| Collection Mémoires et thèses électroniques | |
| AccueilÀ proposNous joindre |
| Chapitre 2 Régulation transcriptionnelle chez Pseudomonas aeruginosa | ||
|---|---|---|
 |  | |
Table des matières
Le chapitre 1 constitue un article de revue soumis pour évaluation à la revue Molecular Microbiology . Cet article regroupe les principales données connues ainsi que les récentes découvertes concernant la régulation transcriptionnelle de Pseudomonas aeruginosa basée sur les facteurs sigma en mettant l’emphase sur les facteurs sigma de type ECF (extracytoplasmic functions). La rédaction de ce manuscrit a été réalisée par moi-même selon une idée originale et de judicieux conseils du Dr Levesque.
Chez Escherichia coli , la transcription est dépendante de 7 facteurs sigma : Un facteur sigma-70 essentiel, σ70 , ainsi que de six facteurs alternatifs dispensables, σ28 , σ32 , σ38 , σ54 , σE et σFecI . La transcription des gènes de E. coli est modulée par la présence de plus de 350 facteurs transcriptionnels de différentes familles souvent décrites pour la première fois chez cette bactérie. Par contre, l’analyse du chromosome de P. aeruginosa a révélé la présence de 24 facteurs sigma parmi les 5570 cadres de lecture ouverts du génome. P. aeruginosa possède aussi un facteur sigma essentiel très homologue à σ70 de E. coli (rpoD) ainsi qu’un seul facteur sigma-54 (rpoN) aussi très homologue à σ54 de E. coli. P. aeruginosa possède également un homologue de σ32, rpoH, impliqué entre autres dans la réponse au choc thermique. Aussi présents sur le chromosome de P. aeruginosa, rpoS, un homologue de σ38 impliqué dans la communication cellule-cellule au début de la phase stationnaire de croissance. Tous les autres facteurs sigma de P. aeruginosa sont de la sous-famille des ECF et regroupe fpvI, pvdS, algU, sigX et 15 autres inconnus.
In eubacteria, the expression of genes is tightly controlled by many transcriptional regulators. Basically, the primary description of all these regulators originated from the well-known bacterium Escherichia coli. The arsenal of transcriptional regulatory proteins of E. coli includes 7 sigma factors which are involved in core RNA polymerase interaction and promoter recognition. E. coli also encodes up to 350 transcriptional regulators from different families to modulate the expression of these genes. Now, five years have passed since the releasing of the genome sequence of the opportunistic pathogen Pseudomonas aeruginosa strain PAO1. Information acquired from the whole sequence of PAO1 revealed the presence of 24 putative sigma factors, 19 of which are from the ECF (extracytoplasmic functions) subfamily. This paper reviews all the actual knowledge regarding the sigma factors of P. aeruginosa with a particular emphasis on the known and unknown ECF members.
Scientific research over the past decade has doubtlessly revealed an awesome quantity of information generated by the genomic effort. To date, the National Center for Biotechnology Information (NCBI) has published 229 complete microbial genomes and 373 genomes in progress since the Haemophilus influenzae sequence was completed in 1995 (http://www.ncbi.nlm.nih.gov/genomes/). In addition to the challenge of understanding the function of all genes annotated in a defined genome, a difficult challenge is coming up-understanding global gene regulation. Since the appearance of new global transcript profiling technologies such as Microarrays or, more recently RNAi-based regulation, we now have the potential to make such an attempt. Comparative genomics could presumably reconstruct metabolic networks. However, the analysis of transcriptional regulatory networks are hindered by the low level of evolutionary conservation of transcriptional regulators (Herrgard et al., 2004). An additional hurdle is the distribution of the genes encoding these proteins widely throughout the genome. The opportunistic pathogen Pseudomonas aeruginosa strain PAO1, encoding one of the largest eubacterial sequenced genomes (6.3Mb), is one of the prototypical examples of the complex network of regulation controlling gene expression. P. aeruginosa is known for its incredible metabolic diversity and capacity for adaptation. As an opportunistic pathogen, P. aeruginosa has a remarkable capacity to infect a large array of life-forms including mammals, insects and plants (Lyczak et al., 2000). The exquisite control of P. aeruginosa gene expression relies on a collection of transcriptional regulators, sigma factors and more precisely, those members of the ECF subfamily. This paper presents a compilation of sigma factors and transcriptional regulators involved in the reconnaissance of gene promoters retrieved from the complete sequence of P. aeruginosa PAO1 strain.
The basic initiation of eubacterial transcription is performed by the multisubunit RNA polymerase (α2ββ’ω) that binds to a dissociable sigma (σ70) factor (Helmann and Chamberlin, 1988). The sigma-70 (70 kDa) factors are responsible for promoter recognition at -10 and -35 bp DNA elements and the melting of double stranded DNA when it binds to the core RNA polymerase (Gruber and Gross, 2003). Escherichia coli possesses one housekeeping sigma subunit that directs most of the transcription during exponential growth and a pool of 6 alternatives sigma factors implicated in the transcription of specific regulons associated with environmental or physiological changes (σ28, σ32, σ38, σ54, σE and σFecI) (Blattner et al., 1997).
On the basis of gene structure and function, members of the sigma-70 family can be divided into four main groups (Paget and Helmann, 2003). The first group consists of the essential primary sigma factors and are closely related to σ70 of E. coli. The second group include members also closely related to σ70 but which are usually dispensable. The proteins from the third group are more distantly homologous to σ70 and are known to activate specific regulons associated with heat-shock or morphological developmental stages such as bacterial sporulation or flagella biogenesis. The fourth group encompasses the largest family and includes the highly divergent extracytoplasmic function factors (ECF) subfamily. This fourth group includes proteins which respond to signals from the extracytoplasmic environment, including detection of misfolded periplasmic proteins (Wosten, 1998).
E. coli has an additional sigma factor, σ54 (54 kDa) which is the only sigma factor that is not homologous to σ70(Merrick, 1993). Although most eubacteria encode multiple proteins of the sigma-70 family, they usually have no more than one representative of the sigma-54 family (Gruber and Gross, 2003). Transcription from σ54 has distinctive characteristics such as the obligatory presence of a cognate ATP-dependent transcriptional activator which interacts with the sigma-54 subunit and a specific DNA sequence (Buck et al., 2000). Hence, transcription from sigma-54 promoters can be entirely turned-off in the absence of the transcriptional activator. σ54-like proteins are widespread among bacteria and are required for many functions such as nitrogen assimilation, phage-shock response and zinc tolerance (Reitzer, 2003). On another level of complexity, E. coli has more than 350 transcriptional regulators that can modulate gene expression to severely control levels of transcripts. These regulators are regrouped in several families based on sequence similarities and information available in literature and include LuxR/UhpA, OmpR, GalR/LacI, LysR, AraC/XylS, ArsR and CRP families (Perez-Rueda and Collado-Vides, 2000).
The regulation of the complex P. aeruginosa genome (5570 ORFs) is presumably orchestrated by a high number of transcriptional regulators and two-component regulatory systems as identified by bioinformatics analysis and genome annotation. In theory, these regulatory elements could explain why this bacterium is retrieved in a wide range of environmental niches. When compared with other bacterial systems, the most striking over-representations in P. aeruginosa are LysR, AraC, ECF sigma factors and two-component regulatory families (Stover et al., 2000). Two-component systems are usually associated with response to environmental changes as well as regulation of expression of virulence traits. Two-component systems are often composed of a sensor kinase and a response regulator which is phosphorylated by the sensor kinase via a transducing signal following an environmental stimulus. The phosphorylated regulator activates the expression of necessary genes for the appropriate response. A recent review described the genetics and biochemistry of these systems. The repertoire of sensor kinases annotated in the PAO1 genome regroups 63 histidine kinases and 64 putative response regulators. A total of 16 atypical kinases, among them 11 with an HPt (Histidine-phosphotransferase) domain and three with independent HPt module have been identified (Rodrigue et al., 2000). The HPt domain, found in many signal transduction proteins, functions as a mediator of the His-Asp phosphorylation (Yaku et al., 1997). These two-component systems based regulation suggests that P. aeruginosa encodes complex control strategies with which to respond to environmental challenges. Regulation of the P. aeruginosa genome is also characterized by the presence of approximately 120 LysR-type regulators most of which have unknown functions (http://www.pseudomonas.com). For instance, PtxR and MvaT, two LysR-type regulators, were recently shown to modulate the expression of toxA, the exotoxin A coding gene (Carty et al., 2003; Westfall et al., 2004). Fifty-nine putative transcriptional regulators are also predicted to possess an AraC/XylS family signature with genes distributed all over into the PAO1 chromosome, including ArgR elements, the arginine-responsive regulator that controls arginine uptake and metabolism (Gallegos et al., 1997; Lu et al., 2004). The other families of regulatory proteins are the LuxR-type including, GacA, LasR and RhlR and GntR, IclR, TetR-type families.
Sigma factor σ54 (or RpoN) is the unique sigma-54 like transcriptional regulator protein found in the P. aeruginosa chromosome to date. To proceedwith initiation of transcription, the RNAP-RpoN complex must participatein an interaction with a transcriptional activator, involvingnucleotide hydrolysis. The Pseudomonas databases predicted 22 proteins having the sigma-54 ATP binding region. Among them, FleQ and FleR are involved in the biosynthesis of flagellin, the major component of the single flagella of P. aeruginosa (Dasgupta et al., 2003), PilR for pilin biosynthesis (Mattick et al., 1996) and AlgB that regulates alginate exopolysaccharide production (Ma et al., 1998).
P. aeruginosa like other gram-negative bacteria has a major sigma-70 factor RpoD that recognizes a large number of promoters controlling expression of housekeeping genes. The RpoD protein has extensive homology with the principal Escherichia coli σ70, indicating that the σ70 has an identical function. It was shown that the P. aeruginosa σ70 can complement a temperature sensitive mutation of the E. coli rpoD gene (Tanaka and Takahashi, 1991). RpoD dependent transcription is essentially performed during the exponential phase of growth (Fujita et al., 1994). The rpoD gene is transcribed from two promoters, PC and PHS. Synthesis of rpoD mRNA from PC is constitutive under both steady-state and heat-shock growth conditions, while that of PHS is transiently induced upon heat-shock (Aramaki and Fujita, 1999). The promoter consensus sequences recognized by RpoD in the -35/-10 region of the transcriptional initiation site are highly similar to those from E. coli σ70. Hence, the E. coli -10 element (TATAAT) and the P. aeruginosa (TAtAAT) are highly similar; whereas the -35 element of P. aeruginosa (TTGaCc) is slightly different from the E. coli consensus (TTGACA) (bases in bold uppercase letters and plain uppercase letters are present in more than 50% and 40% of the sequences, respectively, whereas bases in lower case letters are present in more than 30% of the sequences over 149 RpoD-dependent promoters aligned together)(Ramos, 2004).
Sequence alignment of σ70 like proteins from different genus of eubacteria led to the identification of four highly conserved regions in amino acid composition (Helmann and Chamberlin, 1988). These regions, 1 to 4, have been subdivided further with the accumulation of sequence data (Lonetto et al., 1992). Region 1, the less conserved, is divided into two distinct regions 1.1 and 1.2. Region 1.1 is found only in the primary sigma-70 factors, such as σ70 and RpoD, and was shown to be involved in the modulation of DNA binding and in the efficiency of initiation of transcription. Region 1.2 is probably involved in the formation of an open complex. This region is present in all sigma-70 subfamilies except for the ECF subfamily of proteins (Wilson and Dombroski, 1997). Region 2, the most conserved, is divided into 4 subregions 2.1, 2.3, 2.4 and 2.5. Region 2.1 is involved in core RNAP recognition, region 2.3 in melting of DNA, region 2.4 recognize the -10 element and region 2.5 recognizes a -14/-15 element in E. coli and -16 element in Bacillus subtilis (Harley and Reynolds, 1987; Voskuil et al., 1995). Regions 3 and 4 are both divided into two subregions. Region 3.1 contains a helix-turn-helix DNA-binding motif and the less conserved 3.2 region may be involved in binding the RNAP core enzyme (Zhou and Gross, 1992). Region 4.1 binds transcriptional activators during initiation of transcription and region 4.2 recognizes the -35 element (Harley and Reynolds, 1987). Upon comparison of both, P. aeruginosa RpoD and E. coli σ70, it is noteworthy that regions 2.4 and 4.2, involved in interaction at -10/-35 promoter elements, are 100% identical. The identities found in these regions involved in promoter specificity are in agreement with the cross-recognition of the RpoD-dependent and σ70-dependent promoters assayed to date. Moreover, when necessary, E. coli RNAP responds to activation-repression mechanisms provided that the corresponding regulatory gene is also present (Ramos, 2004).
The similarities in gene regulation between E. coli and P. aeruginosa regarding rpoD and σ70 can be extrapolated with experimental data to another σ70-like factor, RpoH, which shows 61% identity to the σH (sigma 32) protein of E. coli (Benvenisti et al., 1995). In E. coli, σH is responsible for the heat-shock induction response in the upshift of temperature from 30°C to 42°C. σH is required for positive regulation of heat-shock genes as well as the basal expression of more than 20 heat shock proteins, which are molecular chaperones, including DnaK, DnaJ, GrpE, GroEL and GroES as well as specific proteases (Arsene et al., 2000). Transcription of P. aeruginosa rpoH gene was shown to be dependent on AlgU, an homolog of E. coli σE. The AlgU-dependent promoter of rpoH was found to be activated in mucoid mucA mutants, suggesting that conversion to mucoidy and the heat-shock response are co-ordinately regulated in P. aeruginosa (Schurr and Deretic, 1997). A number of σH homologs have been cloned from Gram-negative bacteria that belong to the gamma or the alpha subdivisions of proteobacteria; and it has been reported that the expression of these foreign homologues into E. coli ΔrpoH activated the transcription of DnaK and GroEL from the start sites normally used in E. coli by σH (Nakahigashi et al., 1998). The level of rpoH transcription in P. aeruginosa cells was found to be very low at 30°C but was markedly elevated upon a temperature shift to 42°C, an observation previously made with E. coli. The increased levels of RpoH upon heat shock treatment resulted from both the increased synthesis and stabilization of the normally unstable RpoH protein (Nakahigashi et al., 1998).
Another alternative sigma factor described in several bacterial genera and found in P. aeruginosa is the sigma-28 like FliA (RpoF). The fliA gene was first identified in P. aeruginosa by using an heterologous probe from Salmonella typhimurium. Amino acid sequence analysis revealed that both proteins shared up to 67% similarity. The major function associated with FliA in P. aeruginosa is the control of flagellin biosynthesis. In fact, the P. aeruginosa fliA gene was able to complement the motility defect of an E. coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa nonmotile; it was unable to express the flagellin gene (Starnbach and Lory, 1992). The 5' region of fliC, the structural gene of flagellin, contains potential RpoN-specific promoters as well as a promoter sequence recognized by FliA itself. Analysis of this promoter region as well as transcriptional start site mapping implicated FliA, and not the RpoN consensus sequences as the functional promoter of the flagellin gene (Totten and Lory, 1990). In P. aeruginosa, recent studies on the regulation flagellin synthesis identified the flgM gene encoding the anti-sigma 28 factor. The role for the flgM gene in motility was demonstrated by its inactivation. The beta-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of FlgM with the alternative sigma factor FliA was demonstrated by utilizing the yeast two-hybrid system (Frisk et al., 2002). Molecular mechanisms that control the expression of fliA remain unknown but transcription appears to be constitutive and independent of RpoN or other flagellar regulator such as FleQ or FleR (Dasgupta et al., 2003).
The functions attributed to the RpoS alternative sigma factor in P. aeruginosa has been recently compared to its homolog (σs) in E. coli and shown to be quite different (Venturi, 2003). Moreover, recent data concerning the linking of the RpoS regulon with the quorum sensing circuit makes it one of the best characterized alternative sigma-70 factor in P. aeruginosa (Schuster et al., 2004; Whiteley et al., 2000). Globally, RpoS has effects at the onset of the stationary phase of growth and is clearly involved in the regulation of quorum sensing via the modulation of the transcriptional regulators RhlR and LasR (Latifi et al., 1996). The secretion of autoinducer molecules implicated in the cell-to-cell signaling system of P. aeruginosa also directs the formation of biofilm; all these morphological and physiological changes are RpoS dependent (Davies et al., 1998). RpoS was shown to be involved in secretion of virulence factors such as alginate, exotoxine A, LasA and LasB elastases and exoenzyme S (Hogardt et al., 2004; Sonnleitner et al., 2003; Suh et al., 1999). RpoS is also known to be important for survival under stressful conditions. In fact, stationary-phase cells of RpoS-negative strains were less resistant to the exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol (Cochran et al., 2000; Jorgensen et al., 1999).
In P. aeruginosa, RpoN is the only member of the sigma-54 like family that is a typical case in eubacteria. As a general rule for most alternative sigma factors of P. aeruginosa, the amino acid sequence of the rpoN product from P. aeruginosa shares 67% similarity with the sequence of σ54 of E. coli. Initial observations on RpoN functions were linked solely to nitrogen assimilation. However, the discovery of additional genes transcribed by RNAP complexed with RpoN that are not a necessary part of the nitrogen metabolism pathway suggest novel functions for RpoN (Gussin et al., 1986). In fact, these functions have been identified as motility, transport of nutrients, formation of pili, mucoidy and cell-to-cell signalling (Boucher et al., 2000; Dasgupta et al., 2003; Heurlier et al., 2003; Ishimoto and Lory, 1989; Mattick et al., 1996; Strom and Lory, 1993; Thompson et al., 2003; Totten et al., 1990). By modulating different virulence determinants, RpoN has been strongly linked to the virulence of P. aeruginosa as well as to its ability to efficiently colonize several hosts including mammals, insects, nematodes and plants (Hendrickson et al., 2001). All these RpoN-regulated genes have a consensus promoter sequence of -24(GG)/-12(GC); their expression required at least one transcriptional activator (Thony and Hennecke, 1989). The formation of pili and flagella in P. aeruginosa is under the control of RpoN. All rpoN mutants showed significant reduction of adherence to epithelial cells and tracheobronchial mucin (Chi et al., 1991; Ramphal et al., 1991; Simpson et al., 1992). RpoN mutants have been used as controls defective in adherence in many adhesion experiments and infection models (Comolli et al., 1999). Moreover, RpoN negatively regulates the expression of sadB, an essential gene for surface adhesion (Caiazza and O'Toole, 2004). The interaction of RpoN with PilR, a member of a two-component transcriptional regulatory system, was shown to control the expression of type 4 fimbriae (Mattick et al., 1996). The presence of pilRS and rpoN genes is required for the expression of pilA, the structural gene for type IV pilin, whereas any of these are essential for pilBCD expression (Boyd and Lory, 1996; Koga et al., 1993). Flagella, as well as type IV pili, are also important structures involved in attachment and RpoN was shown to control flagella-based adhesion and motility (Totten et al., 1990). Studies indicate that both motility and adhesion are regulated by a two-component regulatory system called fleRS, which in turn is controlled by another regulator in a cascade that involves rpoN. A fleR mutant possessing pili adheres poorly to mucins, confirming that a flagellar protein and not pili play a major role in adhesion to mucin (Ramphal et al., 1996). Another transcriptional regulator, fleQ, also regulates mucin adhesion and motility in P. aeruginosa. Promoter fusions demonstrated that the expression of fleRS was dependent on RpoN but the expression of fleQ was RpoN-independent (Arora et al., 1997). RpoN coupled with the action of the transcriptional activator FleQ is involved in the expression of fliD, the flagellar cap protein involved in mucin adhesion (Arora et al., 1998; Jyot et al., 2002). RpoN obviously controls the quorum sensing system via a positive regulation of RhlI, the auto-inducer synthase responsible for the synthesis of N-butyryl-L-homoserine lactone (Thompson et al., 2003). Moreover, in rpoN mutants, it was shown that the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth (Heurlier et al., 2003). The RpoN alternative sigma factor is also implicated in the regulation of transporters. Deficiency in RpoN abolished the expression of oprE encoding a channel-forming outer membrane protein under aerobic conditions, but did not affect the expression under anaerobic conditions. One mutation on the putative RpoN recognition site also caused reduction of oprE expression. The regulation of oprE transcription is directly or indirectly controlled by RpoN but also requires some other regulatory proteins bound to the upstream region (Yamano et al., 1998). Hence, the sigma-54 like alternative sigma factor RpoN is implicated at different stages for the regulation of P. aeruginosa virulence factors. Mutations in rpoN in all cases leads to a reduction in virulence; this is visualized as defects in adhesion, motility and cell-to-cell communication observed in several models of infection.
In 2002, JD Helmann published an exhaustive paper on the extracytoplasmic function sigma factors (Helmann, 2002), regrouping the knowledge on the different ECF proteins across most bacterial genera. In 1994, Lonetto et al., described for the first time a novel subgroup of 8 proteins that showed similarity in amino acid sequences including Myxococcus xanthus CarQ, P. aeruginosa AlgU, Pseudomonas syringae HrpL, E. coli sigma E, Alcaligenes eutrophus CnrH, E. coli FecI, Bacillus subtilis SigX and Streptomyces coelicolor sigma E (Lonetto et al., 1994). As mentioned above, the ECF subfamily is a branch of the sigma-70 like sigma factors. ECF are small regulatory proteins that are quite divergent in sequence when compared to other sigma factors. At least three common characteristics are shared among all ECF members. The first one is they often recognize promoter elements with an (AAC) motif in the -35 region. In many cases the ECF sigma factor is co-transcribed with a transmembrane anti-sigma factor having an extracytoplasmic sensory domain and an intracellular inhibitory domain. Finally and most important, they are mainly associated with extracellular functions. Among these functions, we find regulation of periplasmic stress and heat-shock (σE), iron transport (σFecI), metal ion efflux system (CnrH), alginate secretion (AlgU) and synthesis of membrane-localized carotenoids (CarQ). The analysis of completed microbial genomes revealed a correlation between genome size and the number of predicted genes encoding sigma factors. In fact, the model actinomycete S. coelicolor A3(2) (8.7 Mb) encodes an incredible 65 putative sigma factors and at least 45 proteins of the ECF subfamily; these proteins are potentially responsible for a response to disulphide stress, cell wall homeostasis and aerial mycelium development (Bentley et al., 2002). The 6.3 Mb P. aeruginosa genome encodes 24 sigma factors, among which, 19 are from the ECF subfamily. Only three have a characterized function: AlgU is involved in alginate biosynthesis (Schurr et al., 1996), PvdS is involved in iron regulation responsible for biosynthesis of pyoverdine (Cunliffe et al., 1995) and SigX was shown to control the expression of the major outer membrane protein OprF (Brinkman et al., 1999).
AlgU shares a striking 79% sequence similarity with E. coli σE, a heat-shock protein produced in response to growth at elevated temperatures. In P. aeruginosa, AlgU has been primarily identified for its importance in conversion from the non-mucoid to the mucoid phenotypes (Martin et al., 1993a; Schurr et al., 1996). It was also shown to be involved in resistance to oxidative and heat shock stress (Martin et al., 1994; Schurr and Deretic, 1997). AlgU can interact with a DNA region upstream of the algD gene, the key regulatory enzyme in the alginate biosynthesis pathway that leads to the conversion to mucoidy (Martin et al., 1993a). Circumstantial information has accumulated by which non-mucoid strains convert into mucoid strains and are associated with the establishment of chronic lung infections in CF patients (Govan and Deretic, 1996). Four other genes, mucABCD are involved in regulation, (Boucher et al., 1996; Schurr et al., 1996). MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy (Schurr et al., 1996). It has been demonstrated that mucA plays a critical role in the process of mucoidy conversion by (i) the presence of frameshift mutations disrupting the mucA coding region in mucoid cells that were absent in non-mucoid parental strains, (ii) genetic complementation of mucA mutations with the mucA+ gene, (iii) allelic exchanges with specific mutant mucA genes causing conversion to mucoidy in previously non-mucoid cells, and (iv) detection of identical and additional mucA mutations in clinical mucoid strains isolated from the lungs of CF patients. This suggested that the inactivation of mucA results in constitutive expression of the alginate pathway dependent on algU transcription, and that such mutants may be selected in vivo during chronic infections in CF (Martin et al., 1993a). It was also suggested that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. Mathee et al. reported that polymorphonuclear leukocytes and their oxygen radicals can cause the phenotypic and genotypic changes which are typical of the intractable form of P. aeruginosa found in the CF lung (Mathee et al., 1999). MucB is also known to be a negative regulator of AlgU function. The loss of mucB function is sufficient to cause conversion of P. aeruginosa to the mucoid phenotype (Martin et al., 1993b). Recently, the screening of a large P. aeruginosa mutant library constructed by the Signature-Tagged Mutagenesis (STM) approach in the rat lung showed that algU is essential in vivo for maintenance of P. aeruginosa in the rat lung (Potvin et al., 2003). In fact, the conversion to the mucoid phenotype is associated with the maintenance of a long term lung infection in CF patients (Ramsey and Wozniak, 2005). The establishment of these chronic infections in CF lungs was also shown to be mediated by the repression of the type III secretion system via the upregulation of algU in a mucA mutant background (Wu et al., 2004). An exhaustive study from Firoved et al. using transcriptome profiling focused on the analysis of AlgU dependent promoters (GAACTTN16-17TCcaA) by an in silico genomics approach identified 35 new genes regulated by AlgU (sigmulon). They suggested the existence of a previously unknown connection between conversion to mucoidy and the expression of lipoproteins with potential pro-inflammatory activity. This link may be of significance for infections and inflammatory processes in CF (Firoved et al., 2002). Recently, Firoved and Deretic applied microarray analysis on a mucA mutant to identify on whole-genome scale genes that were coinduced with the AlgU sigmulon upon conversion to mucoidy. Transcriptome analysis revealed co-induction of a specific subset of known virulence factors including the lasB elastase gene, the alkaline metalloproteinase gene aprA, and the protease secretion factor genes aprE and aprF as well as toxic factor (cyanide synthase) (Firoved and Deretic, 2003).
PvdS is probably the second most characterized sigma-70 ECF subfamily factor of P. aeruginosa. A number of common characteristics from ECF sigma factors have been shown to be shared by PvdS (Helmann, 2002; Wilson and Lamont, 2000). Nucleotide sequence analysis revealed that pvdS shows considerable similarity to σFecI of E. coli, a positive regulator for transcription of the fec (ferric citrate transport system) operon (Miyazaki et al., 1995). Other members of this subgroup include PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and are all controlled by the Fur (ferric uptake regulator) repressor. In all cases they activate transcription of genes for the biosynthesis or uptake of siderophores (Leoni et al., 2000). The major functions attributed to PvdS, recently reviewed (Vasil and Ochsner, 1999; Visca et al., 2002), included the regulation of pyoverdine siderophore biosynthesis (Cunliffe et al., 1995), exotoxine A (Ochsner et al., 1996) and the endoprotease PrpL (Wilderman et al., 2001). When grown under iron-deficient conditions, P. aeruginosa produces pyoverdine, a fluorescent yellow-green siderophore of low molecular weight. The pvdS gene is required for the expression of these pyoverdine synthesis genes. The pvdS gene is expressed only in iron-starved bacteria and is regulated via the Fur repressor protein (Cunliffe et al., 1995). Fur is a well known and crystallized holorepressor that interacts with the DNA operator sequences (fur box) to tightly control the expression of the iron-regulated subset genes (Ochsner et al., 1995). It was shown that in the presence of a sufficient amount of iron, Fur is able to repress the transcription of pvdS and consequently repressed the expression of pvdA, which encodes a key enzyme of the pyoverdine biosynthetic pathway (Leoni et al., 1996). The production of exotoxine A is also secreted in iron-limiting conditions. The exotoxine A structural gene, toxA, is regulated at the transcriptional level by the gene products of regAB. The expression of both toxA and regAB is repressed under iron-deficient conditions, suggesting a role for Fur in regulation of toxA expression. It was shown that the control of exotoxine A, which is related to iron concentration, is mediated by PvdS in vitro and in lung infections associated with CF (Ochsner et al., 2002). In a pvdS deletion mutant, exotoxine A was produced at low levels of less than 5% compared to wild-type, but still in response to iron starvation (Ochsner et al., 1996). These observations suggested that another regulatory mechanism, in addition to the Fur-PvdS system, was involved in iron regulation of exotoxine A production. This regulator is PtxR, a LysR type transcriptional regulator, which was shown to positively affect the transcription of toxA and regA (Hamood et al., 1996). Iron-regulated transcription of ptxR was demonstrated from a P2 promoter under microaerobic conditions, but not constitutive expression from another promoter called P1, was dependent on pvdS, even under aerobic conditions (Vasil et al., 1998). PtxR and PvdS were also shown to be implicated in the synthesis of pyoverdine via a positive regulation of the pvdABCD operon which is negatively regulated by high-iron conditions (Stintzi et al., 1999). Recently, the analysis of iron starvation response by the GeneChip technology lead to the identification of novel genes involved in the biosynthesis of pyoverdine (Ochsner et al., 2002). Under low-iron conditions, expression of 26 genes or operons was reduced in a pvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes (Ochsner et al., 2002). It is known that pyoverdine is able to chelate extracellular iron and the resulting pyoverdine iron-complexe is transported back into the cell via the FpvA transporter. The binding of iron-free pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning anti-sigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of PvdS (Lamont et al., 2002; Poole et al., 1993). It was shown that FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of pyoverdine complexed to iron significantly increases the activity of FpvI so that production of FpvA is induced. This is the only example of a branched signalling system of this sort and the first example of an anti-sigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI) (Beare et al., 2003).
SigX is the last sigma-70 factor of the ECF subfamily with an attributed function that has been demonstrated experimentally in P. aeruginosa. SigX shares 49% similarity to sigma-W of Bacillus subtilis which is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis (Schobel et al., 2004). The gene encoding OprF, a major outer membrane protein in Pseudomonas sp., was previously thought to be constitutively transcribed from a single RpoD promoter immediately upstream of the gene. In P. aeruginosa as well as in P. fluorescens, the sigX gene is located immediately upstream of oprF and the disruption of sigX in both species significantly reduces oprF expression (Brinkman et al., 1999).
Of the 24 putative and known sigma factors found in the entire PAO1 genome sequence, 15 have no predicted functions except for the common theme of belonging to the ECF subfamily (Table 2). These proteins were found using a keyword search with the Pseudomonas.com website along with the structural feature PS01063 from Prosite. These 15 putative sigma-70 factors, presumably belonging to the ECF subfamily were sorted according to their PA number. In Table 2, we indicated relevant characteristics including the predicted protein molecular mass, as calculated using the Compute pI/Mw program from the ExPASy Proteomics Server (www.expasy.org). We also included in Table 2, characterized and uncharacterized homologous proteins from other bacterial genera, and those found in P. aeruginosa. Homology is expressed as a similarity value (in percentage) including positives and identical residues found by using the BlastP program from National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). As mentioned in the literature, ECF sigma factors are often co-transcribed with a putative, inner-membrane anchored, sensor that could act as a signal transducer and a negative regulator (anti-sigma) (Helmann, 2002). The right portion of Table 2 regroups the main characteristics of these co-transcribed genes that we identify here as anti-sigma factors. PA numbers, molecular mass and homologues were identified and described. Moreover, we added an important set of data concerning putative positions of the transmembrane helix. Using the das program (www.sbc.su.se/~miklos/DAS), that measures hydrophobic potential, we significantly predicted in most cases at least one transmembrane segment. These putative transmembrane helices were confirmed by running topology scans with other known programs from the Expasy Proteomic Server.
The most striking feature of these unknown proteins is the high homology values shared with ECF sigma factors involved in regulation and uptake of ferric citrate (Visca et al., 2002). The ferric citrate transport system of E. coli involves three specific proteins: FecR, the inner-membrane sensor that transduces signals to FecI, the sigma factor, ECF subfamily that bind to core RNAP in the cytoplasm. FecI directs transcription from the promoter upstream of the fecABCDE transport genes. FecA, an outer-membrane protein, was also shown to be essential in the signal cascade (Braun and Braun, 2002). Moreover, transcription of the fecIR regulatory genes and the fec transport genes is repressed by the Fur protein when loaded with iron. Two fecIRA systems have been reported to date in P. aeruginosa, the fiuIRA (PA0470-0472) and fpvIRA. FiuA is an outer membrane receptor protein responsible for the transport of the ferrioxamine, a probable hydroxamate-type ferrisiderophore. FiuA is under the transcriptional control of FiuIR also responsible for signal transduction and transcriptional response to a ferridoxamine cue (Vasil and Ochsner, 1999). As described by Visca et al., 11 of the 15 unknown ECF sigma factors presented in Table 2, excluding PA1351, PA2093, PA2896 and PA3285, possess a significant match with a fur-binding sequence (GATAATGATAATCATTATC) (Visca et al., 2002). Moreover, many of these were experimentally confirmed to be regulated by the Fur repressor (Ochsner and Vasil, 1996). Homologies presented in Table 2 omit highly significant values from other reported systems linked to the metabolism of siderophores such as PupIR from Pseudomonas putida and FpvIR from P. aeruginosa. PupIR regulates the biosynthesis and metabolism of the Pseudobactin BN8 via PupB, which is the pseudobactin receptor. PupI and PupR display significant similarity to the FecI and FecR proteins of E. coli (Koster et al., 1994).
Transcriptome analysis associated with iron starvation revealed that 6 sigma-70 factors of the ECF subfamily were found to be highly regulated by iron starvation as well as their cognate putative transmembrane sensor (Ochsner et al., 2002). These are PA0471-0472, PA1300-1301, PA2467-2468, PA3409-3410, PA3899-3900 and PA4895-4896. Interestingly, none of them were shown to be differentially regulated by PvdS (Ochsner et al., 2002). Another recent study on the iron-starvation response published microarray results according to the iron-response specific to the exponential growth phase, whereas the previously reported work analysed the response during the stationary phase of growth (Palma et al., 2003). Similar results were obtained in both experiments concerning the unknown sigma-70 factors. Briefly, PA0471-0472, PA2467-2468 and PA3899-3900 were upregulated, while sigma-70 factors PA3410 and PA4896 only (not their cognate sensor). New genes identified as upregulated in this study were PA1363, PA1911 and PA2387 (Palma et al., 2003).
By regulating iron influx and export, as well as other cell envelope regulons such as the biosynthesis of alginate and exoproteases, ECF sigma-70 factor-based regulation plays a determining role in successful in vivo implantation (Bashyam and Hasnain, 2004). In order to associate some putative functions to the unknown sigma-70 factors, ECF subfamily of P. aeruginosa, many transcriptome experiments were done using growth in different culture conditions. Mashburn et al., published a study where in P. aeruginosa was shown to utilize Staphylococcus aureus cells as an iron source when co-cultured together in the rat peritoneum (Mashburn et al., 2005). In these experiments, they confirmed the implication of 6 sigma/sensors for the acquisition of iron primarily noted by Ochsner et al. In fact, all of these operons are induced in the rat peritoneum, an iron-starved environment. The same operons were highly repressed when co-cultured with S. aureus cells. PA0149, PA0675 and PA1911-1912 were also shown to be induced in vivo but not when co-cultured with S. aureus (Mashburn et al., 2005). Another study showed that some sigma-70 factors from the ECF subfamily were also important in the interaction with primary normal human airway epithelial (PNHAE) cells (Frisk et al., 2004). In fact, PA1300, PA2468, PA3899 and PA4896 were strongly repressed, as well as many other iron-regulated genes, after 12 h of interaction with the PNHAE cells. This suggested that these epithelial cells can serve also as an iron source for P. aeruginosa (Frisk et al., 2004). It is clear that a subgroup of these sigma-70 factors of unknown function and their cognate sensors (Table 2), not only possess significant homologies to characterized iron uptake regulators, but are highly involved in this kind of regulation.
The recently described VqsR transcriptional regulator of the LuxR family, encoded by PA2591, regulates several virulence factors. Transposon mutation of vqsR was shown to abrogate the production of quorum-sensing autoinducers, the secretion of exoproducts and diminished bacterial virulence in the Caenorhabditis elegans infection model (Juhas et al., 2004). Microarray analysis revealed that 7 ECF sigma factors are upregulated in a vqsR mutant including PA0149, PA0472, PA1350, PA1912, PA2468, PA3899 and PA4896, suggesting the linkage of VqsR with the regulation of iron-based virulence traits (Juhas et al., 2005). Those indirectly regulated by vqsR were also identified to be transcriptionaly promoted by the addition of hydrogen peroxide, except PA1300, PA1350 and PA3410 (Palma et al., 2004). It is known that P. aeruginosa must often overcome a high concentration of oxidants to successfully infect the human host (Palma et al., 2004). This indicated that the response of P. aeruginosa to hydrogen peroxide consists of an upregulation of protective mechanisms such as ECF-type sigma factors coupled to their cognate sensor. Interestingly, from all the unknown sigma factors listed in Table 2, none of them were found to be regulated by the quorum sensing regulon in neither the paper of Schuster et al. nor in the paper of Wagner et al. (Schuster et al., 2003; Wagner et al., 2004).
An exhaustive study on P. aeruginosa genes essential in vivo in the chronic rat lung infection model (Potvin et al., 2003) using the signature-tagged mutagenesis approach (STM) based on PCR screening (Lehoux et al., 2004) identified 148 genes attenuated in vivo. As previously reported, STM2895 was primarily identified to be defective in the secretion of exoproteases (Potvin et al., 2003). PA2895 in Table 2 is a putative transmembrane sensor of the unknown ECF-type sigma factor. In addition to the implication in extracellular function (exoproteases), PA2895 and PA2896 respond to almost all basic characteristics of ECF sigma factors. PA2896 and PA2895 genes are co-transcribed and PA2895 has a transmembrane helix prediction.
The recent data presented in Table 2 concerning the unknown ECF-type sigma factors of P. aeruginosa suggest an implication of several of these sigma factors in the response to iron availability and to hydrogen peroxide. However, the individual role and function of each has to be determined. In his review paper on ECF sigma factors, Helmann suggested different approaches to determine the exact function of an ECF-type transcriptional regulator (Helmann, 2002). First, a mutant strain of the factor under study may have a phenotype, such as PA2895-PA2896, which indicates exoprotease defects, providing indications to a putative function. Second, the identification of the target genes with known functions will suggest a regulation pattern that leads to a visible phenotype. Third, the identification of a specific environmental cue that causes growth defects or any other visible characteristics will also lead to a putative function. These stimuli can be heat, cold, oxidative, metal concentrations, osmolarity, host interaction, nutritional starvation, etc. Many basic techniques from genomics, proteomics and bioinformatics could serve in that kind of investigation.
In conclusion, the 24 sigma factors, more specifically the 19 ECF-type, found distributed all over the genome sequence of P. aeruginosa strain PAO1 certainly provide a key explanation for its remarkable abilities of adaptation. These systems work in association by expressing numerous target gene networks to respond to the different cues that will arise in a defined environment or physiological change. We have identified by informatics analysis in the P. aeruginosa genome sequence all alternative sigma factors and homologues used by the well-known bacterium E. coli. This search includes homologues of the housekeeping sigma factor RpoD, the only sigma-54 RpoN, the stationary phase dependent RpoS, the heat shock RpoH, RpoF for flagella biosynthesis, the pyoverdine siderophore regulator FpvI and RpoE (AlgU) for alginate synthesis and response to heat and oxidative shock. The major difference between these species is that P. aeruginosa encodes 17 additional ECF-type sigma-factors in addition to AlgU and FpvI as compared to E. coli. The well known Pvds which directs the transcriptional control of pyoverdine biosynthesis as well as exotoxine A, the endoprotease PrpL and SigX which has been recently implicated in transport complete the known sigma factor listing. The 15 remaining unknown ECF sigma factors were analysed in silico. Transcriptome data revealed that a majority of the uncharacterized ECF sigma factors were implicated in iron-associated regulation. Even though much of the information still has to be understood, the regulation of the known part of the ECF repertoire, including AlgU, PvdS and FpvI, provides general patterns to the functionality of the regulation-based ECF sigma factors in P. aeruginosa.
Research in RCL’s laboratory was supported by the Canadian Institutes of Health Research. Roger C. Levesque is a scholar of exceptional merit from Le Fonds de Recherche en Santé du Québec, Eric Potvin obtained a studentship from CIHR (STP5394).
Aramaki, H., and Fujita, M. (1999) In vitro transcription analysis of rpoD in Pseudomonas aeruginosa PAO1. FEMS Microbiol Lett 180: 311-316.
Arora, S.K., Ritchings, B.W., Almira, E.C., Lory, S., and Ramphal, R. (1997) A transcriptional activator, FleQ, regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner. J Bacteriol 179: 5574-5581.
Arora, S.K., Ritchings, B.W., Almira, E.C., Lory, S., and Ramphal, R. (1998) The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion. Infect Immun 66: 1000-1007.
Arsene, F., Tomoyasu, T., and Bukau, B. (2000) The heat shock response of Escherichia coli. Int J Food Microbiol 55: 3-9.
Bashyam, M.D., and Hasnain, S.E. (2004) The extracytoplasmic function sigma factors: role in bacterial pathogenesis. Infect Genet Evol 4: 301-308.
Beare, P.A., For, R.J., Martin, L.W., and Lamont, I.L. (2003) Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis. Mol Microbiol 47: 195-207.
Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O'Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., and Hopwood, D.A. (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417: 141-147.
Benvenisti, L., Koby, S., Rutman, A., Giladi, H., Yura, T., and Oppenheim, A.B. (1995) Cloning and primary sequence of the rpoH gene from Pseudomonas aeruginosa. Gene 155: 73-76.
Blattner, F.R., Plunkett, G., 3rd, Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B., and Shao, Y. (1997) The complete genome sequence of Escherichia coli K-12. Science 277: 1453-1474.
Boucher, J.C., Martinez-Salazar, J., Schurr, M.J., Mudd, M.H., Yu, H., and Deretic, V. (1996) Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA. J Bacteriol 178: 511-523.
Boucher, J.C., Schurr, M.J., and Deretic, V. (2000) Dual regulation of mucoidy in Pseudomonas aeruginosa and sigma factor antagonism. Mol Microbiol 36: 341-351.
Boyd, J.M., and Lory, S. (1996) Dual function of PilS during transcriptional activation of the Pseudomonas aeruginosa pilin subunit gene. J Bacteriol 178: 831-839.
Braun, V., and Braun, M. (2002) Iron transport and signaling in Escherichia coli. FEBS Lett 529: 78-85.
Brinkman, F.S., Schoofs, G., Hancock, R.E., and De Mot, R. (1999) Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens. J Bacteriol 181: 4746-4754.
Buck, M., Gallegos, M.T., Studholme, D.J., Guo, Y., and Gralla, J.D. (2000) The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor. J Bacteriol 182: 4129-4136.
Caiazza, N.C., and O'Toole, G.A. (2004) SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14. J Bacteriol 186: 4476-4485.
Carty, N.L., Rumbaugh, K.P., and Hamood, A.N. (2003) Regulation of toxA by PtxR in Pseudomonas aeruginosa PA103. Can J Microbiol 49: 450-464.
Chi, E., Mehl, T., Nunn, D., and Lory, S. (1991) Interaction of Pseudomonas aeruginosa with A549 pneumocyte cells. Infect Immun 59: 822-828.
Cochran, W.L., Suh, S.J., McFeters, G.A., and Stewart, P.S. (2000) Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine. J Appl Microbiol 88: 546-553.
Comolli, J.C., Hauser, A.R., Waite, L., Whitchurch, C.B., Mattick, J.S., and Engel, J.N. (1999) Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect Immun 67: 3625-3630.
Cunliffe, H.E., Merriman, T.R., and Lamont, I.L. (1995) Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor. J Bacteriol 177: 2744-2750.
Dasgupta, N., Wolfgang, M.C., Goodman, A.L., Arora, S.K., Jyot, J., Lory, S., and Ramphal, R. (2003) A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol Microbiol 50: 809-824.
Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H., Costerton, J.W., and Greenberg, E.P. (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280: 295-298.
Firoved, A.M., Boucher, J.C., and Deretic, V. (2002) Global genomic analysis of AlgU (sigma(E))-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis. J Bacteriol 184: 1057-1064.
Firoved, A.M., and Deretic, V. (2003) Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa. J Bacteriol 185: 1071-1081.
Frisk, A., Jyot, J., Arora, S.K., and Ramphal, R. (2002) Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas aeruginosa. J Bacteriol 184: 1514-1521.
Frisk, A., Schurr, J.R., Wang, G., Bertucci, D.C., Marrero, L., Hwang, S.H., Hassett, D.J., and Schurr, M.J. (2004) Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells. Infect Immun 72: 5433-5438.
Fujita, M., Tanaka, K., Takahashi, H., and Amemura, A. (1994) Transcription of the principal sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase. Mol Microbiol 13: 1071-1077.
Gallegos, M.T., Schleif, R., Bairoch, A., Hofmann, K., and Ramos, J.L. (1997) Arac/XylS family of transcriptional regulators. Microbiol Mol Biol Rev 61: 393-410.
Govan, J.R., and Deretic, V. (1996) Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60: 539-574.
Gruber, T.M., and Gross, C.A. (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57: 441-466.
Gussin, G.N., Ronson, C.W., and Ausubel, F.M. (1986) Regulation of nitrogen fixation genes. Annu Rev Genet 20: 567-591.
Hamood, A.N., Colmer, J.A., Ochsner, U.A., and Vasil, M.L. (1996) Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production. Mol Microbiol 21: 97-110.
Harley, C.B., and Reynolds, R.P. (1987) Analysis of E. coli promoter sequences. Nucleic Acids Res 15: 2343-2361.
Helmann, J.D., and Chamberlin, M.J. (1988) Structure and function of bacterial sigma factors. Annu Rev Biochem 57: 839-872.
Helmann, J.D. (2002) The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol 46: 47-110.
Hendrickson, E.L., Plotnikova, J., Mahajan-Miklos, S., Rahme, L.G., and Ausubel, F.M. (2001) Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice. J Bacteriol 183: 7126-7134.
Herrgard, M.J., Covert, M.W., and Palsson, B.O. (2004) Reconstruction of microbial transcriptional regulatory networks. Curr Opin Biotechnol 15: 70-77.
Heurlier, K., Denervaud, V., Pessi, G., Reimmann, C., and Haas, D. (2003) Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1. J Bacteriol 185: 2227-2235.
Hogardt, M., Roeder, M., Schreff, A.M., Eberl, L., and Heesemann, J. (2004) Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS. Microbiology 150: 843-851.
Ishimoto, K.S., and Lory, S. (1989) Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci U S A 86: 1954-1957.
Jorgensen, F., Bally, M., Chapon-Herve, V., Michel, G., Lazdunski, A., Williams, P., and Stewart, G.S. (1999) RpoS-dependent stress tolerance in Pseudomonas aeruginosa. Microbiology 145 ( Pt 4): 835-844.
Juhas, M., Wiehlmann, L., Huber, B., Jordan, D., Lauber, J., Salunkhe, P., Limpert, A.S., von Gotz, F., Steinmetz, I., Eberl, L., and Tummler, B. (2004) Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa. Microbiology 150: 831-841.
Juhas, M., Wiehlmann, L., Salunkhe, P., Lauber, J., Buer, J., and Tummler, B. (2005) GeneChip expression analysis of the VqsR regulon of Pseudomonas aeruginosa TB. FEMS Microbiol Lett 242: 287-295.
Jyot, J., Dasgupta, N., and Ramphal, R. (2002) FleQ, the major flagellar gene regulator in Pseudomonas aeruginosa, binds to enhancer sites located either upstream or atypically downstream of the RpoN binding site. J Bacteriol 184: 5251-5260.
Koga, T., Ishimoto, K., and Lory, S. (1993) Genetic and functional characterization of the gene cluster specifying expression of Pseudomonas aeruginosa pili. Infect Immun 61: 1371-1377.
Koster, M., van Klompenburg, W., Bitter, W., Leong, J., and Weisbeek, P. (1994) Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope. Embo J 13: 2805-2813.
Lamont, I.L., Beare, P.A., Ochsner, U., Vasil, A.I., and Vasil, M.L. (2002) Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 99: 7072-7077.
Latifi, A., Foglino, M., Tanaka, K., Williams, P., and Lazdunski, A. (1996) A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21: 1137-1146.
Lehoux, D.E., Sanschagrin, F., Kukavica-Ibrulj, I., Potvin, E., and Levesque, R.C. (2004) Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies. Methods Mol Biol 266: 289-304.
Leoni, L., Ciervo, A., Orsi, N., and Visca, P. (1996) Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity. J Bacteriol 178: 2299-2313.
Leoni, L., Orsi, N., de Lorenzo, V., and Visca, P. (2000) Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa. J Bacteriol 182: 1481-1491.
Lonetto, M., Gribskov, M., and Gross, C.A. (1992) The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol 174: 3843-3849.
Lonetto, M.A., Brown, K.L., Rudd, K.E., and Buttner, M.J. (1994) Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci U S A 91: 7573-7577.
Lu, C.D., Yang, Z., and Li, W. (2004) Transcriptome analysis of the ArgR regulon in Pseudomonas aeruginosa. J Bacteriol 186: 3855-3861.
Lyczak, J.B., Cannon, C.L., and Pier, G.B. (2000) Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2: 1051-1060.
Ma, S., Selvaraj, U., Ohman, D.E., Quarless, R., Hassett, D.J., and Wozniak, D.J. (1998) Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa. J Bacteriol 180: 956-968.
Martin, D.W., Holloway, B.W., and Deretic, V. (1993a) Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor. J Bacteriol 175: 1153-1164.
Martin, D.W., Schurr, M.J., Mudd, M.H., and Deretic, V. (1993b) Differentiation of Pseudomonas aeruginosa into the alginate-producing form: inactivation of mucB causes conversion to mucoidy. Mol Microbiol 9: 497-506.
Martin, D.W., Schurr, M.J., Yu, H., and Deretic, V. (1994) Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response. J Bacteriol 176: 6688-6696.
Mashburn, L.M., Jett, A.M., Akins, D.R., and Whiteley, M. (2005) Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture. J Bacteriol 187: 554-566.
Mathee, K., Ciofu, O., Sternberg, C., Lindum, P.W., Campbell, J.I., Jensen, P., Johnsen, A.H., Givskov, M., Ohman, D.E., Molin, S., Hoiby, N., and Kharazmi, A. (1999) Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145 ( Pt 6): 1349-1357.
Mattick, J.S., Whitchurch, C.B., and Alm, R.A. (1996) The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa--a review. Gene 179: 147-155.
Merrick, M.J. (1993) In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N). Mol Microbiol 10: 903-909.
Miyazaki, H., Kato, H., Nakazawa, T., and Tsuda, M. (1995) A positive regulatory gene, pvdS, for expression of pyoverdin biosynthetic genes in Pseudomonas aeruginosa PAO. Mol Gen Genet 248: 17-24.
Nakahigashi, K., Yanagi, H., and Yura, T. (1998) Regulatory conservation and divergence of sigma32 homologs from gram-negative bacteria: Serratia marcescens, Proteus mirabilis, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. J Bacteriol 180: 2402-2408.
Ochsner, U.A., Vasil, A.I., and Vasil, M.L. (1995) Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol 177: 7194-7201.
Ochsner, U.A., Johnson, Z., Lamont, I.L., Cunliffe, H.E., and Vasil, M.L. (1996) Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor. Mol Microbiol 21: 1019-1028.
Ochsner, U.A., and Vasil, M.L. (1996) Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. Proc Natl Acad Sci U S A 93: 4409-4414.
Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol Microbiol 45: 1277-1287.
Paget, M.S., and Helmann, J.D. (2003) The sigma70 family of sigma factors. Genome Biol 4: 203.
Palma, M., Worgall, S., and Quadri, L.E. (2003) Transcriptome analysis of the Pseudomonas aeruginosa response to iron. Arch Microbiol 180: 374-379.
Palma, M., DeLuca, D., Worgall, S., and Quadri, L.E. (2004) Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J Bacteriol 186: 248-252.
Perez-Rueda, E., and Collado-Vides, J. (2000) The repertoire of DNA-binding transcriptional regulators in Escherichia coli K-12. Nucleic Acids Res 28: 1838-1847.
Poole, K., Neshat, S., Krebes, K., and Heinrichs, D.E. (1993) Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa. J Bacteriol 175: 4597-4604.
Potvin, E., Lehoux, D.E., Kukavica-Ibrulj, I., Richard, K.L., Sanschagrin, F., Lau, G.W., and Levesque, R.C. (2003) In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 5: 1294-1308.
Ramos, J.-L. (2004) Pseudomonas. Boston: Kluwer Academic/Plenum.
Ramphal, R., Koo, L., Ishimoto, K.S., Totten, P.A., Lara, J.C., and Lory, S. (1991) Adhesion of Pseudomonas aeruginosa pilin-deficient mutants to mucin. Infect Immun 59: 1307-1311.
Ramphal, R., Arora, S.K., and Ritchings, B.W. (1996) Recognition of mucin by the adhesin-flagellar system of Pseudomonas aeruginosa. Am J Respir Crit Care Med 154: S170-174.
Ramsey, D.M., and Wozniak, D.J. (2005) Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Mol Microbiol 56: 309-322.
Reitzer, L. (2003) Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol 57: 155-176.
Rodrigue, A., Quentin, Y., Lazdunski, A., Mejean, V., and Foglino, M. (2000) Two-component systems in Pseudomonas aeruginosa: why so many? Trends Microbiol 8: 498-504.
Schobel, S., Zellmeier, S., Schumann, W., and Wiegert, T. (2004) The Bacillus subtilis sigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC. Mol Microbiol 52: 1091-1105.
Schurr, M.J., Yu, H., Martinez-Salazar, J.M., Boucher, J.C., and Deretic, V. (1996) Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis. J Bacteriol 178: 4997-5004.
Schurr, M.J., and Deretic, V. (1997) Microbial pathogenesis in cystic fibrosis: co-ordinate regulation of heat-shock response and conversion to mucoidy in Pseudomonas aeruginosa. Mol Microbiol 24: 411-420.
Schuster, M., Lostroh, C.P., Ogi, T., and Greenberg, E.P. (2003) Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185: 2066-2079.
Schuster, M., Hawkins, A.C., Harwood, C.S., and Greenberg, E.P. (2004) The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol Microbiol 51: 973-985.
Simpson, D.A., Ramphal, R., and Lory, S. (1992) Genetic analysis of Pseudomonas aeruginosa adherence: distinct genetic loci control attachment to epithelial cells and mucins. Infect Immun 60: 3771-3779.
Sonnleitner, E., Hagens, S., Rosenau, F., Wilhelm, S., Habel, A., Jager, K.E., and Blasi, U. (2003) Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb Pathog 35: 217-228.
Starnbach, M.N., and Lory, S. (1992) The fliA (rpoF) gene of Pseudomonas aeruginosa encodes an alternative sigma factor required for flagellin synthesis. Mol Microbiol 6: 459-469.
Stintzi, A., Johnson, Z., Stonehouse, M., Ochsner, U., Meyer, J.M., Vasil, M.L., and Poole, K. (1999) The pvc gene cluster of Pseudomonas aeruginosa: role in synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS. J Bacteriol 181: 4118-4124.
Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J., Brinkman, F.S., Hufnagle, W.O., Kowalik, D.J., Lagrou, M., Garber, R.L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L., Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier, M.H., Hancock, R.E., Lory, S., and Olson, M.V. (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406: 959-964.
Strom, M.S., and Lory, S. (1993) Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol 47: 565-596.
Suh, S.J., Silo-Suh, L., Woods, D.E., Hassett, D.J., West, S.E., and Ohman, D.E. (1999) Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol 181: 3890-3897.
Tanaka, K., and Takahashi, H. (1991) Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa. Biochim Biophys Acta 1089: 113-119.
Thompson, L.S., Webb, J.S., Rice, S.A., and Kjelleberg, S. (2003) The alternative sigma factor RpoN regulates the quorum sensing gene rhlI in Pseudomonas aeruginosa. FEMS Microbiol Lett 220: 187-195.
Thony, B., and Hennecke, H. (1989) The -24/-12 promoter comes of age. FEMS Microbiol Rev 5: 341-357.
Totten, P.A., Lara, J.C., and Lory, S. (1990) The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J Bacteriol 172: 389-396.
Totten, P.A., and Lory, S. (1990) Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol 172: 7188-7199.
Vasil, M.L., Ochsner, U.A., Johnson, Z., Colmer, J.A., and Hamood, A.N. (1998) The fur-regulated gene encoding the alternative sigma factor PvdS is required for iron-dependent expression of the LysR-type regulator ptxR in Pseudomonas aeruginosa. J Bacteriol 180: 6784-6788.
Vasil, M.L., and Ochsner, U.A. (1999) The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 34: 399-413.
Venturi, V. (2003) Control of rpoS transcription in Escherichia coli and Pseudomonas: why so different? Mol Microbiol 49: 1-9.
Visca, P., Leoni, L., Wilson, M.J., and Lamont, I.L. (2002) Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas. Mol Microbiol 45: 1177-1190.
Voskuil, M.I., Voepel, K., and Chambliss, G.H. (1995) The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. Mol Microbiol 17: 271-279.
Wagner, V.E., Gillis, R.J., and Iglewski, B.H. (2004) Transcriptome analysis of quorum-sensing regulation and virulence factor expression in Pseudomonas aeruginosa. Vaccine 22 Suppl 1: S15-20.
Westfall, L.W., Luna, A.M., San Francisco, M., Diggle, S.P., Worrall, K.E., Williams, P., Camara, M., and Hamood, A.N. (2004) The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression. Microbiology 150: 3797-3806.
Whiteley, M., Parsek, M.R., and Greenberg, E.P. (2000) Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J Bacteriol 182: 4356-4360.
Wilderman, P.J., Vasil, A.I., Johnson, Z., Wilson, M.J., Cunliffe, H.E., Lamont, I.L., and Vasil, M.L. (2001) Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa. Infect Immun 69: 5385-5394.
Wilson, C., and Dombroski, A.J. (1997) Region 1 of sigma70 is required for efficient isomerization and initiation of transcription by Escherichia coli RNA polymerase. J Mol Biol 267: 60-74.
Wilson, M.J., and Lamont, I.L. (2000) Characterization of an ECF sigma factor protein from Pseudomonas aeruginosa. Biochem Biophys Res Commun 273: 578-583.
Wosten, M.M. (1998) Eubacterial sigma-factors. FEMS Microbiol Rev 22: 127-150.
Wu, W., Badrane, H., Arora, S., Baker, H.V., and Jin, S. (2004) MucA-mediated coordination of type III secretion and alginate synthesis in Pseudomonas aeruginosa. J Bacteriol 186: 7575-7585.
Yaku, H., Kato, M., Hakoshima, T., Tsuzuki, M., and Mizuno, T. (1997) Interaction between the CheY response regulator and the histidine-containing phosphotransfer (HPt) domain of the ArcB sensory kinase in Escherichia coli. FEBS Lett 408: 337-340.
Yamano, Y., Nishikawa, T., and Komatsu, Y. (1998) Involvement of the RpoN protein in the transcription of the oprE gene in Pseudomonas aeruginosa. FEMS Microbiol Lett 162: 31-37.
Zhou, Y.N., and Gross, C.A. (1992) How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32. J Bacteriol 174: 7128-7137.
ORF identification number in the P. aeruginosa genome annotated sequence (www.pseudomonas.com)
Molecular weight of ORF’s amino acid sequences calculated using Compute pI/Mw program from the Expasy Proteomics Server (www.expasy.org)
Closest homologues found using the BlastP program from National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). Blast hits values are expressed as percentages of similarity. Hits were selected using three features: closest homologue in other genus characterized or not, closest homologue found in P. aeruginosa genome and closest characterized homologue. BPP, Bordetella parapertussis; BPSS, Burkholderia pseudomallei; ECA, Erwinia carotavora; GLL, Gloeobacter violaceus; NE, Nitrosomonas europaea; PA, Pseudomonas aeruginosa; PP, Pseudomonas putida; RSc, Ralstonia solanacearum
Fig. 1. Conserved regions of σE from E. coli and the related sigma-70 factors from ECF subfamily. Domain 1, which has been implicated in preventing free sigma factors from binding to the promoter, is absent in the ECF subfamily. Domain 2, is the most conserved and contains regions implicated in core binding, DNA melting and interaction with the -10 promoter region. Domain 3 has a very low degree of conservation whereas domain 4 is well conserved containing the helix-turn-helix motif for DNA binding to the -35 promoter region (Figure adapted from Venturi V. published in Pseudomonas, volume 2, chapter 12).
© Éric Potvin, 2007
| | | |
| Chapitre 1 |  | Chapitre 3 Génomique fonctionnelle in vivo de Pseudomonas aeruginosa et criblage à haut débit de nouveaux facteurs de virulence et de cibles antibactériennes |
