SCREENING AND CLONING OF BACTERIAL β-GLUCOSIDASE GENE THAT CAN DEGRADE SALICIN FROM SOME NIF AND VIRULENT BACTERIA
Abstract
β-Glucosidase activity of the most virulent strains of Agrobacterium tumefaciens was found to be much higher than that of the weakly virulent strains. It appears that conversion of coniferin to coniferyl alcohol via bacterially encoded β-glucosidase affects genes induction and thus virulence. If indeed β-glucosidase is a necessary component of the pathway, this will be the first reported evidence of plant signal compound processing by Agrobacterium tumefaciens. The isolation, characterization of β-glucosidase gene from Agrobacterium tumefaciens B3/73 and sequencing of cbgl were done. The encoded enzyme catalyzes controlled hydrolysis of coniferin but not cellobiose (Morris and Morris, 1990).
The Agrobacterium tumefaciens has two β-glucosidase genes cbgl and Cbgl, Cbg1 activity hydrolyzes coniferin but not cellobiose. Agrobacterium tumefaciens B3/73 beta-glucosidase genes Cbgl was cloned and sequenced, cbg1 expressed in Escherichia coli. The 88-kDa predicted product of cbg1 β-Glucosidase is highly similar to another bacterial β-Glucosidase and several fungal β-glucosidases (Morris and Morris, 1990).
To confirm that Agrobacterium tumefaciens has strongly β-glucosidase activity and slight reaction, clones screened showed that two categories were indeed present DNA from individual clones was digested with HindIII and restriction fragment patterns were compared. (Linda et al., 1992).
Two type of pattern were obtained, the relative ability of each gene to cleave coniferin was assessed. Clones containing Agrobacterium tumefaciens B3/73 DNA rapidly and completely hydrolyzed coniferin to coniferyl alcohol. Over the same period, type 2 clones were completely inactive. The different substrate specificities of clones were also evident from their ability to grow on cellobiose. Agrobacterium tumefaciens B3/73 was able to use cellobiose as the sole carbon source. Escherichia coli DH5α and type 1 clones were not able to grow on cellobiose. Other clones were able to utilize cellobiose but grew very slowly. (Linda et al., 1992).
The 5.7 kb HindIlI fragment common to all type 1 clones was purified and ligated into pBR322. Clones with inserts in either orientation were able to cleave X-glucose, indicating that the entire β-glucosidase gene was probably located within this insert. An EcoRI, BamHI, BglII, and PstI restriction map of the insert showed that a 3.5 kb BamHI-PstI fragment with an internal PstI site was found to have the activitty to cleave X-glucose when cloned into pUC19. The sequence surrounding the EcoRI site in the pUC19:3.5 kb BamHI-PstI clone and the sequence were done. (Linda et al., 1992).
Woodward and Wiseman (1982) reported that there are two constitutive, β-glucosidase genes in Agrobacterium tumefaciens B3/73 were appeared. Those represented by clones able to hydrolyze X-glucose and coniferin but not cellobiose. On the other hand, those represented by other clones having lower activity on X-glucose, non activity on coniferin and able to utilize cellobiose for growth.
β -glucosidase, α-glucosidase, and β-galactosidase activities have been reported to be associated with 45 strains of rhizobia, and some cellulolytic and pectinolytic activities have been detected in Rhizobium leguminosarum. (Singh and Singh, 1985).
Several glycosidases have also been detected in Bradyrhizobium lupini A β-glucosidase that is particularly active with cellobiose has been purified from Agrobacterium faecalis. In addition, a β-galactosidase and a β-glucosidase have been purified from the periplasmic space of Rhizobium trifolii, and an endoglucanase gene from Azorhizobium caulinodans has been cloned. (Geelen et al., 1995).
Listeria spp. transcription of bvrB gene was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part of bvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin indicating that the bvr locus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in Listeria. monocytogenes that is involved in environmental regulation of virulence genes (Klaus et al., 1999).
Caulobacter crescentus ability to utilize lactose was examined to obtain an additional genetic tool for study this model organism, Identified a gene lacA that required for growth on lactose as the sole carbon source involved in the catabolism of two glucosides salicin and trehalose., this enzymatic activity is inducible and increased lac expression in the presence of lactose and salicin. (Benjamin et al., 2010).
References
Benjamin, H. A., J. D. Ortiz, J. Manzano and J. C. Chen (2010). Identification of dehydrogenase required for lactose metabolism in Caulobacter crescentus. Appl. Enviro. Microbiol., 76: 3004-3014.
Birnbim, H. C. and J. doly (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acid Res., 7: 1513-1523.
Desomer, M., C. and M. V. Montagu (1991). III egitimate integration of Rodococcus fascians upon electrotranformation as an insertional mutagenesis system. Molecular Microbiology, 5: 2115-2124.
Geelen, D., M. Van Montagu and M. Holsters (1995). Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis. Appl. Environ. Microbiol., 61: 3304-3310.
Gough, C. L., J. M Dow, C. E. Barber and. M. J Daniels (1988). Cloning of two endoglucanase genes of Xanthomonas campestris pv. campestris: analysis of the role of the major endoglucanase in pathogenesis. Mol. Plant Microb. Interact., 1: 275-281.
Klaus Brehm, Mari A. T. R., J. Kreft and J. A. Vazquez-Boland (1999). The bvr Locus of Listeria monocytogenes Mediates Virulence Gene Repression by β-Glucosides. Journal of Bacteriology, 181: 5024-5032.
Linda, A., T. Castle, K. D. Smith and R. O. Morris (1992). Cloning and Sequencing of an Agrobacterium tumefaciens, Glucosidase Gene Involved in Modifying a vir-Inducing Plant Signal Molecule. J. Bacteriol., 174: 478-1486.
Maniatis, T., E. F. Fritsch and J. Sambrook (1982). Molecular cloning. A laboratory Manual. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Perss.
Morris, J. W and R. O. Morris (1990). Identification of an Agrobacterium tumefaciens virulent gene inducer from the Dinoceus gymanosperm pseudo stuga menziesii. Proc. Natl. Acad. Sci., USA, 87: 3612-3618.
Pierre, B. (1983). Detection of cellulase activity polyacrylamid gel using Congo Red Stained, agar replicas. Analytica., l-Biochemistry, 131: 333-336.
Ronlald, M. A. (2004). Microbiological media. Third edition. CRC press Baco-Roton, New York.
Saddler, J. N. (1982). Screening of highly cellulolytic fungi and the action of their cellulase enzymes systems. Enzy. Microb. Technol., 4: 414-418.
Sharp, P. A., B. Sugden and J. Sambrook (1973). Detection of two restriction endonuclease activities in Hamophilus parainflunzae using analytical agarose. Biochemistry, 12: 3055-3063.
Singh, A. P. and J. B. Singh (1985). Differences in α and β-glucosidase and β-galactosidase activity among fast and slow growing species of Rhizobium and Agrobacterium tumefaciens. Microbiol., 43: 169-176.
Walker, D. S., P. J. Reeves and G. P. C. Salmond (1994). The major secreted cellulase, CelV, of Erwinia carotovora subsp. carotovora is an important soft rot virulence factor. Mol. Plant Microbe Interact., 7: 425-431.
William, J. D., F. J. Miller and W. C. Ragsdale. (1988). High Efficiency Transformation of E. coli by high voltage electroporation. Nucleic Acids Research, 16: 6127-6145.
Woodward, J. and A. Wiseman (1982). Fungal and other 1-D-glucosidases their properties and applications. Enzy. Microb. Technol., 4: 73-79.
