Differential expression of two siderophore-dependent iron-acquisition pathways in Erwinia chrysanthemi 3937: characterization of a novel ferrisiderophore permease of the ABC transporter family.
Mahé B,Masclaux C,Rauscher L,Enard C,Expert D
In planta expression of a high-affinity iron-uptake system involving the siderophore chrysobactin in Erwinia chrysanthemi 3937 contributes greatly to invasive growth of this pathogen on its natural host, African violets. A previous study reported that global regulation by iron in this strain was mediated at the transcriptional level via the cbr locus which, when inactivated by insertional mutation, prevents the chrysobactin system from being tightly repressed by FeCl3. Herein, we report the nucleotide sequence of this locus and the functional analysis of its encoded products. Sequence analysis of a 4.8 kb genomic segment of a plasmid encompassing the cbr locus and characterization of the cognate translated products made it possible to uncover a system exhibiting similarity with prokaryotic transporters implicated in the transport of iron complexes. Accordingly, the CbrA product was shown to be the periplasmic component of a permease complex also including two integral membrane proteins, CbrB and CbrC, and the ATP-binding unit CbrD. This system allowed internalization of Fe(III) when supplied to bacterial cells as 59FeCl3 or 59Fe dicitrate, via complexation to a second siderophore recently detected in strain 3937. Most notably, we demonstrate that this second siderophore-mediated iron-acquisition system is operational in bacterial cells grown in the presence of FeCl3. The regulatory effect of cbr was further assessed on a lacZ chrysobactin operon fusion indicating that the transcriptional control exerted by cbr on expression of the chrysobactin system is of homeostatic nature. in conclusion, E. chrysanthemi provides an interesting model in which iron acquisition involves an inductive process resulting in differential expression of two siderophore-mediated pathways in relation to external iron accessibility.
Functional analysis and regulation of the divergent spuABCDEFGH-spuI operons for polyamine uptake and utilization in Pseudomonas aeruginosa PAO1.
Lu Chung-Dar,Itoh Yoshifumi,Nakada Yuji,Jiang Ying
Journal of bacteriology
A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1. This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization). The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants. Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function. Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator.
Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.
Sonnleitner Elisabeth,Valentini Martina,Wenner Nicolas,Haichar Feth el Zahar,Haas Dieter,Lapouge Karine
The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.
Genetic analysis of the histidine utilization (hut) genes in Pseudomonas fluorescens SBW25.
Zhang Xue-Xian,Rainey Paul B
The histidine utilization (hut) locus of Pseudomonas fluorescens SBW25 confers the ability to utilize histidine as a sole carbon and nitrogen source. Genetic analysis using a combination of site-directed mutagenesis and chromosomally integrated lacZ fusions showed the hut locus to be composed of 13 genes organized in 3 transcriptional units: hutF, hutCD, and 10 genes from hutU to hutG (which includes 2 copies of hutH, 1 of which is nonfunctional). Inactivation of hutF eliminated the ability to grow on histidine, indicating that SBW25 degrades histidine by the five-step enzymatic pathway. The 3 hut operons are negatively regulated by the HutC repressor with urocanate (the first intermediate of the histidine degradation pathway) as the physiological inducer. 5'-RACE analysis of transcriptional start sites revealed involvement of both sigma(54) (for the hutU-G operon) and sigma(70) (for hutF); the involvement of sigma(54) was experimentally demonstrated. CbrB (an enhancer binding protein for sigma(54) recruitment) was required for bacterial growth on histidine, indicating positive control of hut gene expression by CbrB. Recognition that a gene (named hutD) encoding a widely distributed conserved hypothetical protein is transcribed along with hutC led to analysis of its role. Mutational and gene fusion studies showed that HutD functions independently of HutC. Growth and fitness assays in laboratory media and on sugar beet seedlings suggest that HutD acts as a governor that sets an upper bound to the level of hut activity.
Expression of the sRNAs CrcZ and CrcY modulate the strength of carbon catabolite repression under diazotrophic or non-diazotrophic growing conditions in Azotobacter vinelandii.
Martínez-Valenzuela Marcela,Guzmán Josefina,Moreno Soledad,Ahumada-Manuel Carlos Leonel,Espín Guadalupe,Núñez Cinthia
Azotobacter vinelandii is a nitrogen-fixing bacterium of the Pseudomonadaceae family that prefers the use of organic acids rather than carbohydrates. Thus, in a mixture of acetate-glucose, glucose is consumed only after acetate is exhausted. In a previous work, we investigated the molecular basis of this carbon catabolite repression (CCR) process under diazotrophic conditions. In the presence of acetate, Crc-Hfq inhibited translation of the gluP mRNA, encoding the glucose transporter in A. vinelandii. Herein, we investigated the regulation in the expression of the small non-coding RNAs (sRNAs) crcZ and crcY, which are known to antagonize the repressing activity of Hfq-Crc. Our results indicated higher expression levels of the sRNAs crcZ and crcY under low CCR conditions (i.e. glucose), in relation to the strong one (acetate one). In addition, we also explored the process of CCR in the presence of ammonium. Our results revealed that CCR also occurs under non-diazotrophic conditions as we detected a hierarchy in the utilization of the supplied carbon sources, which was consistent with the higher expression level of the crcZ/Y sRNAs during glucose catabolism. Analysis of the promoters driving transcription of crcZ and crcY confirmed that they were RpoN-dependent but we also detected a processed form of CrcZ (CrcZ*) in the RpoN-deficient strain derived from a cbrB-crcZ co-transcript. CrcZ* was functional and sufficient to allow the assimilation of acetate.
Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25.
Zhang Xue-Xian,Rainey Paul B
Pseudomonas fluorescens SBW25 is capable of growing on histidine as a sole source of carbon and/or nitrogen. Previous work showed that the two-component regulatory system CbrAB is required for expression of the histidine utilization (hut) locus when histidine is the sole source of carbon and nitrogen. Here, using mutational analysis and transcriptional assays, we demonstrate involvement of a second two-component system, NtrBC. When histidine is the sole carbon source, transcription of the hutU operon is initiated from a sigma54-type promoter and requires CbrB (an enhancer binding protein for sigma54-recruitment). However, when histidine is the sole nitrogen source, the hutU operon is transcribed from a sigma70-type promoter and requires either CbrB or the nitrogen regulator, NtrC. No role was found for the SBW25 homolog of the nitrogen assimilation control protein (NAC). Biolog phenotypic microarray analysis of the ability of the three mutants (DeltacbrB, DeltantrC, and DeltacbrB DeltantrC) to utilize 190 carbon and 95 nitrogen substrates confirmed the central regulatory roles of CbrAB and NtrBC in cellular carbon and nitrogen catabolism: deletion of cbrB abolished growth on 20 carbon substrates; deletion of ntrC eliminated growth on 28 nitrogen substrates. A double cbrB-ntrC mutant was unable to utilize a further 14 nitrogen substrates (including histidine, proline, leucine, isoleucine, and valine). Our data show that CbrAB plays a role in regulation of both carbon and nitrogen catabolism and maintains activity of catabolic pathways under different C:N ratios.
Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA.
Hernández-Arranz Sofía,Sánchez-Hevia Dione,Rojo Fernando,Moreno Renata
RNA (New York, N.Y.)
In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases.
CbrAB-dependent regulation of pcnB, a poly(A) polymerase gene involved in polyadenylation of RNA in Pseudomonas fluorescens.
Zhang Xue-Xian,Liu Yun-Hao,Rainey Paul B
CbrB is a global sigma(54)-dependent regulator required for nutrient acquisition in Pseudomonas. Located downstream of cbrB on the Pseudomonas fluorescens SBW25 chromosome is pcnB, a putative poly(A) polymerase gene. Presence of a sigma(54) promoter in the intergenic region of cbrB and pcnB led to the hypothesis that CbrB regulates pcnB expression in a sigma(54)-dependent manner. Here we show that transcription of pcnB is CbrB dependent. However, 5'-RACE analysis of the pcnB transcript using primers located in the pcnB coding region shows that transcription starts immediately upstream of the putative ATG site at a sigma(70)-like promoter. Deletion of pcnB caused approximately 80% decrease of ployadenylated 23S rRNA; growth of the pcnB mutant was compromised in a range of laboratory media and on sugar beet seedlings. Further 5'-RACE analysis confirmed the existence of the predicted sigma(54) promoter. Genetic analysis showed that the sigma(54) promoter drives expression of crcZ, a homologue of the recently described small RNA from Pseudomonas aeruginosa, in a CbrB-dependent manner. Taken together, our data show that both pcnB and crcZ are part of the CbrB regulon. Moreover, the data draw further attention to the central regulatory role of CbrB and provides a link between mRNA degradation and cellular catabolism.
Lack of CbrB in Pseudomonas putida affects not only amino acids metabolism but also different stress responses and biofilm development.
Amador Cristina I,Canosa Inés,Govantes Fernando,Santero Eduardo
The CbrAB two-component system has been described in certain species of Pseudomonads as a global regulatory system required for the assimilation of several amino acids (e.g. histidine, proline or arginine) as carbon or carbon and nitrogen sources. In this work, we used global gene expression and phenotypic analyses to characterize the roles of the CbrAB system in Pseudomonas putida. Our results show that CbrB is involved in coordination with the nitrogen control system activator, NtrC, in the uptake and assimilation of several amino acids. In addition, CbrB affects other carbon utilization pathways and a number of apparently unrelated functions, such as chemotaxis, stress tolerance and biofilm development. Based on these new findings, we propose that CbrB is a high-ranked element in the regulatory hierarchy of P. putida that directly or indirectly controls a variety of metabolic and behavioural traits required for adaptation to changing environmental conditions.
Pseudomonas aeruginosa gbdR gene is transcribed from a σ54-dependent promoter under the control of NtrC/CbrB, IHF and BetI.
Sánchez Diego Germán,Primo Emiliano David,Damiani María Teresa,Lisa Angela Teresita
Microbiology (Reading, England)
Pseudomonasaeruginosa uses choline as a source of carbon and nitrogen, and also for the synthesis of glycine betaine, an osmoprotectant under stress conditions such as drought and salinity. The transcription factor GbdR is the specific regulator of choline metabolism and it belongs to the Arac/XylS family of transcriptional regulators. Despite the link between choline catabolism and bacterial pathogenicity, gbdR regulation has not been explored in detail. In the present work, we describe how gbdR transcription can be initiated from a σ54-dependent promoter. gbdR transcription can be activated by NtrC in the absence of a preferential nitrogen source, by CbrB in the absence of a preferential carbon source, and by the integration host factor favouring DNA bending. In addition, we found that BetI negatively regulates gbdR expression in the absence of choline. We identified two overlapping BetI binding sites in the gbdR promoter sequence, providing an additional example of σ54-promoter down-regulation. Based on our findings, we propose a model for gdbR regulation and its impact on choline metabolism.
Transcriptional activation of the CrcZ and CrcY regulatory RNAs by the CbrB response regulator in Pseudomonas putida.
García-Mauriño Sofía Muñoz,Pérez-Martínez Isabel,Amador Cristina I,Canosa Inés,Santero Eduardo
The CbrAB two-component system has been described as a high-ranked element in the regulatory hierarchy of Pseudomonas putida that controls a variety of metabolic and behavioural traits required for adaptation to changing environmental conditions. We show that the response regulatory protein CbrB, an activator of σ(N) -dependent promoters, directly controls the expression of the small RNAs CrcZ and CrcY in P. putida. These two RNAs sequester the protein Crc, which is a translational repressor of multiple pathways linked to carbon catabolite repression. We characterized the in vivo and in vitro activation by CbrB at both crcZ and crcY promoters, and identified new DNA sequences where the protein binds. IHF, a co-activator at many σ(N) -dependent promoters, also binds to the promoter regions and contributes to the activation of the sRNAs. CbrB phosphorylation is necessary at physiological activation conditions, but a higher dose of the protein allows in vitro transcriptional activation in its non-phosphorylated form. We also show there is some production of CrcY coming from an upstream promoter independent of CbrB. Thus, CbrAB constitute a global signal transduction pathway integrated in a higher regulatory network that also controls catabolite repression through the expression of the two regulatory RNAs CrcZ and CrcY.
The CbrB Regulon: Promoter dissection reveals novel insights into the CbrAB expression network in Pseudomonas putida.
Barroso Rocío,García-Mauriño Sofía M,Tomás-Gallardo Laura,Andújar Eloísa,Pérez-Alegre Mónica,Santero Eduardo,Canosa Inés
CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN-dependent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multimerisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.
A Pseudomonas putida cbrB transposon insertion mutant displays a biofilm hyperproducing phenotype that is resistant to dispersal.
Amador Cristina I,López-Sánchez Aroa,Govantes Fernando,Santero Eduardo,Canosa Inés
Environmental microbiology reports
The CbrAB two-component system in the Pseudomonads controls a variety of metabolic and behavioural traits required for its adaptation to changing environmental conditions, including the uptake or assimilation of certain carbon sources, and processes such as chemotaxis or stress tolerance. In this work we characterize a miniTn5-luxAB-Km transposon insertion mutant in cbrB (MPO406) in Pseudomonas putida leading to a biofilm overproducing phenotype that is not dispersed when nutrients are depleted. Comparison with a cbrB deletion mutant revealed that all phenotypes previously attributed to CbrB in P. putida correlated in both strains, with the exception of biofilm overproduction and absence of dispersal. We show that in the insertion mutant, the expression of the downstream regulatory RNA CrcZ is upregulated, and also show the presence of a truncated form of CbrB. Also, two additional point mutations in lapG and lapD have been detected in MPO406 by whole genome sequencing. Combination of these effects provides a robust biofilm overproducing phenotype. We present the mutant strain MPO406 as a good candidate to perform bio-production of substances of biotechnological interest or other processes such as bioremediation, which take advantage of immobilized cells on solid surfaces.
The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.
Nishijyo T,Haas D,Itoh Y
A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.
The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti.
Pini Francesco,Frage Benjamin,Ferri Lorenzo,De Nisco Nicole J,Mohapatra Saswat S,Taddei Lucilla,Fioravanti Antonella,Dewitte Frederique,Galardini Marco,Brilli Matteo,Villeret Vincent,Bazzicalupo Marco,Mengoni Alessio,Walker Graham C,Becker Anke,Biondi Emanuele G
Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.
CbrA is a stationary-phase regulator of cell surface physiology and legume symbiosis in Sinorhizobium meliloti.
Gibson Katherine E,Campbell Gordon R,Lloret Javier,Walker Graham C
Journal of bacteriology
Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysaccharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope.
The symbiosis regulator CbrA modulates a complex regulatory network affecting the flagellar apparatus and cell envelope proteins.
Gibson Katherine E,Barnett Melanie J,Toman Carol J,Long Sharon R,Walker Graham C
Journal of bacteriology
Sinorhizobium meliloti participates in a nitrogen-fixing symbiosis with legume plant host species of the genera Medicago, Melilotus, and Trigonella. We recently identified an S. meliloti two-component sensory histidine kinase, CbrA, which is absolutely required to establish a successful symbiosis with Medicago sativa (K. E. Gibson, G. R. Campbell, J. Lloret, and G. C. Walker, J. Bacteriol. 188:4508-4521, 2006). In addition to having a symbiotic defect, the cbrA::Tn5 mutant also has free-living phenotypes that suggest a cell envelope perturbation. Because the bases for these phenotypes are not well understood, we undertook an identification of CbrA-regulated genes. We performed a microarray analysis and compared the transcriptome of the cbrA::Tn5 mutant to that of the wild type. Our global analysis of gene expression identified 162 genes that are differentially expressed in the cbrA::Tn5 mutant, including those encoding proteins involved in motility and chemotaxis, metabolism, and cell envelope function. With regard to those genes with a known role in symbiosis, we observed increased expression of nine genes with overlapping functions in bacterial invasion of its host, which suggests that the mutant could be competent for invasion. Since these CbrA-repressed genes are vital to the invasion process, it appears that down-regulation of CbrA activity is important at this stage of nodule development. In contrast, our previous work showed that CbrA is required for bacteria to establish themselves within the host as nitrogen-fixing symbionts. Therefore, we propose a model in which CbrA functions as a developmental switch during symbiosis.
Glucose uptake in Azotobacter vinelandii occurs through a GluP transporter that is under the control of the CbrA/CbrB and Hfq-Crc systems.
Quiroz-Rocha Elva,Moreno Renata,Hernández-Ortíz Armando,Fragoso-Jiménez Juan Carlos,Muriel-Millán Luis Felipe,Guzmán Josefina,Espín Guadalupe,Rojo Fernando,Núñez Cinthia
Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon source. In this study, we show that GluP (Avin04150), annotated as an H-coupled glucose-galactose symporter, is the glucose transporter in A. vinelandii. This protein, which is widely distributed in bacteria and archaea, is uncommon in Pseudomonas species. We found that expression of gluP was under catabolite repression control thorugh the CbrA/CbrB and Crc/Hfq regulatory systems, which were functionally conserved between A. vinelandii and Pseudomonas species. While the histidine kinase CbrA was essential for glucose utilization, over-expression of the Crc protein arrested cell growth when glucose was the sole carbon source. Crc and Hfq proteins from either A. vinelandii or P. putida could form a stable complex with an RNA A-rich Hfq-binding motif present in the leader region of gluP mRNA. Moreover, in P. putida, the gluP A-rich Hfq-binding motif was functional and promoted translational inhibition of a lacZ reporter gene. The fact that gluP is not widely distributed in the Pseudomonas genus but is under control of the CbrA/CbrB and Crc/Hfq systems demonstrates the relevance of these systems in regulating metabolism in the Pseudomonadaceae family.
The sensor kinase CbrA is a global regulator that modulates metabolism, virulence, and antibiotic resistance in Pseudomonas aeruginosa.
Yeung Amy T Y,Bains Manjeet,Hancock Robert E W
Journal of bacteriology
Pseudomonas aeruginosa is an opportunistic pathogen that possesses a large arsenal of virulence factors enabling the pathogen to cause serious infections in immunocompromised patients, burn victims, and cystic fibrosis patients. CbrA is a sensor kinase that has previously been implied to play a role with its cognate response regulator CbrB in the metabolic regulation of carbon and nitrogen utilization in P. aeruginosa. Here it is demonstrated that CbrA and CbrB play an important role in various virulence and virulence-related processes of the bacteria, including swarming, biofilm formation, cytotoxicity, and antibiotic resistance. The cbrA deletion mutant was completely unable to swarm while exhibiting an increase in biofilm formation, supporting the inverse regulation of swarming and biofilm formation in P. aeruginosa. The cbrA mutant also exhibited increased cytotoxicity to human lung epithelial cells as early as 4 and 6 h postinfection. Furthermore, the cbrA mutant demonstrated increased resistance toward a variety of clinically important antibiotics, including polymyxin B, ciprofloxacin, and tobramycin. Microarray analysis revealed that under swarming conditions, CbrA regulated the expression of many genes, including phoPQ, pmrAB, arnBCADTEF, dnaK, and pvdQ, consistent with the antibiotic resistance and swarming impairment phenotypes of the cbrA mutant. Phenotypic and real-time quantitative PCR (RT-qPCR) analyses of a PA14 cbrB mutant suggested that CbrA may be modulating swarming, biofilm formation, and cytotoxicity via CbrB and that the CrcZ small RNA is likely downstream of this two-component regulator. However, as CbrB did not have a resistance phenotype, CbrA likely modulates antibiotic resistance in a manner independent of CbrB.
CbrA is a flavin adenine dinucleotide protein that modifies the Escherichia coli outer membrane and confers specific resistance to Colicin M.
Helbig Stephanie,Hantke Klaus,Ammelburg Moritz,Braun Volkmar
Journal of bacteriology
Colicin M (Cma) is a protein toxin produced by Escherichia coli that kills sensitive E. coli cells by inhibiting murein biosynthesis in the periplasm. Recombinant plasmids carrying cbrA (formerly yidS) strongly increased resistance of cells to Cma, whereas deletion of cbrA increased Cma sensitivity. Transcription of cbrA is positively controlled by the two-component CreBC system. A ΔcreB mutant was highly Cma sensitive because little CbrA was synthesized. Treatment of CbrA-overproducing cells by osmotic shock failed to render cells Cma sensitive because the cells were resistant to osmotic shock. In a natural environment with a growth-limiting nutrient supply, cells producing CbrA defend themselves against colicin M synthesized by competing cells. Isolated CbrA is a protein with noncovalently bound flavin adenine dinucleotide. Sequence comparison and structure prediction assign the closest relative of CbrA with a known crystal structure as digeranylgeranyl-glycerophospholipid reductase of Thermoplasma acidophilum. CbrA is found in Escherichia coli, Citrobacter, and Salmonella bongori but not in other enterobacteria. The next homologs with the highest identity (over 50%) are found in the anaerobic Clostridium botulinum group 1 and a few other Firmicutes.
Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida.
Valentini Martina,García-Mauriño Sofía M,Pérez-Martínez Isabel,Santero Eduardo,Canosa Inés,Lapouge Karine
Microbiology (Reading, England)
The CbrA/B system in pseudomonads is involved in the utilization of carbon sources and carbon catabolite repression (CCR) through the activation of the small RNAs crcZ in Pseudomonas aeruginosa, and crcZ and crcY in Pseudomonas putida. Interestingly, previous works reported that the CbrA/B system activity in P. aeruginosa PAO1 and P. putida KT2442 responded differently to the presence of different carbon sources, thus raising the question of the exact nature of the signal(s) detected by CbrA. Here, we demonstrated that the CbrA/B/CrcZ(Y) signal transduction pathway is similarly activated in the two Pseudomonas species. We show that the CbrA sensor kinase is fully interchangeable between the two species and, moreover, responds similarly to the presence of different carbon sources. In addition, a metabolomics analysis supported the hypothesis that CCR responds to the internal energy status of the cell, as the internal carbon/nitrogen ratio seems to determine CCR and non-CCR conditions. The strong difference found in the 2-oxoglutarate/glutamine ratio between CCR and non-CCR conditions points to the close relationship between carbon and nitrogen availability, or the relationship between the CbrA/B and NtrB/C systems, suggesting that both regulatory systems sense the same sort or interrelated signal.
The Sinorhizobium meliloti sensor histidine kinase CbrA contributes to free-living cell cycle regulation.
Sadowski Craig S,Wilson Daniel,Schallies Karla B,Walker Graham,Gibson Katherine E
Microbiology (Reading, England)
Sinorhizobium meliloti is alternately capable of colonizing the soil as a free-living bacterium or establishing a chronic intracellular infection with its legume host for the purpose of nitrogen fixation. We previously identified the S. meliloti two-component sensor histidine kinase CbrA as playing an important role in regulating exopolysaccharide production, flagellar motility and symbiosis. Phylogenetic analysis of CbrA has highlighted its evolutionary relatedness to the Caulobacter crescentus sensor histidine kinases PleC and DivJ, which are involved in CtrA-dependent cell cycle regulation through the shared response regulator DivK. We therefore became interested in testing whether CbrA plays a role in regulating S. meliloti cell cycle processes. We find the loss of cbrA results in filamentous cell growth accompanied by cells that contain an aberrant genome complement, indicating CbrA plays a role in regulating cell division and possibly DNA segregation. S. meliloti DivK localizes to the old cell pole during distinct phases of the cell cycle in a phosphorylation-dependent manner. Loss of cbrA results in a significantly decreased rate of DivK polar localization when compared with the wild-type, suggesting CbrA helps regulate cell cycle processes by modulating DivK phosphorylation status as a kinase. Consistent with a presumptive decrease in DivK phosphorylation and activity, we also find the steady-state level of CtrA increased in cbrA mutants. Our data therefore demonstrate that CbrA contributes to free-living cell cycle regulation, which in light of its requirement for symbiosis, points to the potential importance of cell cycle regulation for establishing an effective host interaction.
Sinorhizobium meliloti CtrA Stability Is Regulated in a CbrA-Dependent Manner That Is Influenced by CpdR1.
Schallies Karla B,Sadowski Craig,Meng Julia,Chien Peter,Gibson Katherine E
Journal of bacteriology
UNLABELLED:CbrA is a DivJ/PleC-like histidine kinase of DivK that is required for cell cycle progression and symbiosis in the alphaproteobacterium Sinorhizobium meliloti. Loss of cbrA results in increased levels of CtrA as well as its phosphorylation. While many of the known Caulobacter crescentus regulators of CtrA phosphorylation and proteolysis are phylogenetically conserved within S. meliloti, the latter lacks the PopA regulator that is required for CtrA degradation in C. crescentus. In order to investigate whether CtrA proteolysis occurs in S. meliloti, CtrA stability was assessed. During exponential growth, CtrA is unstable and therefore likely to be degraded in a cell cycle-regulated manner. Loss of cbrA significantly increases CtrA stability, but this phenotype is restored to that of the wild type by constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated (CpdR1(D53A)). Addition of CpdR1(D53A) fully suppresses cbrA mutant cell cycle defects, consistent with regulation of CtrA stability playing a key role in mediating proper cell cycle progression in S. meliloti. Importantly, the cbrA mutant symbiosis defect is also suppressed in the presence of CpdR1(D53A). Thus, regulation of CtrA stability by CbrA and CpdR1 is associated with free-living cell cycle outcomes and symbiosis. IMPORTANCE:The cell cycle is a fundamental process required for bacterial growth, reproduction, and developmental differentiation. Our objective is to understand how a two-component signal transduction network directs cell cycle events during free-living growth and host colonization. The Sinorhizobium meliloti nitrogen-fixing symbiosis with plants is associated with novel cell cycle events. This study identifies a link between the regulated stability of an essential response regulator, free-living cell cycle progression, and symbiosis.
Requirement of the Pseudomonas aeruginosa CbrA sensor kinase for full virulence in a murine acute lung infection model.
Yeung Amy T Y,Janot Laure,Pena Olga M,Neidig Anke,Kukavica-Ibrulj Irena,Hilchie Ashley,Levesque Roger C,Overhage Joerg,Hancock Robert E W
Infection and immunity
Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of respiratory tract and other nosocomial infections. The sensor kinase CbrA is a central regulator of carbon and nitrogen metabolism and in vitro also regulates virulence-related processes in P. aeruginosa. Here, we investigated the role of CbrA in two murine models of infection. In both peritoneal infections in leukopenic mice and lung infection models, the cbrA mutant was less virulent since substantially larger numbers of cbrA mutant bacteria were required to cause the same level of infection as wild-type or complemented bacteria. In contrast, in the chronic rat lung model the cbrA mutant grew and persisted as well as the wild type, indicating that the decrease of in vivo virulence of the cbrA mutant did not result from growth deficiencies on particular carbon substrates observed in vitro. In addition, a mutant in the cognate response regulator CbrB showed no defect in virulence in the peritoneal infection model, ruling out the involvement of certain alterations of virulence properties in the cbrA mutant including defective swarming motility, increased biofilm formation, and cytotoxicity, since these alterations are controlled through CbrB. Further investigations indicated that the mutant was more susceptible to uptake by phagocytes in vitro, resulting in greater overall bacterial killing. Consistent with the virulence defect, it took a smaller number of Dictyostelium discoideum amoebae to kill the cbrA mutant than to kill the wild type. Transcriptional analysis of the cbrA mutant during D. discoideum infection led to the conclusion that CbrA played an important role in the iron metabolism, protection of P. aeruginosa against oxidative stress, and the regulation of certain virulence factors.
Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction.
Zhang Xue-Xian,Gauntlett Jonathan C,Oldenburg Darby G,Cook Gregory M,Rainey Paul B
Journal of bacteriology
UNLABELLED:CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA. This, coupled with the findings of [(3)H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction. IMPORTANCE:Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in Pseudomonas represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.
Two-component system CbrA/CbrB controls alginate production in Azotobacter vinelandii.
Quiroz-Rocha Elva,Bonilla-Badía Fernando,García-Aguilar Valentina,López-Pliego Liliana,Serrano-Román Jade,Cocotl-Yañez Miguel,Guzmán Josefina,Ahumada-Manuel Carlos L,Muriel-Millán Luis Felipe,Castañeda Miguel,Espín Guadalupe,Nuñez Cinthia
Microbiology (Reading, England)
Azotobacter vinelandii, belonging to the Pseudomonadaceae family, is a free-living bacterium that has been considered to be a good source for the production of bacterial polymers such as alginate. In A. vinelandii the synthesis of this polymer is regulated by the Gac/Rsm post-transcriptional regulatory system, in which the RsmA protein binds to the mRNA of the biosynthetic algD gene, inhibiting translation. In several Pseudomonas spp. the two-component system CbrA/CbrB has been described to control a variety of metabolic and behavioural traits needed for adaptation to changing environmental conditions. In this work, we show that the A. vinelandii CbrA/CbrB two-component system negatively affects alginate synthesis, a function that has not been described in Pseudomonas aeruginosa or any other Pseudomonas species. CbrA/CbrB was found to control the expression of some alginate biosynthetic genes, mainly algD translation. In agreement with this result, the CbrA/CbrB system was necessary for optimal rsmA expression levels. CbrA/CbrB was also required for maximum accumulation of the sigma factor RpoS. This last effect could explain the positive effect of CbrA/CbrB on rsmA expression, as we also showed that one of the promoters driving rsmA transcription was RpoS-dependent. However, although inactivation of rpoS increased alginate production by almost 100 %, a cbrA mutation increased the synthesis of this polymer by up to 500 %, implying the existence of additional CbrA/CbrB regulatory pathways for the control of alginate production. The control exerted by CbrA/CbrB on the expression of the RsmA protein indicates the central role of this system in regulating carbon metabolism in A. vinelandii.
CbrA Mediates Colicin M Resistance in Escherichia coli through Modification of Undecaprenyl-Phosphate-Linked Peptidoglycan Precursors.
Barreteau Hélène,Patin Delphine,Bouhss Ahmed,Blanot Didier,Mengin-Lecreulx Dominique,Touzé Thierry
Journal of bacteriology
Colicin M is an enzymatic bacteriocin produced by some strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, (formerly ), was shown to protect cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. and assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. Overexpression of the chromosomal gene allows to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of to generate reduced forms of C-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.
Unraveling the role of the CbrA histidine kinase in the signal transduction of the CbrAB two-component system in Pseudomonas putida.
Monteagudo-Cascales Elizabet,García-Mauriño Sofía M,Santero Eduardo,Canosa Inés
The histidine kinase CbrA of the CbrAB two-component system of Pseudomonas putida is a key element to recognise the activating signal and mediate auto- and trans-phosphorylation of the response element CbrB. CbrA is encoded by the gene cbrA which is located downstream of a putative open reading frame we have named cbrX. We describe the role of the CbrX product in the expression of CbrA and show there is translational coupling of the genes. We also explore the role of the transmembrane (TM) and PAS domains of CbrA in the signal recognition. A ΔcbrXA mutant lacking its TM domains is uncoupled in its growth in histidine and citrate as carbon sources, but its overexpression restores the ability to grow in such carbon sources. In these conditions ΔTM-CbrA is able to respond to carbon availability, thus suggesting an intracellular nature for the signal sensed.
Transport and kinase activities of CbrA of Pseudomonas putida KT2440.
Wirtz Larissa,Eder Michelle,Schipper Kerstin,Rohrer Stefanie,Jung Heinrich
The CbrA/CbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central role in carbon catabolite repression in Pseudomonas species. CbrA is composed of a domain with similarity to proteins of the solute/sodium symporter family (SLC5) and domains typically found in bacterial sensor kinases. Here, the functional properties of the sensor kinase CbrA and its domains are analyzed at the molecular level using the system of the soil bacterium P. putida KT2440 as a model. It is demonstrated that CbrA can bind and transport L-histidine. Transport is specific for L-histidine and probably driven by an electrochemical proton gradient. The kinase domain is not required for L-histidine uptake by the SLC5 domain of CbrA, and has no significant impact on transport kinetics. Furthermore, it is shown that the histidine kinase can autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SLC5 domain is not essential for these activities but appears to modulate the autokinase activity. A phosphatase activity of CbrA is not detected. None of the activities is significantly affected by L-histidine. The results demonstrate that CbrA functions as a L-histidine transporter and sensor kinase.
Negative transcriptional control of iron transport in Erwinia chrysanthemi involves an iron-responsive two-factor system.
Expert D,Sauvage C,Neilands J B
Systemic virulence of the phytopathogen Erwinia chrysanthemi 3937 requires a functional iron assimilation system which, in this enterobacterium, is mediated by the siderophore chrysobactin and the outer membrane transport protein Fct. We investigated the regulation of this system by iron. No direct similarity with the Escherichia coli fur gene was found. Insertional mutagenesis allowed isolation of a regulatory mutant which expressed chrysobactin and two other high-affinity iron transport systems previously characterized in strain 3937, regardless of the iron level. RNA/DNA hybridization analysis established that regulation of chrysobactin by iron occurs at the transcriptional level. From a wild-type gene library, a recombinant cosmid able to restore normal regulation in the mutant strain was isolated. By generating a series of subclones and mini-Mulac insertions, we identified a regulatory locus (cbr) extending beyond c. 2.5kb which encodes two polypeptides, CbrA and CbrB, with molecular weights of 34,000 and 55,000 respectively. Functional analysis of the locus suggests that the cognate genes cbrA and cbrB are clustered within an operon. Their expression was studied through chromosomal lac gene fusions, in the presence of plasmid-borne wild-type constructions, under high- and low-iron conditions. In summary, the data show that in the presence of iron, cbr negatively regulates the chrysobactin biosynthetic and transport genes, while under conditions of depletion, cbr is subject to negative autogeneous regulation.
Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two-component systems.
Zhou Lu,Lei Xiang-He,Bochner Barry R,Wanner Barry L
Journal of bacteriology
Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.
Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa.
Li Wei,Lu Chung-Dar
Journal of bacteriology
The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.
Effect of metabolic imbalance on expression of type III secretion genes in Pseudomonas aeruginosa.
Rietsch Arne,Wolfgang Matthew C,Mekalanos John J
Infection and immunity
The type III secretion system is a dedicated machinery used by many pathogens to deliver toxins directly into the cytoplasm of a target cell. Expression and secretion of the type III effectors are triggered by cell contact. In Pseudomonas aeruginosa and Yersinia spp., expression can be triggered in vitro by removing calcium from the medium. The mechanism underlying either mode of regulation is unclear. Here we characterize a transposon insertion mutant of P. aeruginosa PAO1 that displays a marked defect in cytotoxicity. The insertion is located upstream of several genes involved in histidine utilization and impedes the ability of PAO1 to intoxicate eukaryotic cells effectively in a type III-dependent fashion. This inhibition depends on the presence of histidine in the medium and appears to depend on the excessive uptake and catabolism of histidine. The defect in cytotoxicity is mirrored by a decrease in exoS expression. Other parameters such as growth or piliation are unaffected. The cytotoxicity defect is partially complemented by an insertion mutation in cbrA that also causes overexpression of cbrB. The cbrAB two-component system has been implicated in sensing and responding to a carbon-nitrogen imbalance. Taken together, these results suggest that the metabolic state of the cell influences expression of the type III regulon.
Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species.
Sonnleitner Elisabeth,Haas Dieter
Applied microbiology and biotechnology
Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.
Two small RNAs, CrcY and CrcZ, act in concert to sequester the Crc global regulator in Pseudomonas putida, modulating catabolite repression.
Moreno Renata,Fonseca Pilar,Rojo Fernando
The Crc protein is a translational repressor that recognizes a specific target at some mRNAs, controlling catabolite repression and co-ordinating carbon metabolism in pseudomonads. In Pseudomonas aeruginosa, the levels of free Crc protein are controlled by CrcZ, a sRNA that sequesters Crc, acting as an antagonist. We show that, in Pseudomonas putida, the levels of free Crc are controlled by CrcZ and by a novel 368 nt sRNA named CrcY. CrcZ and CrcY, which contain six potential targets for Crc, were able to bind Crc specifically in vitro. The levels of CrcZ and CrcY were low under conditions generating a strong catabolite repression, and increased strongly when catabolite repression was absent. Deletion of either crcZ or crcY had no effect on catabolite repression, but the simultaneous absence of both sRNAs led to constitutive catabolite repression that compromised growth on some carbon sources. Overproduction of CrcZ or CrcY significantly reduced repression. We propose that CrcZ and CrcY act in concert, sequestering and modulating the levels of free Crc according to metabolic conditions. The CbrA/CbrB two-component system activated crcZ transcription, but had little effect on crcY. CrcY was detected in P. putida, Pseudomonas fluorescens and Pseudomonas syringae, but not in P. aeruginosa.
Metabolism and the Evolution of Social Behavior.
Boyle Kerry E,Monaco Hilary T,Deforet Maxime,Yan Jinyuan,Wang Zhe,Rhee Kyu,Xavier Joao B
Molecular biology and evolution
How does metabolism influence social behavior? This fundamental question at the interface of molecular biology and social evolution is hard to address with experiments in animals, and therefore, we turned to a simple microbial system: swarming in the bacterium Pseudomonas aeruginosa. Using genetic engineering, we excised a locus encoding a key metabolic regulator and disrupted P. aeruginosa's metabolic prudence, the regulatory mechanism that controls expression of swarming public goods and protects this social behavior from exploitation by cheaters. Then, using experimental evolution, we followed the joint evolution of the genome, the metabolome and the social behavior as swarming re-evolved. New variants emerged spontaneously with mutations that reorganized the metabolome and compensated in distinct ways for the disrupted metabolic prudence. These experiments with a unicellular organism provide a detailed view of how metabolism-currency of all physiological processes-can determine the costs and benefits of a social behavior and ultimately influence how an organism behaves towards other organisms of the same species.
The expression of the genes involved in leucine catabolism of Pseudomonas aeruginosa is controlled by the transcriptional regulator LiuR and by the CbrAB/Crc system.
Díaz-Pérez Alma Laura,Núñez Cinthia,Meza Carmen Victor,Campos-García Jesús
Research in microbiology
Pseudomonas aeruginosa metabolizes leucine through the leucine/isovalerate utilization pathway, whose enzymes are encoded in the liuRABCDE gene cluster (liu). In this study, we investigated the role of the LiuR protein in the liu cluster regulation. Our results indicated that liu expression is regulated at the transcriptional level by LiuR. Mobility shift assays using purified recombinant His-tagged LiuR showed that it was able to bind at the promoter region of liuR, in a dose-dependent manner. Results revealed that expression of the liu operon is subjected to carbon catabolite repression control (CCR); protein LiuD was strongly expressed in the presence of leucine, but it was repressed in the presence of glucose or succinate. Furthermore, this CCR control was dependent on LiuR as in the liuR mutant the LiuD protein was strongly expressed in all the carbon sources tested. In agreement with this result, in the absence of the Crc protein, LiuD was expressed independently of the carbon source used, whereas in a cbrB mutant its expression was severely impaired. The results indicated that the liu cluster is subjected to a coordinated transcriptional and translational regulation by the LiuR repressor and by the CbrAB/Crc system, respectively, in response to the available carbon source.
Catabolite repression in Pseudomonas aeruginosa PAO1 regulates the uptake of C4 -dicarboxylates depending on succinate concentration.
Valentini Martina,Lapouge Karine
In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at μM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased.
Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa.
Sonnleitner Elisabeth,Abdou Laetitia,Haas Dieter
Proceedings of the National Academy of Sciences of the United States of America
In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.
Intrinsic and Extrinsic Aspects on Biofilms.
Melo Roberta T,Mendonça Eliane P,Monteiro Guilherme P,Siqueira Mariana C,Pereira Clara B,Peres Phelipe A B M,Fernandez Heriberto,Rossi Daise A
Frontiers in microbiology
Biofilm represents a way of life that allows greater survival of microorganisms in hostile habitats. is able to form biofilms and on surfaces at several points in the poultry production chain. Genetic determinants related to their formation are expressed differently between strains and external conditions are decisive in this respect. Our approach combines phylogenetic analysis and the presence of seven specific genes linked to biofilm formation in association with traditional microbiology techniques, using Mueller Hinton and chicken juice as substrates in order to quantify, classify, determine the composition and morphology of the biomass of simple and mixed biofilms of 30 strains. It also evaluates the inhibition of its formation by biocides commonly used in industry and also by zinc oxide nanoparticles. Genetic analysis showed high heterogeneity with the identification of 23 pulsotypes. Despite the diversity, the presence of , and genes in all strains shows the high potential for biofilm formation. This ability was only expressed in chicken juice, where they presented phenotype of a strong biofilm producer, with a mean count of 7.37 log CFU/mL and an ultrastructure characteristic of mature biofilm. The composition of simple and mixed biofilms was predominantly composed by proteins. The exceptions were found in mixed biofilms with , which includes a carbohydrate-rich matrix, lower ability to sessile form in chicken juice and compact architecture of the biofilm, this aspects are intrinsic to this species. Hypochlorite, chlorhexidine, and peracetic acid were more effective in controlling viable cells of in biofilm, but the existence of tolerant strains indicates exposure to sublethal concentrations and development of adaptation mechanisms. This study shows that in chicken juice presents greater potential in producing mature biofilms.
Gene-centric metegenome analysis reveals diversity of Pseudomonas aeruginosa biofilm gene orthologs in fresh water ecosystem.
Anupama Rani,Mukherjee Amitava,Babu Subramanian
Metagenomic analysis of biofilm forming bacteria in environmental samples remains challenging due to the non-availability of gene sequences of most of the uncultivable bacteria. Sequences of Pseudomonas aeruginosa PAO1-UW genes involved either directly or indirectly in biofilm formation were analyzed using BLASTn to obtain matching sequences from different strain, species and genus. Conserved regions in the functional domain of the amino acid sequences were used to design common primers for direct PCR analysis of freshwater metagenomes. Seven key genes such as aceA, clpP, typA, cbrA, phoR, rpoS and gacA involved in biofilm formation were validated. The ortholog genes belonged to wide range of Pseudomonas sp. indicating the diversity of biofilm genes and the conservation of protein functional domains. The approach would also help in analyzing the expression of biofilm genes in different bacteria of freshwater systems for monitoring toxic contaminations such as organic or inorganic pollutants.
Pseudomonas donghuensis HYS virulence towards Caenorhabditis elegans is regulated by the Cbr/Crc system.
Xie Guanfang,Zeng Man,You Jia,Xie Zhixiong
Pseudomonas donghuensis HYS is the type strain of a recently identified species, P. donghuensis, which has pathogenic potential with an unclear virulence mechanism. In this study, we used Caenorhabditis elegans as a host to explore the virulence mechanism of P. donghuensis HYS. Based on a correlation between P. donghuensis HYS virulence and its repellence property, we identified 68 potential virulence-related genes, among them the Cbr/Crc system, which regulates the virulence of prokaryotic microorganisms. Slow-killing assays indicated that cbrA, cbrB, or specific sRNA-encoding genes all affected P. donghuensis virulence positively, whereas crc affected it negatively. Transcriptome analyses demonstrated that the Cbr/Crc system played an important role in the pathogenesis of P. donghuensis. In addition, experiments using the worm mutant KU25 pmk-1(km25) showed a correlation between P. donghuensis HYS virulence and the PMK-1/p38 MAPK pathway in C. elegans. In conclusion, our data show that Crc plays a novel role in the Cbr/Crc system, and the P. donghuensis virulence phenotype therefore differs from that of P. aeruginosa. This process also involves C. elegans innate immunity. These findings significantly increase the available information about Cbr/Crc-based virulence mechanisms in the genus Pseudomonas.
Metabolite profiling of the cold adaptation of Pseudomonas putida KT2440 and cold-sensitive mutants.
Dethlefsen Sarah,Jäger Christian,Klockgether Jens,Schomburg Dietmar,Tümmler Burkhard
Environmental microbiology reports
Free-living bacteria such as Pseudomonas putida are frequently exposed to temperature shifts and non-optimal growth conditions. We compared the transcriptome and metabolome of the cold adaptation of P. putida KT2440 and isogenic cold-sensitive transposon mutants carrying transposons in their cbrA, cbrB, pcnB, vacB, and bipA genes. Pseudomonas putida changes the mRNA expression of about 43% of all annotated open reading frames during this initial phase of cold adaptation, but only a small number of 6-93 genes were differentially expressed at 10°C between the wild-type strain and the individual mutants. The spectrum of metabolites underwent major changes during cold adaptation particularly in the mutants. Both the KT2440 strain and the mutants increased the levels of the most abundant sugars and amino acids which were more pronounced in the cold-sensitive mutants. All mutants depleted their pools for core metabolites of aromatic and sugar metabolism, but increased their pool of polar amino acids which should be advantageous to cope with the cold stress.
Role of a local transcription factor in governing cellular carbon/nitrogen homeostasis in Pseudomonas fluorescens.
Naren Naran,Zhang Xue-Xian
Nucleic acids research
Autoactivation of two-component systems (TCSs) can increase the sensitivity to signals but inherently cause a delayed response. Here, we describe a unique negative feedback mechanism enabling the global NtrB/NtrC regulator to rapidly respond to nitrogen starvation over the course of histidine utilization (hut) in Pseudomonas fluorescens. NtrBC directly activates transcription of hut genes, but overexpression will produce excess ammonium leading to NtrBC inactivation. To prevent this from occurring, the histidine-responsive repressor HutC fine-tunes ntrBC autoactivation: HutC and NtrC bind to the same operator site in the ntrBC promoter. This newly discovered low-affinity binding site shows little sequence similarity with the consensus sequence that HutC recognizes for substrate-specific induction of hut operons. A combination of genetic and transcriptomic analysis indicated that both ntrBC and hut promoter activities cannot be stably maintained in the ΔhutC background when histidine fluctuates at high concentrations. Moreover, the global carbon regulator CbrA/CbrB is involved in directly activating hut transcription while de-repressing hut translation via the CbrAB-CrcYZ-Crc/Hfq regulatory cascade. Together, our data reveal that the local transcription factor HutC plays a crucial role in governing NtrBC to maintain carbon/nitrogen homeostasis through the complex interactions between two TCSs (NtrBC and CbrAB) at the hut promoter.
CrcZ and CrcX regulate carbon source utilization in Pseudomonas syringae pathovar tomato strain DC3000.
Filiatrault Melanie J,Stodghill Paul V,Wilson Janet,Butcher Bronwyn G,Chen Hanrong,Myers Christopher R,Cartinhour Samuel W
Small non-coding RNAs (ncRNAs) are important components of many regulatory pathways in bacteria and play key roles in regulating factors important for virulence. Carbon catabolite repression control is modulated by small RNAs (crcZ or crcZ and crcY) in Pseudomonas aeruginosa and Pseudomonas putida. In this study, we demonstrate that expression of crcZ and crcX (formerly designated psr1 and psr2, respectively) is dependent upon RpoN together with the two-component system CbrAB, and is influenced by the carbon source present in the medium in the model plant pathogen Pseudomonas syringae pv tomato DC3000. The distribution of the members of the Crc ncRNA family was also determined by screening available genomic sequences of the Pseudomonads. Interestingly, variable numbers of the Crc family members exist in Pseudomonas genomes. The ncRNAs are comprised of three main subfamilies, named CrcZ, CrcX and CrcY. Most importantly the CrcX subfamily appears to be unique to all P. syringae strains sequenced to date.
Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.
Abdou Laetitia,Chou Han-Ting,Haas Dieter,Lu Chung-Dar
Journal of bacteriology
In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.