Induction of the pho regulon and polyphosphate synthesis against spermine stress in Pseudomonas aeruginosa.

Growth of Pseudomonas aeruginosa on spermine requires a functional γ-glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to ß-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C599 T mutation at the phoU gene that results in Ser200 Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild-type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.

Molecular characterization and regulation of operons for asparagine and aspartate uptake and utilization in Pseudomonas aeruginosa.

Pseudomonas aeruginosa can utilize proteogenic amino acids as the sole source of carbon and nitrogen. In particular, utilization of l-Asp and l-Asn is insensitive to carbon catabolite repression as strong growth remains in the mutants devoid of the essential CbrAB activators of most catabolic genes. Transcriptome analysis was conducted to identify genes for the catabolism, uptake and regulation of these two amino acids. Gene inactivation and growth phenotype analysis established two asparaginases AsnA and AsnB for the degradation of l-Asn to l-Asp, whereas only AnsB is required for the deamidation of d-Asn to d-Asp. While d-Asp is a dead-end product, conversion of l-Asp to fumarate is catalysed by an aspartase AspA as further evidenced by enzyme kinetics. The results of measuring promoter-lacZ expression in vivo and mobility shift assays in vitro demonstrated that asnR and aspR encode two transcriptional regulators in response to l-Asn and l-Asp, respectively, for the induction of the ansPA operon and the aspA gene. Exogenous l-Glu also caused induction of the aspA gene, most likely due to its conversion to l-Asp by the aspartate transaminase AspC. Expression of several transporters were found inducible by l-Asn and/or l-Asp, including AatJQMP for acid amino acids, DctA and DctPQM for C4-dicarboxylates, and PA5530 for C5-dicarboxylates. In summary, a complete pathway and regulation for l-Asn and l-Asp catabolism was established in this study. Cross induction of three transport systems for dicarboxylic acids may provide a physiological explanation for the insensitivity of l-Asn and l-Asp utilization to carbon catabolite repression.

Molecular characterization of LhpR in control of hydroxyproline catabolism and transport in Pseudomonas aeruginosa PAO1.

Utilization of hydroxy-l-proline (l-Hyp) in Pseudomonas aeruginosa requires conversion of l-Hyp to d-Hyp followed by the d-Hyp dehydrogenase pathway; however, the molecular mechanism in control of l-Hyp catabolism and transport was not clear. DNA microarray analysis revealed twelve genes in two adjacent loci that were induced by exogenous l-Hyp and d-Hyp. The first locus includes lhpABFE encoding a Hyp epimerase (LhpA) and d-Hyp dehydrogenase (LhpBEF), while the second locus codes for a putative ABC transporter (LhpPMNO), a protein of unknown function (LhpH), Hyp/Pro racemase (LhpK) and two enzymes in l-Hyp catabolism (LhpC and LhpG). Proximal to these two loci, lhpR encodes a transcriptional regulator of the AraC family. The importance of these genes on l-Hyp catabolism was supported by growth phenotype analysis on knockout mutants. Induction of the lhpA and lhpP promoters by exogenous l-Hyp and d-Hyp was demonstrated by the measurement of ß-galactosidase activities from promoter-lacZ fusions in PAO1, and no induction could be detected in the ΔlhpR mutant. Induction of the lhpA promoter by d-Hyp was completely abolished in the lhpA lhpK double mutant devoid of two epimerases, while the induction effect of l-Hyp remained unchanged. The purified His-tagged LhpR binds specifically to the lhp promoter regions, and formation of nucleoprotein complexes is affected by the presence of l-Hyp but not d-Hyp. Putative LhpR binding sites were deduced from serial deletions and comparative genomic sequence analysis. In summary, expression of lhp genes for Hyp catabolism and uptake requires the transcriptional activator LhpR and l-Hyp as the signalling compound.

Molecular characterization of lysR-lysXE, gcdR-gcdHG and amaR-amaAB operons for lysine export and catabolism: a comprehensive lysine catabolic network in Pseudomonas aeruginosa PAO1.

Among multiple interconnected pathways for l-Lysine catabolism in pseudomonads, it has been reported that Pseudomonas aeruginosa PAO1 employs the decarboxylase and the transaminase pathways. However, up until now, knowledge of several genes involved in operation and regulation of these pathways was still missing. Transcriptome analyses coupled with promoter activity measurements and growth phenotype analyses led us to identify new members in l-Lys and d-Lys catabolism and regulation, including gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for d-Lys catabolism, lysR-lysXE for putative l-Lys efflux and lysP for putative l-Lys uptake. The gcdHG operon encodes an acyl-CoA transferase (gcdG) and glutaryl-CoA dehydrogenase (gcdH) and is under the control of the transcriptional activator GcdR. Growth on l-Lys was enhanced in the mutants of lysX and lysE, supporting the operation of l-Lys efflux. The transcriptional activator LysR is responsible for l-Lys specific induction of lysXE and the PA4181-82 operon of unknown function. The putative operator sites of GcdR and LysR were deduced from serial deletions and comparative genomic sequence analyses, and the formation of nucleoprotein complexes was demonstrated with purified His-tagged GcdR and LysR. The amaAB operon encodes two enzymes to convert pipecolate to 2-aminoadipate. Induction of the amaAB operon by l-Lys, d-Lys and pipecolate requires a functional AmaR, supporting convergence of Lys catabolic pathways to pipecolate. Growth on pipecolate was retarded in the gcdG and gcdH mutants, suggesting the importance of glutarate in pipecolate and 2-aminoadipate utilization. Furthermore, this study indicated links in the control of interconnected networks of lysine and arginine catabolism in P. aeruginosa.

The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 µM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution.

Functional characterization of the dguRABC locus for D-Glu and d-Gln utilization in Pseudomonas aeruginosa PAO1.

D-Glu, an essential component of peptidoglycans, can be utilized as a carbon and nitrogen source by Pseudomonas aeruginosa. DNA microarrays were employed to identify genes involved in D-Glu catabolism. Through gene knockout and growth phenotype analysis, the divergent dguR-dguABC (D-Glu utilization) gene cluster was shown to participate in D-Glu and D-Gln catabolism and regulation. Growth of the dguR and dguA mutants was abolished completely on D-Glu or retarded on D-Gln as the sole source of carbon and/or nitrogen. The dguA gene encoded a FAD-dependent D-amino acid dehydrogenase with d-Glu as its preferred substrate, and its promoter was specifically induced by exogenous D-Glu and D-Gln. The function of DguR as a transcriptional activator of the dguABC operon was demonstrated by promoter activity measurements in vivo and by mobility shift assays with purified His-tagged DguR in vitro. Although the DNA-binding activity of DguR did not require D-Glu, the presence of D-Glu, but not D-Gln, in the binding reaction was found to stabilize a preferred nucleoprotein complex. The presence of a putative DguR operator was revealed by in silica analysis of the dguR-dguA intergenic regions among Pseudomonas spp. and binding of DguR to a highly conserved 19 bp sequence motif was further demonstrated. The dguB gene encodes a putative enamine/imine deaminase of the RidA family, but its role in D-Glu catabolism remains to be determined. Whilst a lesion in dguC encoding a periplasmic solute binding protein only affected growth on D-Glu slightly, expression of the previously characterized AatJMQP transporter for acidic l-amino acid uptake was found inducible by D-Glu and essential for D-Glu utilization. In summary, the findings of this study supported DguA as a new member of the FAD-dependent d-amino acid dehydrogenase family, and DguR as a D-Glu sensor and transcriptional activator of the dguA promoter.

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion.

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.

A novel Pseudomonas aeruginosa strain with an oprD mutation in relation to a nosocomial respiratory infection outbreak in an intensive care unit.

Seven imipenem-resistant Pseudomonas aeruginosa isolates were recovered from the sputum samples of pneumonia patients in southwestern China. They had identical antibiotic resistance patterns and indistinguishable pulsed-field gel electrophoresis profiles. Nucleotide sequence analysis revealed a 4-bp (AGTC) insertion in the oprD gene, resulting in a frameshift in the cognate open reading frame. These isolates became imipenem susceptible when the chromosomal oprD lesion was complemented, indicating that the 4-bp insertion in the oprD gene resulted in imipenem resistance.

Functional characterization of the potRABCD operon for spermine and spermidine uptake and regulation in Staphylococcus aureus.

Spermine, a potent bactericidal polyamine, exerts a strong synergistic effect with ß-lactams against methicillin-resistant Staphylococcus aureus. Transcriptome analysis revealed that the putative potRABCD operon for polyamine uptake and regulation exhibited significant fold change upon exposure to exogenous spermine. Properties of the PotABCD transporter in polyamine uptake were studied using wild-type and the pot deletion mutant. It was found that spermidine and spermine, but not putrescine, were the preferred substrates for the Pot system of high affinity. The PotR protein was purified from a recombinant strain of Escherichia coli, and binding of PotR to the pot regulatory region was demonstrated by electromobility shift assays. In summary, these results support the physiological function of PotR in regulation of the expression of PotABCD for spermidine and spermine uptake in S. aureus.

Characterization of Staphylococcus aureus responses to spermine stress.

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA). To explore the Spm-based antibacterial targets in S. aureus, time course-dependent transcriptome analysis was conducted on Mu50 (MRSA) in the absence and presence of Spm. Genes in the sigB regulon and most ATP-producing pathways were found down-regulated when exposure to high dose Spm. In contrast, a number of genes for iron acquisition and regulation showed significant induction, indicating a specific connection between Spm and iron-depletion. The tetM gene for tetracycline (Tc) resistance exhibited most significant fold change among the listed genes. It was specifically upregulated by Tc and Spm but not by other ribosome-targeted drugs or other polyamines; however, such induction of tetM cannot confer resistance to Spm. A set of genes for osmotic balance, including kdpABCDE for potassium ion uptake and regulation, was also induced by Spm stress. Addition of KCl or NaCl, but not high concentration sucrose, was found to increase Spm MIC over 30-fold. In summary, transcriptome analysis demonstrated a specific pattern of response upon Spm exposure, suggesting Spm may alter the intracellular iron status and suppress the SigB regulon to exert its toxicity.

Time-related transcriptome analysis of B. subtilis 168 during growth with glucose.

Gene expression in Bacillus subtilis from late exponential to stationary phase was monitored by DNA microarrays with samples taken from the culture in LB broth with glucose supplement to prevent sporulation. Three major patterns of gene expression as revealed in this study were consistent to the expression profiling of PerR/Spx regulons and three major sigma factors-SigA, SigB, and SigW. Expression of most SigA-dependent house-keeping genes was significantly decreased and remained at low levels in the stationary phase. The sigB gene and additional genes of the SigB regulon for stress response exhibited a distinct pattern of transient induction with a peak in transition phase. The majority of induced genes after cessation of SigB-dependent surge were subjected to regulation by SigW, PerR, and Spx in response to oxidative stress. No induction of spo0A and skfA regulons supports the suppression of sporulation and cannibalism processes in the stationary phase by glucose supplement. In summary, these results depicted complicated strategies by cells to adapt changes from the fast growing exponential phase toward the stationary phase. The absence of programmed cell death and sporulation greatly facilitated data analysis and the identification of distinct expression patterns in the stationary phase of growth in B. subtilis.

Functional characterization of the agtABCD and agtSR operons for 4-aminobutyrate and 5-aminovalerate uptake and regulation in Pseudomonas aeruginosa PAO1.

Growth of Pseudomonas aeruginosa on diamines cadaverine, putrescine, and diaminopropane requires the γ-glutamylation pathway to convert these diamines into δ-aminovalerate (AMV), γ-aminobutyrate (GABA), and ß-alanine. From DNA microarrays experiments the agtABCD operon (PA0603-0606) encoding components for an ABC transporter system was found inducible by exogenous AMV, GABA, and ß-alanine, but not by diamines. Induction of the agtABCD operon was abolished in the mutants of upstream agtS (PA0600) or agtR (PA0601) genes encoding the membrane-anchored sensor and the response regulator of a two-component regulatory system, respectively. Growth phenotype analysis supports the physiological functions of these agt genes on utilization of AMV and GABA. Through measurements of ß-galactosidase activities from an agtA::lacZ fusion, the requirement of a functional AgtS in control of the induction effect by exogenous AMV and GABA was further substantiated. The recombinant hexa-hisidine tagged agtR was constructed and purified to demonstrate its specific interactions with the agtA promoter region by electrophoretic mobility shift assays. In summary, this study establishes the functions of agtSR and agtABCD operons in AMV and GABA uptake, and provides a potential linkage between AMV/GABA metabolism and polymicrobial infection through the recently reported function of agtR in sensing of peptidoglycan shed by gram-positive bacteria (Korgaonkar et al., Proc Natl Acad Sci USA 110:1059-1064, 2013).

Molecular characterization of PauR and its role in control of putrescine and cadaverine catabolism through the γ-glutamylation pathway in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the γ-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utilization) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4, and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activation of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mutant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauA genes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR to the promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT and CAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT was more proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauA promoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly establishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the γ-glutamylation pathway.

Differential expression of secretion machinery during bacterial growth: SecY and SecF decrease while SecA increases during transition from exponential phase to stationary phase.

Transcription of many house-keeping genes, including secY and some other sec genes, decreases in the transition from the exponential phase to the stationary phase (feast to famine) in Bacillus subtilis. Unexpectedly and in contradiction to earlier reports, enhanced transcription was observed for another group of sec genes, including secA which codes for an essential ATPase for protein secretion. Consistent with the transcription data, the SecA protein of B. subtilis increases significantly in the stationary phase. Immunoblot analyses of Sec proteins during the transition in Escherichia coli also revealed the pronounced decreases of SecY and SecF and the increase of SecA, resulting in drastic increases of SecA/SecY and SecA/SecF ratios from exponential to stationary phases. The differential expression of Sec proteins in the stationary phase suggests the possibility of specific physiological functions.

A PBP 2 mutant devoid of the transpeptidase domain abolishes spermine-ß-lactam synergy in Staphylococcus aureus Mu50.

Exogenous spermine was reported to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by ß-lactams through a strong synergistic effect of unknown nature. Spermine alone also exerts an antimicrobial activity against S. aureus in a pH-dependent manner. MIC measurements revealed stronger effects of spermine under alkaline conditions, suggesting the nucleophilic property of spermine instead of its positive charge as the cause of adverse effects. A spontaneous suppressor mutant (MuM) of MRSA Mu50 was selected for spermine resistance and conferred complete abolishment of spermine-ß-lactam synergy. In comparison to that in Mu50, the spermine MIC in MuM remained constant (64 mM) at pH 6 to 8; however, MuM, a heat-sensitive mutant, also grew in a very narrow pH range. Furthermore, MuM acquired a unique phenotype of vancomycin-spermine synergy. Genome resequencing revealed a 7-bp deletion in pbpB, which results in a truncated penicillin-binding protein 2 (PBP 2) without the transpeptidase domain at the C terminus while the N-terminal transglycosidase domain remains intact. The results of fluorescent Bocillin labeling experiments confirmed the presence of this defective PBP 2 in MuM. All the aforementioned phenotypes of MuM were reverted to those of Mu50 after complementation by the wild-type pbpB carried on a recombinant plasmid. The anticipated changes in cell wall metabolism and composition in MuM were evidenced by observations that the cell wall of MuM was more susceptible to enzyme hydrolysis and that MuM exhibited a lower level of autolytic activities. Pleiotropic alterations in gene expression were revealed by microarray analysis, suggesting a remarkable flexibility of MuM to circumvent cell wall damage by triggering adaptations that are complex but completely different from that of the cell wall stress stimulon. In summary, these results reveal phenotypic changes and transcriptome adaptations in a unique pbpB mutant and provide evidence to support the idea that exogenous spermine may perturb normal cell wall formation through its interactions with PBP 2.

γ-glutamyl Spermine Synthetase PauA2 as a potential target of antibiotic development against Pseudomonas aeruginosa.

Polyamines are absolute requirements for cell growth. When in excess, Pseudomonas aeruginosa possesses six γ-glutamylpolyamine synthetases (GPSs) encoded by the pauA1-pauA7 genes to initiate polyamine catabolism. Recently, the pauA2 mutant was reported to lose the capability to grow on spermine (Spm) and spermidine (Spd) as sole carbon and nitrogen sources. Although this mutant grew normally in defined minimal medium and LB broth, growth was completely abolished by the addition of Spm or Spd. These two compounds exert a bactericidal effect (Spm > Spd) on the mutants as demonstrated by MIC measurements (over 500-fold reduction) and time-killing curves. Spm toxicity in the pauA2 mutant was attenuated when the major uptake system was further deleted from the strain, suggesting cytoplasmic targets of toxicity. In addition, the synergistic effect of Spm and carbenicillin in the wild-type strain PAO1 was diminished in mutants without functional PauA2. Furthermore, Spm MIC was reduced by 8-fold when the Spm uptake system was deleted from the wild-type strain, suggesting a second target of Spm toxicity in the periplasm. Experiments were also conducted to test the hypothesis that native Spm and Spd in human serum may be sufficient to kill the pauA2 mutant. Growth of the mutant was completely inhibited by 40% (vol/vol) human serum, whereas the parental strain required 80%. Colony counts indicated that the mutant but not the parent was in fact killed by human plasma. In addition, carbenicillin MIC against the mutant was reduced by 16-fold in the presence of 20% human serum while that of the parental strain remained unchanged. Taking PauA2 as the template, sequence comparison indicates that putative PauA2 homologues are widespread in a variety of Gram-negative bacteria. In summary, this study reveals the importance of GPS in alleviation of polyamine toxicity when in excess, and it provides strong support to the feasibility of GPS as a molecular target for new antibiotic development.

Arginine racemization by coupled catabolic and anabolic dehydrogenases.

D-amino acids exist in living organisms as specialized components of many different machineries. Biosynthesis of D-amino acids from racemization of predominant L-enantiomers is catalyzed by a single enzyme. Here, we report the finding of a novel 2-component amino acid racemase for D-to-L inversion in D-arginine metabolism of Pseudomonas aeruginosa. From DNA microarray analysis, the putative dauBAR operon (for D-arginine utilization) of unknown functions was found to be highly induced by D-arginine. The importance of the dau operon in D-arginine metabolism was demonstrated by the findings that strains with a lesion at dauA or dauB failed to use D-arginine as sole carbon source. Two lines of evidence suggest that DauA and DauB are required for D-to-L racemization of arginine. First, growth complementation of an L-arginine auxotroph by D-arginine was abolished by a lesion at dauA or dauB. Second, D-arginine induced L-arginine-specific genes in the parental strain PAO1 but not in its dauA or dauB mutants. This hypothesis was further supported by activity measurements of the purified enzymes: DauA catalyzes oxidative deamination of D-arginine into 2-ketoarginine and ammonia, and DauB is able to use 2-ketoarginine and ammonia as substrates and convert them into L-arginine in the presence of NADPH or NADH. Thus, we propose that DauA and DauB are coupled catabolic and anabolic dehydrogenases to perform D-to-L racemization of arginine, which serves as prerequisite of D-arginine utilization through L-arginine catabolic pathways.

Functional characterization of seven γ-Glutamylpolyamine synthetase genes and the bauRABCD locus for polyamine and ß-Alanine utilization in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa and many other bacteria can utilize biogenic polyamines, including diaminopropane (DAP), putrescine (Put), cadaverine (Cad), and spermidine (Spd), as carbon and/or nitrogen sources. Transcriptome analysis in response to exogenous Put and Spd led to the identification of a list of genes encoding putative enzymes for the catabolism of polyamines. Among them, pauA1 to pauA6, pauB1 to pauB4, pauC, and pauD1 and pauD2 (polyamine utilization) encode enzymes homologous to Escherichia coli PuuABCD of the γ-glutamylation pathway in converting Put into GABA. A series of unmarked pauA mutants was constructed for growth phenotype analysis. The results revealed that it requires specific combinations of pauA knockouts to abolish utilization of different polyamines and support the importance of γ-glutamylation for polyamine catabolism in P. aeruginosa. Another finding was that the list of Spd-inducible genes overlaps almost completely with that of Put-inducible ones except the pauA3B2 operon and the bauABCD operon (ß-alanine utilization). Mutation analysis led to the conclusion that pauA3B2 participate in catabolism of DAP, which is related to the aminopropyl moiety of Spd, and that bauABCD are essential for growth on ß-alanine derived from DAP (or Spd) catabolism via the γ-glutamylation pathway. Measurements of the pauA3-lacZ and bauA-lacZ expression indicated that these two promoters were differentially induced by Spd, DAP, and ß-alanine but showed no apparent response to Put, Cad, and GABA. Induction of the pauA3 and bauA promoters was abolished in the bauR mutant. The recombinant BauR protein was purified to demonstrate its interactions with the pauA3 and bauA regulatory regions in vitro. In summary, the present study support that the γ-glutamylation pathway for polyamine utilization is evolutionarily conserved in E. coli and Pseudomonas spp. and is further expanded in Pseudomonas to accommodate a more diverse metabolic capacity in this group of microorganisms.

Regulation and characterization of the dadRAX locus for D-amino acid catabolism in Pseudomonas aeruginosa PAO1.

D-amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In Pseudomonas aeruginosa, the dadAX operon, encoding the D-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for D- and L-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all D-amino acids tested as substrates except D-Glu and D-Gln. DadA also showed comparable k(cat) and K(m) values on D-Ala and several D-amino acids. dadRAX knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of L-Ala, L-Trp, D-Ala, and a specific set of D-amino acids as sole nitrogen sources was abolished in the dadA mutant and/or severely hampered in the dadR mutant while growth yield on D-amino acids was surprisingly improved in the dadX mutant. The dadA promoter was induced by several L-amino acids, most strongly by Ala, and only by D-Ala among all tested D-amino acids. Enhanced growth of the dadX mutant on D-amino acids is consistent with the finding that the dadA promoter was constitutively induced in the dadX mutant, where exogenous D-Ala but not L-Ala reduced the expression. Binding of DadR to the dadA regulatory region was demonstrated by electromobility shift assays, and the presence of L-Ala but not D-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the dadA regulatory region was demonstrated in vitro, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation in vivo. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed D-amino acid dehydrogenase activity of broad substrate specificity and the physiological functions of DadRAX in catabolism of several D-amino acids and support L-Ala as the signal molecule for induction of the dadAX genes through DadR binding to several putative operator sites.

Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.

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.