Ecf, an Alternative Sigma Factor from Neisseria gonorrhoeae , Controls Expression of msrAB , Which Encodes Methionine Sulfoxide Reductase

Abstract

A DNA microarray was used to identify genes transcribed in Neisseria gonorrhoeae using Ecf, an alternative sigma factor. No differences between the transcriptional profiles of strain FA1090 and a mutant where ecf had been inactivated could be detected when both were grown in vitro. We therefore constructed a gonococcal strain in which Ecf can be overexpressed. Some differentially expressed genes are clustered with ecf on the genome and appear to form a single transcriptional unit. Expression of the gene encoding MsrAB, which possesses methionine sulfoxide reductase activity, was also dependent on Ecf, suggesting that the regulon responds to oxidative damage. Western blotting confirmed that the increased level of MsrAB protein is dependent on the presence of Ecf.Bacterial sigma factors are essential components of the RNA polymerase holoenzyme and determine promoter selectivity and specificity. Bacteria usually contain at least one essential sigma factor, sigma-70, which is necessary for cell viability, as well as a number of accessory sigma factors that are often involved in responses to environmental stimuli or the phase of growth. The relative amount of RNA polymerase holoenzyme containing each sigma factor determines the amplitude of the expression of a specific collection of genes.We have searched the genome sequence of Neisseria gonorrhoeae strain FA1090 (GenBank accession number AE004969) for the presence of genes encoding sigma factors. As expected, there is a gene, rpoD, encoding sigma-70 (NGO0999). Only two intact genes encoding alternative sigma factors, rpoH (NGO0288) and ecf (NGO1944), were found. Laskos et al. (11) have shown that there is also an inactive RpoN-like sigma factor, RLS, although this sequence feature (NGO1766) has not been annotated as such in the publicly available annotations of the N. gonorrhoeae strain FA1090 genome sequence (GenBank accession number AE004969; annotations at www .stdgen.lanl.gov, cmr.tigr.org/tigr-scripts/CMR/CmrHomePage .cgi, and www.ncbi.nlm.nih.gov/genomes/lproks.cgi). The ecf gene encodes a member of the extracytoplasmic function (ECF) family of sigma factors. As the name suggests, ECF sigma factors from different bacterial species appear to respond specifically to a variety of extracytoplasmic stimuli. Characteristically, their activity is controlled by anti-sigma factors, and they control relatively small regulons (1). In most examples of this system investigated to date, the ECF sigma factors regulate not only their own expression but also that of the genes encoding the cognate anti-sigma factor, which are located in the same operon.We have used DNA microarrays to assess the role of the sigma factor Ecf in gonococci and show that it controls a small regulon which contains the msrAB gene, which encodes an unusual methionine sulfoxide reductase. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strain DH5␣ [F Ϫ endA1 thi-1 hsdR17 supE44 relA1 ⌬l...

Impact of Moderate Temperature Changes on Neisseria meningitidis Adhesion Phenotypes and Proteome

“…And yet, our finding that N. meningitidis seems to possess a functional E response to membrane stress is in conflict with reports that suggested a function of this sigma factor/anti-sigma factor system outside the periplasm (49,50). The E response in N. gonorrhoeae was suggested to be triggered by oxidative stress (63), and the levels of expression of the proteins MsrAB, AniA, and NspA, which are known to be regulated by E (49,63), were unchanged in our proteomic analysis. However, the latter studies (49,50,63) were all conducted using N. meningitidis grown to log phase, whereas we used mature overnight cultures for our analyses here, which might explain potential differences in the outcomes with respect to apparent E -dependent effects during distinct stages of N. meningitidis growth.…”

“…The E response in N. gonorrhoeae was suggested to be triggered by oxidative stress (63), and the levels of expression of the proteins MsrAB, AniA, and NspA, which are known to be regulated by E (49,63), were unchanged in our proteomic analysis. However, the latter studies (49,50,63) were all conducted using N. meningitidis grown to log phase, whereas we used mature overnight cultures for our analyses here, which might explain potential differences in the outcomes with respect to apparent E -dependent effects during distinct stages of N. meningitidis growth. Although we could show NMB2132 (NHBA) and NMB1969 (NalP) to be regulated on the transcript level, there was no effect of E in their regulation ( Fig.…”

New Functional Identity for the DNA Uptake Sequence in Transformation and Its Presence in Transcriptional Terminators

“…This may also influence expression levels of various proteins and provide a means of fine tuning such. Indeed, a recent report by Gunesekere and coworkers suggests that an inverted repeat sequence inside a gene cluster in N. gonorrhoeae allows partial read-through, resulting in two transcripts of different lengths (26). Although this particular inverted repeat found downstream of the gene of NGO1947 does not contain a DUS, it is comparable in length and has an identical GϩC content to a DUS-IR (up-stem sequence, 5Ј-AAGCCGCGUUUU-3Ј).…”

Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum

“…Reverse transcription was performed as described elsewhere (Gunesekere et al, 2006), except for the use of 1 mg RNA with 5.5 mg random hexamers. Reactions were stopped by heating to 70 uC for 15 min.…”