Proline utilization system is required for infection by the pathogenic ?-proteobacterium Brucella abortus.
ABSTRACT: Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a ?putR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.
Project description:Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.
Project description:Cellular metabolism recently emerged as a central player modulating the bacterial cell cycle. The Alphaproteobacterium Caulobacter crescentus appears as one of the best models to study these connections, but its metabolism is still poorly characterized. Considering that it lives in oligotrophic environments, its capacity to use amino-acids is often critical for its growth. Here, we characterized the C. crescentus PutA bi-functional enzyme and showed that it is required for the utilization of proline as a carbon source. We also found that putA transcription and proline utilization by PutA are strictly dependent on the Lrp-like PutR activator. The activation of putA by PutR needs proline, which most likely acts as an effector molecule for PutR. Surprisingly, we also observed that an over-production of PutR leads to cell elongation in liquid medium containing proline, while it inhibits colony formation even in the absence of proline on solid medium. These cell division and growth defects were equally pronounced in a ?putA mutant background, indicating that PutR can play other roles beyond the control of proline catabolism. Altogether, these findings suggest that PutR might connect central metabolism with cell cycle processes.
Project description:Proline utilization A (PutA) from Escherichia coli is a membrane-associated trifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate and moonlights as a transcriptional regulator. As a regulatory protein, PutA represses transcription of the put regulon, which contains the genes encoding PutA and the proline transporter PutP. The binding of proline to the proline dehydrogenase active site and the subsequent reduction of the flavin induce high affinity membrane association of PutA and relieve repression of the put regulon, thereby causing PutA to switch from its regulatory to its enzymatic role. Here, we present evidence suggesting that residues of the ?3-?3 loop of the proline dehydrogenase domain (??)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. Mutation of the conserved residues Asp370 and Glu372 in the ?3-?3 loop abrogates the ability of proline to induce functional membrane association. Both in vitro lipid/membrane binding assays and in vivo cell-based assays demonstrate that mutagenesis of Asp370 (D370N/A) or Glu372 (E372A) dramatically impedes PutA functional switching. The crystal structures of the proline dehydrogenase domain mutants PutA86-630D370N and PutA86-630D370A complexed with the proline analogue l-tetrahydro-2-furoic acid show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. Rather, these results suggest that the ?3-?3 loop may be involved in transmitting the status of the proline dehydrogenase active site and flavin redox state to the distal membrane association domain.
Project description:The Vibrio vulnificus putAP genes encoding a proline dehydrogenase and a proline permease are transcribed in the same direction. Proline dehydrogenase activity and the level of putA transcript were determined to reach a maximum in exponential phase and were then repressed when growth slowed down. Northern blotting and primer extension analyses revealed that transcription of putAP genes results in two different transcripts, transcript A (putA transcript) and transcript AP (putAP transcript). Expression of putAP genes was directed by two promoters, promoter P(putA) and promoter P(putAP). A crp null mutation decreased proline dehydrogenase activity and the level of the put transcripts, indicating that transcription of putAP is under the positive control of cyclic AMP receptor protein. Proline dehydrogenase and the level of both put transcripts were increased by proline but repressed by glutamate. In contrast, the level of transcript A, not transcript AP, increased when proline dehydrogenase was induced by NaCl. Since P(putA) activity, not P(putAP) activity, was increased by NaCl, it is apparent that transcript A and transcript AP are transcribed through P(putA) and P(putAP), respectively. Cells challenged with NaCl and various hyperosmotic stresses accumulated higher levels of glutamate than control cells, indicating that glutamate is a compatible solute in V. vulnificus.
Project description:Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and ?1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs.
Project description:Pseudomonas aeruginosa PAO1 utilizes proline as the sole source of carbon and nitrogen via a bifunctional enzyme (the putA gene product) that has both proline dehydrogenase (EC 188.8.131.52) and pyrroline 5-carboxylate dehydrogenase (EC 184.108.40.206) activities. We characterized the pruR-putAP loci encoding the proline catabolic system of this strain. In contrast to the putA and putP (encoding proline permease) genes of other gram- negative bacteria, which are located at divergent or separate loci, Northern blotting demonstrated that the two genes form an operon in strain PAO1. While the phylogenetic lineage of the PutP protein of strain PAO1 was related to that of the origin (80% identity to the P. putida counterpart), PutA of PAO1 (PutA(PAO)) was rather distantly related (47% identity) to the P. putida counterpart. Moreover, unlike the PutA proteins of P. putida and enteric bacteria, PutA(PAO) appeared to lack a regulatory function. Upstream of the putAP operon, the divergent PA0781 gene specified a hypothetical outer membrane protein with a molecular weight of 74,202. This gene appeared to be dispensable for proline utilization as indicated by the normal growth of a knockout mutant of PA0781 on medium containing proline. The pruR (proline utilization regulator) gene immediately upstream of PA0781 encoded a transcriptional activator of the AraC/XylS protein family and mediated the proline-responsive expression of putAP. Primer extension studies identified a PruR-dependent promoter responsive to proline in the 5'-flanking region of putA. Thus, the proline utilization system of P. aeruginosa differs from that of P. putida with respect to putA structure, the organization of the putAP genes, and the regulatory mechanism of putA expression.
Project description:Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.
Project description:Proline has important roles in multiple biological processes such as cellular bioenergetics, cell growth, oxidative and osmotic stress response, protein folding and stability, and redox signaling. The proline catabolic pathway, which forms glutamate, enables organisms to utilize proline as a carbon, nitrogen, and energy source. FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent glutamate semialdehyde dehydrogenase (GSALDH) convert proline to glutamate in two sequential oxidative steps. Depletion of PRODH and GSALDH in humans leads to hyperprolinemia, which is associated with mental disorders such as schizophrenia. Also, some pathogens require proline catabolism for virulence. A unique aspect of proline catabolism is the multifunctional proline utilization A (PutA) enzyme found in Gram-negative bacteria. PutA is a large (>1000 residues) bifunctional enzyme that combines PRODH and GSALDH activities into one polypeptide chain. In addition, some PutAs function as a DNA-binding transcriptional repressor of proline utilization genes. This review describes several attributes of PutA that make it a remarkable flavoenzyme: (1) diversity of oligomeric state and quaternary structure; (2) substrate channeling and enzyme hysteresis; (3) DNA-binding activity and transcriptional repressor function; and (4) flavin redox dependent changes in subcellular location and function in response to proline (functional switching).
Project description:Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to its enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the epsilon-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline-reduced, membrane-associated form. The 2.15 A resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane-binding domain include major reorganization of the flavin ribityl chain, severe (35 degrees ) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5) atom, Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the beta1-alphal loop to complete the active site.
Project description:Proline is converted to glutamate in two successive steps by the proline utilization A (PutA) flavoenzyme in gram-negative bacteria. PutA contains a proline dehydrogenase domain that catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidation of proline to delta1-pyrroline-5-carboxylate (P5C) and a P5C dehydrogenase domain that catalyzes the NAD+-dependent oxidation of P5C to glutamate. Here, we characterize PutA from Helicobacter hepaticus (PutA(Hh)) and Helicobacter pylori (PutA(Hp)) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutA(Hh) and PutA(Hp) lack DNA binding activity, in contrast to PutA from Escherichia coli (PutA(Ec)), which both regulates and catalyzes proline utilization. PutA(Hh) and PutA(Hp) display catalytic activities similar to that of PutA(Ec) but have higher oxygen reactivity. PutA(Hh) and PutA(Hp) exhibit 100-fold-higher turnover numbers (approximately 30 min(-1)) than PutA(Ec) (<0. 3 min(-1)) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutA(Hh) forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutA(Hh) and PutA(Hp) was probed by oxidative stress studies in E. coli. Expression of PutA(Ec) and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutA(Hp) and PutA(Hh) expression dramatically reduces E. coli cell survival and is correlated with relatively lower proline levels and increased hydrogen peroxide formation. The discovery of reduced oxygen species formation by PutA suggests that proline catabolism may influence redox homeostasis in the ecological niches of these Helicobacter species.