1887

Abstract

In serovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible for -inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion of (STM4418) led to a severe growth defect, and deletion of (STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genes Bioluminescence-based reporter assays demonstrated a strong induction of their promoters P and P in the presence of MI but not of glucose. Deletion of , which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of P and P, indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed in . Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Using -[1,2-[H](N)]inositol, a saturable uptake activity of IolT1 with a value between 0.49 and 0.79 mM was determined in DH5 expressing IolT1, in serovar Typhimurium strain 14028, and in mutant 14028 Δ. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.032250-0
2010-01-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/1/128.html?itemId=/content/journal/micro/10.1099/mic.0.032250-0&mimeType=html&fmt=ahah

References

  1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  2. Anderson, W. A. & Magasanik, B. ( 1971; ). The pathway of myo-inositol degradation in Aerobacter aerogenes. Conversion of 2-deoxy-5-keto-d-gluconic acid to glycolytic intermediates. J Biol Chem 246, 5662–5675.
    [Google Scholar]
  3. Bäumchen, C., Krings, E., Bringer, S., Eggeling, L. & Sahm, H. ( 2009; ). myo-Inositol facilitators IolT1 and IolT2 enhance d-mannitol formation from d-fructose in Corynebacterium glutamicum. FEMS Microbiol Lett 290, 227–235.
    [Google Scholar]
  4. Berman, T. & Magasanik, B. ( 1966; ). The pathway of myo-inositol degradation in Aerobacter aerogenes. Ring scission. J Biol Chem 241, 807–813.
    [Google Scholar]
  5. Boutte, C. C., Srinivasan, B. S., Flannick, J. A., Novak, A. F., Martens, A. T., Batzoglou, S., Viollier, P. H. & Crosson, S. ( 2008; ). Genetic and computational identification of a conserved bacterial metabolic module. PLoS Genet 4, e1000310 [CrossRef]
    [Google Scholar]
  6. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  7. Datsenko, K. A. & Wanner, B. L. ( 2000; ). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.[CrossRef]
    [Google Scholar]
  8. Fry, J., Wood, M. & Poole, P. S. ( 2001; ). Investigation of myo-inositol catabolism in Rhizobium leguminosarum bv. viciae and its effect on nodulation competitiveness. Mol Plant Microbe Interact 14, 1016–1025.[CrossRef]
    [Google Scholar]
  9. Gauchat-Feiss, D., Frey, J., Belet, M. & Deshusses, J. ( 1985; ). Cloning of genes involved in myo-inositol transport in a Pseudomonas sp. J Bacteriol 162, 324–327.
    [Google Scholar]
  10. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef]
    [Google Scholar]
  11. Kawsar, H. I., Ohtani, K., Okumura, K., Hayashi, H. & Shimizu, T. ( 2004; ). Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens. FEMS Microbiol Lett 235, 289–295.[CrossRef]
    [Google Scholar]
  12. Klumpp, J. & Fuchs, T. M. ( 2007; ). Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages. Microbiology 153, 1207–1220.[CrossRef]
    [Google Scholar]
  13. Kreutzenbeck, P., Kröger, C., Lausberg, F., Blaudeck, N., Sprenger, G. A. & Freudl, R. ( 2007; ). Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities. J Biol Chem 282, 7903–7911.[CrossRef]
    [Google Scholar]
  14. Krings, E., Krumbach, K., Bathe, B., Kelle, R., Wendisch, V. F., Sahm, H. & Eggeling, L. ( 2006; ). Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on l-lysine formation. J Bacteriol 188, 8054–8061.[CrossRef]
    [Google Scholar]
  15. Kröger, C. & Fuchs, T. M. ( 2009; ). Characterization of the myo-inositol utilization island of Salmonella enterica serovar Typhimurium. J Bacteriol 191, 545–554.[CrossRef]
    [Google Scholar]
  16. Law, C. J., Maloney, P. C. & Wang, D. N. ( 2008; ). Ins and outs of major facilitator superfamily antiporters. Annu Rev Microbiol 62, 289–305.[CrossRef]
    [Google Scholar]
  17. Legakis, N. J., Papavassiliou, J. T. & Xilinas, M. E. ( 1976; ). Inositol as a selective substrate for the growth of klebsiellae and serratiae. Zentralbl Bakteriol Orig A 235, 453–458.
    [Google Scholar]
  18. Link, A. J., Phillips, D. & Church, G. M. ( 1997; ). Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol 179, 6228–6237.
    [Google Scholar]
  19. McIlvaine, T. C. ( 1921; ). A buffer solution for colorimetric comparison. J Biol Chem 49, 183–186.
    [Google Scholar]
  20. Miwa, Y. & Fujita, Y. ( 2001; ). Involvement of two distinct catabolite-responsive elements in catabolite repression of the Bacillus subtilis myo-inositol (iol) operon. J Bacteriol 183, 5877–5884.[CrossRef]
    [Google Scholar]
  21. Primrose, S. B. & Ronson, C. W. ( 1980; ). Polyol metabolism by Rhizobium trifolii. J Bacteriol 141, 1109–1114.
    [Google Scholar]
  22. Reber, G., Belet, M. & Deshusses, J. ( 1977; ). myo-Inositol transport system in Pseudomonas putida. J Bacteriol 131, 872–875.
    [Google Scholar]
  23. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  24. Schaaf, S. & Bott, M. ( 2007; ). Target genes and DNA-binding sites of the response regulator PhoR from Corynebacterium glutamicum. J Bacteriol 189, 5002–5011.[CrossRef]
    [Google Scholar]
  25. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. ( 1990; ). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60–89.
    [Google Scholar]
  26. Van de Peer, Y. & De Wachter, R. ( 1994; ). treecon for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
    [Google Scholar]
  27. Van Dyk, T. K. & Rosson, R. A. ( 1998; ). Photorhabdus luminescens luxCDABE promoter probe vectors. Methods Mol Biol 102, 85–95.
    [Google Scholar]
  28. Yebra, M. J., Zuniga, M., Beaufils, S., Perez-Martinez, G., Deutscher, J. & Monedero, V. ( 2007; ). Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol. Appl Environ Microbiol 73, 3850–3858.[CrossRef]
    [Google Scholar]
  29. Yoshida, K. I., Aoyama, D., Ishio, I., Shibayama, T. & Fujita, Y. ( 1997; ). Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis. J Bacteriol 179, 4591–4598.
    [Google Scholar]
  30. Yoshida, K. I., Shibayama, T., Aoyama, D. & Fujita, Y. ( 1999; ). Interaction of a repressor and its binding sites for regulation of the Bacillus subtilis iol divergon. J Mol Biol 285, 917–929.[CrossRef]
    [Google Scholar]
  31. Yoshida, K., Yamamoto, Y., Omae, K., Yamamoto, M. & Fujita, Y. ( 2002; ). Identification of two myo-inositol transporter genes of Bacillus subtilis. J Bacteriol 184, 983–991.[CrossRef]
    [Google Scholar]
  32. Yoshida, K., Yamaguchi, M., Ikeda, H., Omae, K., Tsurusaki, K. & Fujita, Y. ( 2004; ). The fifth gene of the iol operon of Bacillus subtilis, iolE, encodes 2-keto-myo-inositol dehydratase. Microbiology 150, 571–580.[CrossRef]
    [Google Scholar]
  33. Yoshida, K., Yamaguchi, M., Morinaga, T., Kinehara, M., Ikeuchi, M., Ashida, H. & Fujita, Y. ( 2008; ). myo-Inositol catabolism in Bacillus subtilis. J Biol Chem 283, 10415–10424.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.032250-0
Loading
/content/journal/micro/10.1099/mic.0.032250-0
Loading

Data & Media loading...

Supplements

vol. , part 1, pp. 128 - 138

Primers used in this study [PDF](39 KB)



PDF

vol. , part 1, pp. 128 - 138

[PDF](10 KB)



PDF
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