The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa

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.     

Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.     

Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.     

Effect of glutamate transport and catabolism on symbiotic effectiveness in Rhizobium leguminosarum bv. phaseoli

Seven Tn5 induced mutants unable to use glutamate as sole carbon and nitrogen source were isolated from the effective Rhizobium leguminosarum bv. phaseoli strain P121-R. As indicated by restriction and hybridisation analysis, all the mutants arose from a single Tn5 insertion in the chromosome. The ¹⁴C-glutamate uptake rate of the mutants was 76 to 88% lower than that of strain P121-R. Inoculation of Phaseolus vulgaris cv. Labrador with these mutants significantly decreased shoot dry matter yield and the total nitrogen content respectively, as compared to inoculation with the parental strain P121-R. All the mutants formed nodules, however they were smaller, white to greenish and approximately 30% less numerous than those formed by strain P121-R. These observations suggest that glutamate transport and catabolism in R. leguminosarum bv. phaseoli P121-R may play an important role in the establishment of an effective symbiosis in field bean. None of the mutants isolated was an auxotroph. All mutants were unable to grow on aspartate suggesting that glutamate and aspartate, probably have the same transporter as indicated in Rhizobium meliloti and in Bacillus subtilis. All mutants readily used glutamine, proline, arginine as sole carbon and nitrogen source, but grew more slowly than the wild type strain. On the other hand, all the mutants were impaired in growth on histidine and -aminobutyrate as sole carbon and nitrogen source. As the catabolism of these amino acids occurs predominantly through glutamate, our results indicate that mutants are also impaired in their ability to use histidine and -aminobutyrate as a nitrogen source. Our results also suggest that other amino acids catabolized through the glutamate pathways may be an additional important carbon source for bacteroids in nodules.     

Utilization of arginine by Klebsiella aerogenes.

Klebsiella aerogenes utilized arginine as the sole source of carbon or nitrogen for growth. Arginine was degraded to 2-ketoglutarate and not to succinate, since a citrate synthaseless mutant grows on arginine as the only nitrogen source. When glucose was the energy source, all four nitrogen atoms of arginine were utilized. Three of them apparently did not pass through ammonia but were transferred by transamination, since a mutant unable to produce glutamate by glutamate synthase or glutamate dehydrogenase utilized three of four nitrogen atoms of arginine. Urea was not involved as intermediate, since a unreaseless mutant did not accumulate urea and grew on arginine as efficiently as the wild-type strain. Ornithine appeared to be an intermediate, because cells grown either on glucose and arginine or arginine alone could convert arginine in the presence of hydroxylamine to ornithine. This indicates that an amidinotransferase is the initiating enzyme of arginine breakdown. In addition, the cells contained a transaminase specific for ornithine. In contrast to the hydroxylamine-dependent reaction, this activity could be demonstrated in extracts. The arginine-utilizing system (aut) is apparently controlled like the enzymes responsible for the degradation of histidine (hut) through induction, catabolite repression, and activation by glutamine synthetase.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

2-Aminoethylphosphonate utilization in Pseudomonas putida BIRD-1 is controlled by multiple master regulators

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.     

Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with lesions in gene amdT are pleiotropically affected in their ability to utilize a wide variety of nitrogen sources in the presence of glucose. Ability to utilize a number of these compounds as sole sources of carbon and nitrogen is not altered. One of these mutants, amdT102, has properties consistent with it being derepressed for glucose repression of the utilization of most (but not all) nitrogen sources. The amdT102 mutant can grow strongly on histidine, lysine and cystine as sole nitrogen sources while the wild type strain grows extremely poorly on these amino acids. Similar but less extreme effects apply to many other nitrogen sources. The amdT19 mutant is unable to utilize most nitrogen sources in the presence of glucose, suggesting that it is subject to greatly increased repression of nitrogen source utilization. The amdT mutants are not affected in their ability to use many compounds as sole carbon sources. Carbon sources other than glucose also affect utilization of nitrogen sources in the amdT mutants.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

ntrB基因对Agrobacterium sp.ATCC31749合成胞外多糖的调控研究

利用三亲本接合方法将含有ntrB基因两端同源序列的自杀载体pJQ-ntrB-cat导入土壤杆菌(Agrobacterium sp.ATCC 31749)中,获得了ntrB基因突变株.结果表明,ntrB突变株对NH4Cl和KNO3的利用能力有所下降.当分别以谷氨酸和谷氨酰胺为氮源时,ntrB突变株生长状况与野生菌相同.n...     

Analysis of an L-histidinol-utilizing mutant of Pseudomonas aeruginosa.

Transductional analysis was applied to the Pseudomonas aeruginosa mutant PAO14 (hnc-1). This mutant can utilize L-histidinol as sole source of carbon and nitrogen and has a 60-fold increased histidinol dehydrogenase (HDH) content (Dhawale, Creaser & Loper, 1972). Transductional analysis was carried out using 18 histidine-requiring mutants to see where the hnc-1 locus maps in relation to the structural genes of histidine biosynthesis. The hnc-1 marker cotransduced with group IV genes at 97 to 100 % and not at all with group I, which is known to be the structural gene for HDH. The data obtained in the studies of Km (histidinol) and Km (NAD), and the effect of pH and temperature on the HDH activity from PAO1 and PAO14 are in full agreement with the genetic data that the hnc-1 mutation is not in the structural gene for HDH. It is suggested that hnc-1 may be a mutation in a regulatory gene affecting HDH synthesis in PAO14 and may map close to his-IV whose function in histidine biosynthesis is not known.     

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.     

Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli.

The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.     

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.