1887

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 ΔputR 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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000490
2017-07-08
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/163/7/970.html?itemId=/content/journal/micro/10.1099/mic.0.000490&mimeType=html&fmt=ahah

References

  1. Celli J. The changing nature of the Brucella-containing vacuole. Cell Microbiol 2015;17:951–958 [CrossRef][PubMed]
    [Google Scholar]
  2. Godfroid J, Scholz HC, Barbier T, Nicolas C, Wattiau P et al. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Prev Vet Med 2011;102:118–131 [CrossRef][PubMed]
    [Google Scholar]
  3. Roop RM, Gaines JM, Anderson ES, Caswell CC, Martin DW. Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host. Med Microbiol Immunol 2009;198:221–238 [CrossRef][PubMed]
    [Google Scholar]
  4. Halvorson H. Utilization of single L-amino acids as sole source of carbon and nitrogen by bacteria. Can J Microbiol 1972;18:1647–1650 [CrossRef][PubMed]
    [Google Scholar]
  5. Chubukov V, Gerosa L, Kochanowski K, Sauer U. Coordination of microbial metabolism. Nat Rev Microbiol 2014;12:327–340 [CrossRef][PubMed]
    [Google Scholar]
  6. Price CT, Al-Quadan T, Santic M, Rosenshine I, Abu Kwaik Y. Host proteasomal degradation generates amino acids essential for intracellular bacterial growth. Science 2011;334:1553–1557 [CrossRef][PubMed]
    [Google Scholar]
  7. Gouzy A, Poquet Y, Neyrolles O. Amino acid capture and utilization within the Mycobacterium tuberculosis phagosome. Future Microbiol 2014;9:631–637 [CrossRef][PubMed]
    [Google Scholar]
  8. Barel M, Ramond E, Gesbert G, Charbit A. The complex amino acid diet of Francisella in infected macrophages. Front Cell Infect Microbiol 2015;5:9 [CrossRef][PubMed]
    [Google Scholar]
  9. Gerhardt P, Tucker LA, Wilson JB. The nutrition of brucellae: utilization of single amino acids for growth. J Bacteriol 1950;59:777–782[PubMed]
    [Google Scholar]
  10. Ronneau S, Moussa S, Barbier T, Conde-Álvarez R, Zuniga-Ripa A et al. Brucella, nitrogen and virulence. Crit Rev Microbiol 2016;42:507–525 [CrossRef][PubMed]
    [Google Scholar]
  11. Kohler S, Foulongne V, Ouahrani-Bettache S, Bourg G, Teyssier J et al. The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell. Proc Natl Acad Sci USA 2002;99:15711–15716 [CrossRef][PubMed]
    [Google Scholar]
  12. Tanner JJ. Structural biology of proline catabolism. Amino Acids 2008;35:719–730 [CrossRef][PubMed]
    [Google Scholar]
  13. Ratzkin B, Roth J. Cluster of genes controlling proline degradation in Salmonella typhimurium. J Bacteriol 1978;133:744–754[PubMed]
    [Google Scholar]
  14. Newell SL, Brill WJ. Mutants of Salmonella typhimurium that are insensitive to catabolite repression of proline degradation. J Bacteriol 1972;111:375–382[PubMed]
    [Google Scholar]
  15. Cho K, Winans SC. The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated. Mol Microbiol 1996;22:1025–1033 [CrossRef][PubMed]
    [Google Scholar]
  16. Keuntje B, Masepohl B, Klipp W. Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein. J Bacteriol 1995;177:6432–6439 [CrossRef][PubMed]
    [Google Scholar]
  17. Jafri S, Evoy S, Cho K, Craighead HG, Winans SC. An Lrp-type transcriptional regulator from Agrobacterium tumefaciens condenses more than 100 nucleotides of DNA into globular nucleoprotein complexes. J Mol Biol 1999;288:811–824 [CrossRef][PubMed]
    [Google Scholar]
  18. Soto MJ, Jiménez-Zurdo JI, van Dillewijn P, Toro N. Sinorhizobium meliloti putA gene regulation: a new model within the family Rhizobiaceae. J Bacteriol 2000;182:1935–1941 [CrossRef][PubMed]
    [Google Scholar]
  19. Caswell CC, Gaines JM, Roop RM 2nd. The RNA chaperone Hfq independently coordinates expression of the VirB type IV secretion system and the LuxR-type regulator BabR in Brucella abortus 2308. J Bacteriol 2012;194:3–14 [CrossRef][PubMed]
    [Google Scholar]
  20. Bellaire BH, Elzer PH, Hagius S, Walker J, Baldwin CL et al. Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle. Infect Immun 2003;71:1794–1803 [CrossRef][PubMed]
    [Google Scholar]
  21. Miller JH. Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1972; pp.352–355
    [Google Scholar]
  22. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45 [CrossRef][PubMed]
    [Google Scholar]
  23. Sheehan LM, Budnick JA, Blanchard C, Dunman PM, Caswell CC. A LysR-family transcriptional regulator required for virulence in Brucella abortus is highly conserved among the α-proteobacteria. Mol Microbiol 2015;98:318–328 [CrossRef][PubMed]
    [Google Scholar]
  24. Sheehan LM, Budnick JA, Roop RM 2nd, Caswell CC. Coordinated zinc homeostasis is essential for the wild-type virulence of Brucella abortus. J Bacteriol 2015;197:1582–1591 [CrossRef][PubMed]
    [Google Scholar]
  25. Dunigan DD, Waters SB, Owen TC. Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity. Biotechniques 1995;19:640–649[PubMed]
    [Google Scholar]
  26. Luo M, Gamage TT, Arentson BW, Schlasner KN, Becker DF et al. Structures of proline utilization A (PutA) reveal the fold and functions of the aldehyde dehydrogenase superfamily domain of unknown function. J Biol Chem 2016;291:24065–24075 [CrossRef][PubMed]
    [Google Scholar]
  27. Gee JM, Valderas MW, Kovach ME, Grippe VK, Robertson GT et al. The Brucella abortus Cu, Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice. Infect Immun 2005;73:2873–2880 [CrossRef][PubMed]
    [Google Scholar]
  28. Krishnan N, Becker DF. Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress. J Bacteriol 2006;188:1227–1235 [CrossRef][PubMed]
    [Google Scholar]
  29. Krishnan N, Doster AR, Duhamel GE, Becker DF. Characterization of a Helicobacter hepaticus putA mutant strain in host colonization and oxidative stress. Infect Immun 2008;76:3037–3044 [CrossRef][PubMed]
    [Google Scholar]
  30. Zhang L, Alfano JR, Becker DF. Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli. J Bacteriol 2015;197:431–440 [CrossRef][PubMed]
    [Google Scholar]
  31. Menzel R, Roth J. Enzymatic properties of the purified PutA protein from Salmonella typhimurium. J Biol Chem 1981;256:9762–9766[PubMed]
    [Google Scholar]
  32. Menzel R, Roth J. Purification of the putA gene product. A bifunctional membrane-bound protein from Salmonella typhimurium responsible for the two-step oxidation of proline to glutamate. J Biol Chem 1981;256:9755–9761[PubMed]
    [Google Scholar]
  33. Ostrovsky de Spicer P, Maloy S. PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc Natl Acad Sci USA 1993;90:4295–4298 [CrossRef][PubMed]
    [Google Scholar]
  34. Muro-Pastor AM, Ostrovsky P, Maloy S. Regulation of gene expression by repressor localization: biochemical evidence that membrane and DNA binding by the PutA protein are mutually exclusive. J Bacteriol 1997;179:2788–2791 [CrossRef][PubMed]
    [Google Scholar]
  35. Jiménez-Zurdo JI, García-Rodríguez FM, Toro N. The Rhizobium meliloti putA gene: its role in the establishment of the symbiotic interaction with alfalfa. Mol Microbiol 1997;23:85–93 [CrossRef][PubMed]
    [Google Scholar]
  36. van Dillewijn P, Soto MJ, Villadas PJ, Toro N. Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation. Appl Environ Microbiol 2001;67:3860–3865 [CrossRef][PubMed]
    [Google Scholar]
  37. van Dillewijn P, Villadas PJ, Toro N. Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa. Appl Environ Microbiol 2002;68:4201–4208 [CrossRef][PubMed]
    [Google Scholar]
  38. Nakajima K, Inatsu S, Mizote T, Nagata Y, Aoyama K et al. Possible involvement of putA gene in Helicobacter pylori colonization in the stomach and motility. Biomed Res 2008;29:9–18 [CrossRef][PubMed]
    [Google Scholar]
  39. Cheng Z, Lin M, Rikihisa Y. Ehrlichia chaffeensis proliferation begins with NtrY/NtrX and PutA/GlnA upregulation and CtrA degradation induced by proline and glutamine uptake. MBio 2014;5:e02141-14 [CrossRef][PubMed]
    [Google Scholar]
  40. Marr AG, Olsen CB, Unger HS, Wilson JB. The oxidation of glutamic acid by Brucella abortus. J Bacteriol 1953;66:606–610[PubMed]
    [Google Scholar]
  41. Carrica MC, Fernandez I, Martí MA, Paris G, Goldbaum FA. The NtrY/X two-component system of Brucella spp. acts as a redox sensor and regulates the expression of nitrogen respiration enzymes. Mol Microbiol 2012;85:39–50 [CrossRef][PubMed]
    [Google Scholar]
  42. Mirabella A, Yañez Villanueva RM, Delrue RM, Uzureau S, Zygmunt MS et al. The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength. Microbiology 2012;158:2642–2651 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000490
Loading
/content/journal/micro/10.1099/mic.0.000490
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error