains only varies 10-fold and is found in amounts greater than those predicted from the beta-galactosidase levels. The observation that the beta-galactosidase and thiogalactoside transacetylase levels are not directly proportional, that the lacZ messenger ribonucleic acid (mRNA) levels are not proportional to the beta-galactosidase activity, that, at least for the one fusion strain tested, the SuA polarity suppressor does not affect the beta-galactosidase level, and that, in all but one strain, the beta-galactosidase activity appears to reside in normal beta-galactosidase molecules suggests that the disproportionately low production of beta-galactosidase is due to a decrease in the frequency of translation initiation of lacZ mRNA in these strains. Several mechanisms are proposed to explain this decrease. Some possible bases for the disproportional production of beta-galactosidase and thiogalactoside transacetylase are also described. The preferred explanation for these disproportional enzyme levels is that only a fraction of the full complement of ribosomes need initiate translation at lacZ for the functional synthesis of lac mRNA to occur and that once the lac ribonucleic acid is made a full complement of ribosomes can bind at internal translation initiation sites at Y and A.     

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.     

Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis.

sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.     

The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5'-translational operator mRNA

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16S ribosomal RNA (rRNA) as well as a translational operator located in the 5'-untranslated region (5'-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5'-UTR of the BS15 gene and beta-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5'-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.     

Iron transport of Escherichia coli K-12: involvement of the colicin B receptor and of a citrate-inducible protein.

It was shown that feuB mutants (defective in ferric enterochelin uptake) were unable to adsorb colicin B. In addition, they were missing one of the three outer-membrane proteins which are over produced in strains grown in iron-deficient, extracted medium. Thus this protein (the feuB protein) is probably the receptor for colicin B and functions in enterochelin-mediated iron transport. The feuB gene was located by P1 transduction at approximately 72.5 min on the Escherichia coli K-12 genetic map and thus maps separately from the other genes concerned with the enterochelin system. The outer membranes of various strains grown in the presence of 1 mM citrate contained a high level of a protein which was present in very small amounts when citrate was absent from the growth medium. This protein was most easily observed in feuB mutants grown in the presence of citrate, since on polyacrylamide gels it ran in a similar position to the feuB protein, which is missing in these mutants. The relationship of this citrate-inducible protein to the inducible citrate-dependent iron uptake system is discussed.