Genetic mapping of rpoD implicates the major sigma factor of Bacillus subtilis RNA polymerase in sporulation initiation

Summary We have mapped the chromosomal locus of rpoD, which encodes the major sigma factor of Bacillus subtilis RNA polymerase. The rpoD locus lay between aroD and lys, tightly linked to dnaE and inseparable from crsA. Marker order in this region was acf-aroD-dnaE-rpoD(crsA)-spoOG-lys. By transformation using cloned donor DNA from the rpoD region, we identified the gene immediately upstream of rpoD as dnaE, which coded for a 62,000 dalton protein essential for DNA replication. Both dnaE and rpoD were transcribed in the same direction, counterclockwise on the chromosome. The gene functions and organization in the rpoD region are thus similar to those of the E. coli sigma operon. We also used transformation to identify crsA47 as a mutation within the sigma coding region itself. The crsA alteration of sigma renders the sporulation process insensitive to glucose catabolite repression, and also restores sporulation ability to strains carrying early-blocked spoOE, spoOF, and spoOK mutations. Thus the major sigma factor and these spoO gene products directly or indirectly affect the same cellular function.     

A new mutation rpoD800, affecting the sigma subunit of E. coli RNA polymerase is allelic to two other sigma mutants

Summary We have characterized a new mutation rpoD800 affecting the sigma gene of E. coli. Upon transfer to high temperature, a strain with the rpoD800 mutation ceases growth within 30 min. We find that this mutation renders sigma about 10-fold more thermolabile than the wild type sigma at 45°C in vitro. We have compared the temperature profile for inactivation of wild type and mutant sigma and find that the mutant inactivates at a temperature about 9° C lower than does the wild type.The chromosomal locus affected by rpoD800 is shown to be allelic to the locus affected by the spontaneous mutants ts285 and alt-1. All three mutations result in altered sigma and in altered growth at high temperature. We argue that the single locus affected is the structural gene for the sigma subunit of E. coli RNA polymerase.     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.     

The rpoN Gene of Enterococcus faecalis Directs Sensitivity to Subclass IIa Bacteriocins

The sigma54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding sigma54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that sigma54 is especially involved in sensitivity to subclass IIa bacteriocins. Received: 5 May 2000 / Accepted 28 June 2000     

Partial characterization of anrpoD-like gene oflactoccocus lactis subsp.lactis ML3 with a polymerase chain reaction-based approach

With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of anrpoD-like gene ofLactoccocus lactis subsp.lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical RpoD box of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs inPediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, andLactococcus lactis subsp.cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.     

Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS, whose product is similar to a sigma factor

In many species of actinomycetes, carotenogenesis can be photoinduced. The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency. In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration. We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S. setonii ISP5395 cells the capacity to synthesize carotenoids. Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis. We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S. setonii ISP5395.     

Processing of cell‐surface signalling anti‐sigma factors prior to signal recognition is a conserved autoproteolytic mechanism that produces two functional domains

Cell‐surface signalling (CSS) enables Gram‐negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti‐sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP‐mediated proteolysis of the anti‐sigma factors is key to σ^ECF activation. Using the Pseudomonas aeruginosa FoxR anti‐sigma factor, we show here that RseP is responsible for the generation of an N‐terminal tail that likely contains pro‐sigma activity. Furthermore, it has been reported previously that this anti‐sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti‐sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti‐sigma factors.     

Perpetuation of the hereditary sigma virus in populations of its host,Drosophila melanogaster. Geographical analysis of correlated polymorphisms

In natural populations of Drosophila melanogaster, about 10% of the individuals are infected by a virus, sigma, which is not contagious but is transmitted through gametes. These populations are also regularly polymorphic for two alleles, O and P, of a locus ref(2)P; the P allele interferes with the multiplication of the virus. Two viral Types are found in populations, differing in their sensitivity to the P allele. Many samples of flies have been collected in different parts of the world and for each of them, the P frequency has been measured and the viral Type determined. A clear geographical differentiation appears for both these traits; they present a mutual adaptation leading to relatively low frequencies of infected flies in natural populations. Most viruses are only known from highly selected laboratory strains. The observations reported in this paper give evidence of the self restraint exercised by the sigma virus at the population level; they indicate that the characteristics of wild viral clones are likely to differ from those of laboratory strains and also from one population to another.The sigma virus is comparable to other genetical elements, that can be more efficiently transmitted than a mendelian allele, such as transposable elements. The discussion illustrates some of the factors involved in the perpetuation of such elements in a population and points out the difficulty of taking them all into consideration in theoretical models dealing with their perpetuation.     

Drosophila genes which intervene in multiplication of sigma virus (author''s transl)]

Summary Distinction between Drosophila strains, differing their capacity for supporting multiplication of sigma virus, arises essentially from comparison of the incubation time after inoculation of a viral suspension. This is the most general and the most useful characteristic. By this mean five allelic differences with the reference Drosophila strain Oregon have been found. Corresponding genes, ref(1)H, ref(2)M, ref(2)P, ref(3)O and ref(3)D are located all over Drosophila chromosomes. The specific spectra of viral strains sensitive to the one or the other allele was determined for each gene.Some characteristic properties of flies in which the virus has been brought by injection or heredity were compared between heterozygotes and homozygotes for the permissive and for the non permissive allele:time of incubation as a function of the size of the inoculum,probability of initiating infection,kinetics of the virus multiplication in inoculated fly,efficiency of a viral genome brought by a spermatozoa in infecting an egg,perpetuation of the carrier state of sigma virus in germ line cells of stabilized females or males and in somatic cells.The properties concerning the perpetuation of sigma virus carrier state allow to distinguish two classes of viral functions in which the considered ref gene product can intervene: 1) functions necessary for viral genome replication and, of course, for perpetuation of carrier state, 2) other functions, (late functions — necessary for maturation - and functions necessary for cell penetration of inoculated virus).Homozygotes for each of the two alleles of a gene which acts on incubation time can show no difference in one property which is specific of a differenciated cell type only because the considered gene is not expressed in the cell type involved. Conversely genes can exist which act on such a property and which have no action on incubation time. Probably such a gene has been discovered; it intervenes in the transmission of sigma virus by stabilized males; this gene is named ref(3)V.Discussion of all the properties of flies homozygotes for each allele permits us to conclude that ref(1)H, ref(2)M, ref(2)P, ref(3)D and possibly ref(3)V genes (if this last gene intervenes directly in sigma's physiology) are involved in a function necessary for replication. No conclusive evidence has been found for ref(3)O, still it seems to intervene in a late function. Problems of functional interactions between products of the first five ref genes have been mentioned.