1887

Microbiology Society logo

Volume 160, Issue 10

Functional characterization of the locus for -Glu and -Gln utilization in PAO1 Free

Abstract

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

  • Received:
  • Accepted:
  • Published Online:
Funding
This study was supported by the:
  • National Science Foundation (Award NSF0950217)
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.081141-0
2014-10-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/10/2331.html?itemId=/content/journal/micro/10.1099/mic.0.081141-0&mimeType=html&fmt=ahah

References

  1. Doublet P., van Heijenoort J., Bohin J. P., Mengin-Lecreulx D. ( 1993). The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity. J Bacteriol 175:2970–2979[PubMed]
     [Google Scholar]
  2. Farinha M. A., Kropinski A. M. ( 1990). Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol 172:3496–3499[PubMed]
     [Google Scholar]
  3. Fu G., Yuan H., Li C., Lu C. D., Gadda G., Weber I. T. ( 2010). Conformational changes and substrate recognition in Pseudomonas aeruginosa d-arginine dehydrogenase. Biochemistry 49:8535–8545 [View Article][PubMed]
     [Google Scholar]
  4. Galakatos N. G., Daub E., Botstein D., Walsh C. T. ( 1986). Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination. Biochemistry 25:3255–3260 [View Article][PubMed]
     [Google Scholar]
  5. Haas D., Holloway B. W., Schamböck A., Leisinger T. ( 1977). The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa. Mol Gen Genet 154:7–22 [View Article][PubMed]
     [Google Scholar]
  6. Hashim S., Kwon D. H., Abdelal A., Lu C. D. ( 2004). The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa. J Bacteriol 186:3848–3854 [View Article][PubMed]
     [Google Scholar]
  7. He W., Li C., Lu C. D. ( 2011). Regulation and characterization of the dadRAX locus for d-amino acid catabolism in Pseudomonas aeruginosa PAO1. J Bacteriol 193:2107–2115 [View Article][PubMed]
     [Google Scholar]
  8. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. ( 1998). A broad-host-range Flp–FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [View Article][PubMed]
     [Google Scholar]
  9. Johnson D. A., Tetu S. G., Phillippy K., Chen J., Ren Q., Paulsen I. T. ( 2008). High-throughput phenotypic characterization of Pseudomonas aeruginosa membrane transport genes. PLoS Genet 4:e1000211 [View Article][PubMed]
     [Google Scholar]
  10. Kolodkin-Gal I., Romero D., Cao S., Clardy J., Kolter R., Losick R. ( 2010). d-Amino acids trigger biofilm disassembly. Science 328:627–629 [View Article][PubMed]
     [Google Scholar]
  11. Lam H., Oh D. C., Cava F., Takacs C. N., Clardy J., de Pedro M. A., Waldor M. K. ( 2009). d-Amino acids govern stationary phase cell wall remodeling in bacteria. Science 325:1552–1555 [View Article][PubMed]
     [Google Scholar]
  12. Lambrecht J. A., Flynn J. M., Downs D. M. ( 2012). Conserved YjgF protein family deaminates reactive enamine/imine intermediates of pyridoxal 5′-phosphate (PLP)-dependent enzyme reactions. J Biol Chem 287:3454–3461 [View Article][PubMed]
     [Google Scholar]
  13. Li C., Lu C. D. ( 2009a). Arginine racemization by coupled catabolic and anabolic dehydrogenases. Proc Natl Acad Sci U S A 106:906–911 [View Article][PubMed]
     [Google Scholar]
  14. Li C., Lu C. D. ( 2009b). Unconventional integration of the bla gene from plasmid pIT2 during ISlacZ/hah transposon mutagenesis in Pseudomonas aeruginosa PAO1. Curr Microbiol 58:472–477 [View Article][PubMed]
     [Google Scholar]
  15. Li C., Yao X., Lu C. D. ( 2010). Regulation of the dauBAR operon and characterization of d-amino acid dehydrogenase DauA in arginine and lysine catabolism of Pseudomonas aeruginosa PAO1. Microbiology 156:60–71 [View Article][PubMed]
     [Google Scholar]
  16. Lu C. D., Yang Z., Li W. ( 2004). Transcriptome analysis of the ArgR regulon in Pseudomonas aeruginosa. J Bacteriol 186:3855–3861 [View Article][PubMed]
     [Google Scholar]
  17. Nishijyo T., Park S. M., Lu C. D., Itoh Y., Abdelal A. T. ( 1998). Molecular characterization and regulation of an operon encoding a system for transport of arginine and ornithine and the ArgR regulatory protein in Pseudomonas aeruginosa. J Bacteriol 180:5559–5566[PubMed]
     [Google Scholar]
  18. Singh B., Röhm K. H. ( 2008). Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system. Microbiology 154:797–809 [View Article][PubMed]
     [Google Scholar]
  19. Sonawane A., Klöppner U., Derst C., Röhm K. H. ( 2003). Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase. Arch Microbiol 179:151–159[PubMed]
     [Google Scholar]
  20. Wild J., Hennig J., Lobocka M., Walczak W., Kłopotowski T. ( 1985). Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K12. Mol Gen Genet 198:315–322 [View Article][PubMed]
     [Google Scholar]
  21. Winsor G. L., Lam D. K., Fleming L., Lo R., Whiteside M. D., Yu N. Y., Hancock R. E., Brinkman F. S. ( 2011). Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39:Database issueD596–D600 [View Article][PubMed]
     [Google Scholar]
  22. Wu H. M., Kuan Y. C., Chu C. H., Hsu W. H., Wang W. C. ( 2012). Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants. PLoS ONE 7:e48301 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.081141-0
Loading
Functional characterization of the dguRABC locus for d-Glu and d-Gln utilization in Pseudomonas aeruginosa PAO1
Microbiology 160, 2331 (2014); https://doi.org/10.1099/mic.0.081141-0
/content/journal/micro/10.1099/mic.0.081141-0
/content/journal/micro/10.1099/mic.0.081141-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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