SCREENING AND CLONING OF BACTERIAL β-GLUCOSIDASE GENE THAT CAN DEGRADE SALICIN FROM SOME NIF AND VIRULENT BACTERIA

-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).


SCREENING AND CLONING OF BACTERIAL β-GLUCOSIDASE GENE THAT CAN DEGRADE SALICIN FROM SOME NIF AND VIRULENT BACTERIA HANAN H. AHMED
Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City, Minufiya University.Egypt -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).
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 β 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, βglucosidase 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 βgalactosidase 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).

Media used
The media used were carried out according to Ronlald (2004).The bacteria were grown at 28C for 48 hours.
Escherichia coli HC1061 was used as cloning host was grown on LB medium g/l the composition of the media is agar 15 g., bacto tryptone 20 g., bacto yeast 5 g., NaCl 0.2 g., pH 7.5 supplemented with Carbenicillin (100 g/ml) antibiotic stock.William et al. (1988) the composition of the final concentration 200 g/ml Carbenicillin is 2500x stock Carbenicillin, 500 mg/ml in water, add 200 l/500 ml.

Screening of β-glucosidase activity
Very sensitive test for detecting colonies of cellulase producing microorganisms was used.For βglucosidase enzyme assay bacterial colonies or spots of bacterial culture were grown 24 hours on appropriate agar media containing 0.2% Salicin used as a source of carbon Pierre (1983).Salicin containing plates were stained with Congo Red 0.1%, after two to three washes with 1M NaCl, the appearance of a clear halo indicated glucosidase activity.

Bacterial
DNA Preparation: chromosomal DNA was isolated from Bacterial isolates as described by Desomer et al. (1991).
Gel electrophoresis: 1% Agarose gel electrophoresis was performed as described by Sharp et al. (1973).

Construction and screening of genomic library 1-Restriction endonucleases enzymes used:
The DNA was partially digested with restriction enzymes used according to the manufacturer , s instructions standard conditions to EcoRI, or SamI, HincII, SalI and HindIII were used.Total DNA digestion by the Restriction enzymes the volumes used were 5 µl, DNA, 2 µl, Restriction enzymes, 1 µl, RNase enzyme, 3 µl, buffer, 14 µl, deionized water then incubated for 30 min.

Elution and Purification of DNA fragment from agarose gel
The obtained product by the for 30 min.and treated with calf intestine alkaline phosphatase.

Ligation 2-5 kbp fragment with pUC19 vector
Genomic DNA was prepared from the bacterial isolates according to Maniatis et al. (1982).The DNA was partially digested with EcoRI, SamI, and the 2 to 5 kb fragments were isolated by electroelution from agarose gel.pUC19 plasmid DNA was digested with EcoRI, treated with calf intestine alkaline phosphatase, fragments were used in the ligation mixture 20 µl DNA (insert), 2 µl pUC19, 1 µl T 4 ligase DNA enzyme, 6 µl ligase DNA buffer.The ligation mixture was kept at 16C overnight.

Preparation and transformation of electrocompetent cells
Ttransformation of E. coli bacteria by electroporation was carried out according to William et al. (1988).

Color selection
To made LB plus cb antibiotic plate add 40µl (20 mg/ml) of a stock solution of X -gal solution was added, 4 µl (200 mg/ml) of a stock solution of (IPTG) and cb antibiotic directly into the medium when you pour the plates, place them at 4C for several hours to enhance were incubated until developed blue color.

Extraction of plasmids from transformed positive clones
Preparation of plasmids from transformed positive clones for mapping of β-glucosidase gene cleaving site on the pUC19 plasmid was carried out according to Birnboim and Doy (1979).

The polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) was conducted to ensure the presence of the gene of interest (2 and 3 kb fragment) in the extracted DNA from recombinant plasmid digested with EcoRI restriction enzyme.Forward and reverse primers were used in the reaction and designed to anneal with pUC19.The sequence of both primers was as followed: Forward primer: GTTAAACGACGGCCAGTG Reverse primer: CAGGAAACAGCTATGACC

Procedure
The PCR program involved an initially step of denaturation at 94C for 4 min.;followed by 40 cycles of amplification involving denaturation step at 94C for 1 min., primers annealing to the template realised at 55C for 1 min., extension step at 72C for 1 min.and final extension at 72C for ten min.The PCR DNA products were separated in (1%) agrose gel and the bands were stained by ethidium bromide and detected on UV transilluminator and photographed.

The efficacy of bacterial isolates to produced β-glucosidase enzyme
Four Egyptian isolates of bacterial isolates namely Rhizobium leguminosarum bv.Trifloii, Azorhizobium caulinodans, Rhodococcus fascins and Agrobacterium tumefaciens were tested for their ability to use salicin as a sole source carbon.The detection was carried out by appearing a clear halo zone around the colonies which stained with Congo Red. Figure (1) clearly show the capability of Rhizobium leguminosarum bv.Trifloii, and Agrobacterium tumefaciens to produce β-glucosidase enzyme as shown in plates (4 and 5).(Saddler et al., 1982;Geelen et al., 1995).The screening of βglucosidase, cellobiohydrolase, endogluconase and polygalacturonase activities showed the clear formation of haloes around bacterial colonies or spots after overnight incubation on appropriate agar medium, plates stained with Congo Red 0.1% after two or three washes with 1 M NaCl the appearance of a clear halo indicated enzyme activity.Klaus et al. (1999) studied bvrB transcription in Listeria monocytogenes in the presence of glucose (control) or the βglucosides cellobiose, salicin, and arbutin.The level of bvrB expression was significantly higher in the presence of cellobiose and salicin, The Listeria monocytogenes bvrB gene induced by cellobiose and salicin but not by arbutin.The results indicated 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 L. monocytogenes that is involved in environmental regulation of virulence genes.In general, cellulolytic organisms produce cellulases to break down cellulose to cellobiose and glucose, which are used as carbon sources.Because Azorhizobium caulinodans cannot grow on glucose or cellobiose, it is very unlikely that the Egl enzyme would serve this purpose.Cellulases might also assist in the release of host plant nutrients, as suggested for Xanthomonas campestris, Erwinia carotovora (Gough et al., 1988) and Pseudomonas solanacearum (Walker et al., 1994).

Plasmid extraction from transformed clones
The new constructs of pUC19 plasmid were introduced into E. coli HC1061 strain by electroporation.Selection of colonies was done by plate assay.These plates LB media contained IPTG with X-gal substrate and carbanicilin.The blue and white colonies obtained were selected.Transformed colonies were confirmed using EcoRI in which the expected band sizes was detected as illustrated in Fig. (3).It was shown that digesting the plasmid from E. coli DH1061 white clones with EcoRI restriction enzyme resulted in the production of 2 and 3 kb (insert) fragments.Linda et al. (1992) studied the Agrobacterium tumefaciens the EcoRI site in the pUC19 3.5-kb BamHI-PstI clone and the sequence upstream of the BamHI site.Sequence data (3,710 nucleotides) were obtained for 176 nucleotides upstream of the BamHI site through the downstream PstI site.There are at least two constitutive, β-glucosidase genes in A. tumefaciens B3/73.Those represented by two types at clones were able to hydrolyze X-glucose and coniferin but not cellobiose.On the other hand, those represented by two types of clones had lower activity on X-glucose and no activity on coniferin and were able to utilize cellobiose for growth.Azorhizobium caulinodans ORS571, cell extracts were tested for the presence of β-glucosidase and cellobiohydrolase activities, using chromophoric substrates.Hydrolysis of the substrates was not observed, suggesting the absence of exoglucanases.Subcloning into pUC19and expression into E. coli assigned the Egl activity to a 3 kb DNA fragment.This fragment, which was sufficient to confer CMC degradation, (Geelen et al., 1995) Preparation of recombinant plasmid DNA from E. coli HC1061 clones and digestion with the same enzyme showed that insert of about 2-3 kb was present (Fig. 4).Electroelution of the fragments insert (separately) ligated into pUC19 plasmid and transformed into E. coliHC1061.
Selection derived recombinant clones from each transformation from the white clones from each transformed E. coliHC1061 and using Congo Red plate assay and recombinant clones expressing βglucosidase activity by clear halo formation around the clones the result indicated Rhizobium leguminosarum have at least two βglucosidase genes (Fig. 5).

Agrobacterium tumefaciens the
EcoRI site in the pUC19 3.5-kb BamHI-PstI clone and the sequence upstream of the BamHI site in the pBR322:5.7-kbHindIII plasmid were obtained by doublestranded plasmid sequencing with synthetic oligonucleotide primers.Sequence data (3,710 nucleotides) were obtained for 176 nucleotides upstream of the BamHI site through the downstream PstI site.There appear to be at least two constitutive, β-glucosidase genes in A. tumefaciens B3/73.Those represented by type 1 clones were able to hydrolyze Xglucose and coniferin but not cellobiose.On the other hand, those represented by type 2 clones had lower activity on Xglucose and no activity on coniferin and were able to utilize cellobiose for growth.(Linda et al., 1992).

6-PCR confirmation
Selected clones were checked for the presence of about 2 and 3 kb and fragment of β-glucosidase gene.PCR confirmed the presence of about 2 and 3 kb fragment β-glucosidase gene in recombinant pUC19 vector.Two PCR primers were used forword and reverse primers annealing to pUC19 based on published data available in the Gene Bank.Using these primers, a PCR product fragment with a MW. of about 2 and 3 kb was obtained.As shown in Fig. (6).The recombinant plasmids showed the expected band size for βglucosidase gene (2 and 3 kbp) there appear to be at least two βglucosidase genes in Rhizobium leguminosarum bv.Trifloii able to utilize Salicin.Linda et al. (1992) found that there is two β-glucosidases in Agrobacterium tumefaciens B3/73.There appear to be at least two constitutive, βglucosidase genes in A. tumefaciens B3/73.Those represented by type 1 clones were able to hydrolyze X-glucose and coniferin but not cellobiose.On the other hand, those represented by two types of clones had lower activity on X-glucose and no activity on coniferin and were able to utilize cellobiose for growth.3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) digested with EcoRI restriction enzyme and λ phage PstI as a marker.

SUMMARY
Electroporation at high voltage was used by a cell suspention of very low conductivity 10 µl of daylilies ligation mixture, 90 µl of Escherichia coli HC1061 were added.The previous volume was put in precool cuvatte without air bubbles, plasmid was introduced into , β-glucosidase activity by replicaplating and exposing the replicated colonies to top agar containing Salicin.

Figure ( 2
Figure (2) showed the digested genomic DNA from Agrobacterium The white E. coli HC1061 recombinant clones, carrying a βglucosidase gene from Agrobacterium tumefacien and Rhizobium leguminosarum bv.Trifloii were selected using plate assay grown overnight on LB medium containing Salicin, followed by Congo Red staining.The recombinant clones were characterized by a large halo formation around the white clones E.coli HC1061.(Fig. 3).Other white E. coli clones but relatively with no activity.Specific fluidity by recombinant plasmidencoded β-glucosidase, Salicin hydrolysis from E. coli carrying recombinant plasmid.The E. coli positive clones showed clones on LB medium with Salicin to repeat and confirm the Congo Red stain, the positive E. coli clones were again active.

Fig
Fig. (3): Screening of recombinant plasmids origin of glucosidase genes activity in the transformed clones, No 1 is Agrobacterium tumefacien, No 2 is Rhizobium leguminosarum bv Trifloii.

Fig
Fig. (5): Verification and Screening of plasmids origin of glucosidase genes activity in the transformed clones, No 1 is 2kbp fragment, No2 is 3kbp fragment of Rhizobium leguminosarum bv.Trifloii.