Identification of a novel anti-σE factor in Neisseria meningitidis

BMC Microbiology IRdeseearnchtairftiiccleation of a novel anti-E factor in Neisseria meningitidis Carla Th P Hopman 0 Dave Speijer 1 Arie van der Ende 0 Yvonne Pannekoek 0 0 Academic Medical Center, Center for Infection and Immunity Amsterdam (CINIMA), Department of Medical Microbiology , Amsterdam , the Netherlands 1 Academic Medical Center, Department of Medical Biochemistry , Amsterdam , the Netherlands Background: Fine tuning expression of genes is a prerequisite for the strictly human pathogen Neisseria meningitidis to survive hostile growth conditions and establish disease. Many bacterial species respond to stress by using alternative factors which, in complex with RNA polymerase holoenzyme, recognize specific promoter determinants. E, encoded by rpoE (NMB2144) in meningococci, is known to be essential in mounting responses to environmental challenges in many pathogens. Here we identified genes belonging to the E regulon of meningococci. Results: We show that meningococcal E is part of the polycistronic operon NMB2140-NMB2145 and autoregulated. In addition we demonstrate that E controls expression of methionine sulfoxide reductase (MsrA/MsrB). Moreover, we provide evidence that the activity of E is under control of NMB2145, directly downstream of rpoE. The protein encoded by NMB2145 is structurally related to anti-sigma domain (ASD) proteins and characterized by a zinc containing anti- factor (ZAS) motif, a hall mark of a specific class of Zn2+-binding ASD proteins acting as anti- factors. We demonstrate that Cys residues in ZAS, as well as the Cys residue on position 4, are essential for anti-E activity of NMB2145, as found for a minority of members of the ZAS family that are predicted to act in the cytoplasm and responding to oxidative stimuli. However, exposure of cells to oxidative stimuli did not result in altered expression of E. Conclusions: Together, our results demonstrate that meningococci express a functional transcriptionally autoregulated E factor, the activity of which is controlled by a novel meningococcal anti- factor belonging to the ZAS family. - Background RNA polymerase holoenzyme, consisting of a 5-subunit core RNA polymerase (2') and a dissociable subunit, sigma (), initiates bacterial transcription. The factor contains many of the promoter recognition determinants and several factors each recognizing their specific class of promoter sequences have been described [1-5]. In general, in exponentially growing bacteria transcription is initiated by RNA polymerase carrying the housekeeping , known as 70 [6]. Alternative factors mediate transcription of regulons activated under specific environmental conditions [7,8]. The activity of many alternative s is inhibited by a specific anti- factor. In a wide variety of bacterial species the factor E,, also known as extracytoplasmic factor or ECF, belonging to the group IV s, is essential in mounting responses to environmental challenges such as oxidative stress, heat shock, and misfolding of membrane proteins [9,10]. In addition, E is of importance for virulence of bacterial pathogens [11-22]. The regulon size of E varies widely among bacterial species studied, ranging from 89 unique E controlled transcription units in E. coli and related bacteria [23] to a relatively small regulon of 5 genes in Neisseria gonorrhoeae [24]. In most examples, the gene encoding E (rpoE) is located in an autoregulated operon that also contains, directly downstream of rpoE, the gene encoding its cognate anti-E factor [25-28]. Extensive sequence analysis showed that about one third (1265/3600) of known and predicted anti-group IV factors, encoded in a gene cluster with a group IV (with only one exception), contain a conserved structural N-terminal fold, recently described as the anti-sigma domain (ASD) [26]. Typically, the ASD is in the N-terminus, oriented towards the cytoplasm, preceding a C-terminal transmembrane 588 nt rpoE 2144-01 segment. However, 20% of the 1265 ASD containing proteins are not predicted to contain a transmembrane spanning C-terminal domain [26]. Among these, 95% (227/ 248) are characterized by the presence of an invariant Hisx3Cysx2Cys sequence motif important for anti-sigma activity, co-ordinating Zn2+, described as the zinc containing anti- factor (ZAS) group IV anti-s proteins [29]. ASD proteins and ASD proteins containing the ZAS motif are predicted to bind specifically to s and inhibit their activities [25-28]. The strictly human pathogen Neisseria meningitidis colonizes the nasopharynx of approximately 10 to 30% of the population. In rare instances colonization results in invasive disease leading to life-threatening septicemia and meningitis [30]. Meningococci possess a variety of genes involved in adaptation to specific changes in the environment encountered in the host [31-36]. In addition to nutrient limitation, meningococci are also exposed to massive amounts of reactive oxygen species produced by host defenses [37,38]. Fine tuning expression of genes required to survive hostile growth conditions is a prerequisite for the meningococcus to establish disease. All four publicly available, completely sequenced genomes of N. meningitidis contain a gene (NMA0233, NMB2144, NMC2123 and NMCC2103) encoding a protein with homology to E, the factor involved in stress responses [39-42]. In this study we explored the E regulon of N. meningitidis. In addition, we provide evidence that the expression of E (encoded by NMB2144) in meningococci is autoregulated and that its activity is under control of a protein encoded directly downstream of rpoE. This protein, encoded by NMB2145, is structurally related to ASD proteins and contains the ZAS motif (His30x3Cys34x2Cys37). We demonstrate that the Cys residues in the ZAS motif, as well as a Cys on position 4, are important (Cys4 and C37) or essential (Cys34) for anti-E activity of NMB2145. Results The gene cluster containing rpoE is transcribed as a polycistronic operon and transcriptionally regulated by E In many bacterial species, rpoE is part of an autoregulated polycistronic operon also encoding its cognate antisigma factor [25-28]. In meningococci, NMB2144 is annotated as rpoE, encoding a protein with a molecular weight of approximately 23 kDa, 98% identical to the E orthologue of N. gonorrhoeae [24] and 28% identical to E of E. coli. Meningococcal rpoE is part of a 3 kb cluster of genes NMB2140 through NMB2145 (Fig.1a) having a genomic arrangement similar to that found in N. gonorrhoeae [24]. All genes, except NMB2144, are annotated as hypothetical proteins. The minimal spacing found in the cluster suggests co-transcription of its genes. Figure 1 Transcriptional analysis of the NMB2140-NMB2145 region. A) Schematic representation of the organization of the NMB2140-NMB2145 region. Genes are indicated as open arrows that show the orientation and relative sizes of the putative ORFs. Primers used in RT-PCR are indicated by closed arrows. Sizes o (...truncated)