Structural characterization of the bglH gene encoding a beta-glucosidase-like enzyme in an endophytic Bacillus pumilus strain

A beta-glucosidase-like enzyme-encoding gene (bglH) of an endophytic Bacillus pumilus strain (CL16) was cloned using a shotgun genomic library constructed in Escherichia coli. The nucleotide sequence of the entire cloned fragment (2484 bp) was determined and characterized. An incomplete open reading frame (ORF) of 534 bp (ORF1) designated bglP and a complete ORF of 1419 bp (ORF2) designated bglH, located in the fragment, are organized in an operon. The protein deduced from 1419 bp (ORF2) had 472 amino acid residues without a characteristic signal peptide sequence, suggesting that the enzyme is localized in the cytoplasm. The amino acid sequence deduced from bglH gene had high similarity with β-glucosidases from the glycosyl hydrolase family 1. Over-expression of the B. pumilus bglH gene in E. coli showed a 54 kDa protein whose identity was confirmed by mass spectrometry (MALDI-TOF).

Cellulose comprises the major carbohydrate polymer of the plant cell wall.It is an unbranched polymer composed of anhydro-1,4-D-glucopyranoside units linked by β-glucosidic bonds.Enzymatic degradation of cellulose within the polysaccharide matrix of the cell wall requires the synergism of multiple enzymes such as the cellulases, exo-(cellobiohydrolase) and endo-β-1,4-glucanases, and β-glucosidases (cellobiases) (Knauf and Moniruzzaman, 2004).The β-glucosidases are widespread in microorganisms where they metabolize various carbohydrate substrates, including cellobiose, produced as a consequence of cellulose hydrolysis, and aromatic β-glucosides such as arbutin and salicin that are produced by a variety of plants (Tajima et al., 2001;Spiridonov and Wilson, 2001;Park et al., 2002;Marques et al., 2003;An et al., 2005).
Considerable polymorphism in β-glucosidase forms, functions and kinetics has been reported (Ogunseitan, 2003).Although several cellulolytic enzymes released by phytopathogens have already been well-characterized, knowledge on the uptake and hydrolysis of carbohydrates by endophytic microorganisms is limited.Endophyte microorganisms colonize inner plant tissues, living symbiotically with the host species (Azevedo et al., 2000).These microorganisms have been investigated as a source of new genes and proteins for use in industrial processes (Stamford et al., 2001(Stamford et al., , 2002;;Pleban et al., 1997;Reddy et al., 1996;Moy et al., 2002;Lima et al., 2004).
Recently, 15 endophytic strains of Bacillus spp.isolated from Citrus were evaluated for cellulolytic activity (Lima et al., 2004).The Bacillus pumilus strain CL16 showed high cellulase activity and was selected for further studies.We have cloned the β-1,4-endoglucanase eglA gene from strain CL16 and expressed it in E. coli.The endo-1,4-β-glucanase EglA has high thermostability, an important feature in biotechnical processes that require high temperatures (Lima et al., 2004).
During the study described in the present paper we isolated and characterized a new locus of β-glucoside sugar utilization genes from the endophytic B. pumilus CL16 strain.
This fragment was successfully used as a probe for screening the bglH gene in a shotgun genomic library constructed from B. pumilus strain CL16 (Lima et al., 2004).Hybridization was done using the DIG High Prime DNA Labeling and Detection Starter Kit II, according to the manufacturer's instructions (Roche, Germany).Pre-hybridization (30 min) and hybridization (overnight) steps were at 42 ºC.Only one positive transformant was recovered from 2400 colonies.The recombinant plasmid isolated from this clone was denoted pMH2.The presence of the bglH gene was confirmed by two steps of sequencing.Firstly, using the M13 primers (Amersham Biosciences, Germany) and then with a set of new primers, GLICO1 F (5'-TCCAgAgATTCTTggACAAgT-3'), GLICO2 R (5'-CACTTggAACAAATTggTgATg-3') and GLm F (5'-gCATAAgCACggAATTgAgTC3-3') designed specifically for a bgl internal segment.
Two open reading frames (ORF) were found to compose the insert: an incomplete ORF of 534 bp (ORF1) and a complete one with 1419 bp (ORF2), which presented high similarity with the bglP and bglH genes, respectively, both from B. subtilis subsp.subtilis strain 168 (Kunst et al., 1997).The bglP gene from B. subtilis encodes an aryl-βglucoside-specific enzyme II of the phosphoenolpyruvate sugar: phosphotransferase system (PTS), whereas the activity of BglH from B. subtilis was only recently directly demonstrated (Setlow et al., 2004).These authors showed that the bglH gene from B. subtilis encodes an arylphospho-β-D-glucosidase and that this gene was induced by aryl-β-D-glucosides.
The β-glucoside utilization pathways that rely upon the PTS for carbohydrate uptake have been characterized in several bacteria (Krüger and Hecker, 1995;Lai et al., 1997;Brown and Thomson, 1998;Brehm et al., 1999;Marasco et al., 1998;An et al., 2005), but not in B. pumilus.According to An et al. (2005) the discovery of new PTS-related sequences in bacterial genomes continues, and suggests that PTS enzymes might have additional unknown functions.
The nucleotide sequence of the entire insert (2484 bp) and the deduced protein sequence of the bglH gene from B. pumilus are shown in Figure 1.The ORF1 was upstream from ORF2, and separated by a 24 bp sequence.The absence of a promoter sequence between the ORFs identified by us, and their similarity with the operon described for B. subtilis strain 168 (Entrez Gene-NCBI server) suggest that they are organized into an operon.The nucleotide sequence downstream from ORF2 was compared to other sequences deposited at NCBI GenBank (BLASTX) but no similarity could be identified.
We found that the 1419-nucleotide-long ORF2 had a GC content of 41.6%.It was preceded by a potential ribosome binding site (AGGAGG) that was 9 bp upstream from the putative ATG start codon but, as expected, with no adjacent promoter sequence.Downstream from the TAA stop codon, no sequence resembling a rho-independent transcriptional terminator could be identified.The protein deduced from the ORF-complete sequence had 472 amino acid residues with an estimated molecular mass of 53.9 kDa and a isoelectric point of 4.97.The CDSearch program (NCBI server) revealed that BglH had a single domain consisting of the glycosyl hydrolase family 1 (GH1) sequence, covering 464 residues from amino acid 4 to 468.
The deduced B. pumilus BglH amino acid sequence was compared to other BglH sequences deposited at NCBI Bogas et al. 101 GenBank (Figure 2) and found to be highly  (Withers and Aebersold, 1995).
The absence of a signal peptide sequence (SignalP 3.0 program) and the lack of potential transmembrane regions (TMPred program) in the BglH sequence suggest that the enzyme is localized in the cytoplasm, as is the case for most bacterial β-glucosidases (Bhatia et al., 2002 and references therein).
We constructed an expression plasmid for the overproduction of β-glucosidase (Bgl) by amplifying the bgl open reading frame (ORF) using the polymerase chain reaction (PCR) and the MHF (5'-CACCATgAACAAgTT AgAAAAAACAT-3') and MHR (5'-TTAgTAATCCAA ATgTTCCCCATTTg5-3') primer pair.For DNA polymerization the AccuPrime Pfx enzyme (Invitrogen, USA) was used.The amplified product was cloned into the pENTR/ SD/D-TOPO plasmid producing the entry vector of the Gateway Cloning System (Invitrogen, USA).From the entry vector the bgl gene was transferred to the expression vector pET-DEST42 by in vitro site-specific recombination.The recombinant expression plasmid containing the bgl gene was named the pAB1 plasmid.The cloned fragment was completely sequenced to confirm that no mutations were introduced during the amplification procedures.
Transformed Escherichia coli BL21 CodonPlus (DE3) cells harboring the pAB1 expression vector were grown on Luria-Bertani medium (LB) supplemented with 250 μg mL -1 ampicillin and incubated at 37ºC until the log phase (OD 600nm = 0.2).After induction with 0.5 mmol L -1 IPTG (isopropyl-beta-D-thiogalactopyranoside) a strong band of 54 kDa was detected by sodium dodecyl sulfate polyacrylamide gel electrophoreses (SDS-PAGE) analysis (Laemmli, 1970), suggesting the over-expression of the B. pumilus bglH gene in E. coli (Figure 3).This molecular mass is consistent with that predicted from the amino acid sequence of the BglH protein.The over-expressed protein was completely purified and subjected to digestion with trypsin followed by peptide fingerprint analysis by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), in order to confirm the protein identity.The provided peptide masses were equivalent to the ones predicted, confirming that the overexpressed 54 kDa-band was in fact the BglH protein of B. pumilus.
Crude protein extracts obtained from the E. coli cells harboring the pAB1 plasmid were assayed for activity against ρ-nitrophenyl-β-D-glucopyranoside and cellobiose.Each assay consisted of 0.5 mL 5 mM of ρ-nitro-phenyl-β-D-glucopyranoside or 0.2% of cellobiose as substrate, 0.1 mL of 50 mM phosphate buffer (pH 5.8 to 7.5) or Mc'Ilvaine buffer (pH 3 to 7) and 0.05 mL of crude enzyme.The mixture was incubated for 1 h at 37 ºC and the activity of β-glucosidase towards ρ-nitrophenyl-β-D-glucopyranoside was estimated by measuring the amount of ρ-nitrophenol released at 400 nm.The activity of β-glucosidase toward cellobiose was estimated by measuring the glucose released by the glucose oxidase method (Glucose Enzyme Color Kit, Bio Diagnostica, Brazil).The enzymatic activity of Bgl against ρ-nitrophenyl-β-D-glucopyranoside was assayed in Mc'Ilvaine buffer (pH 7.0) using the same protocol.Little activity against synthetic aryl-β-D-glucosides and no activity against cellobiose were observed.The highest activity against ρ-nitrophenyl-β-D- glucopyranoside (0.106 μmol min -1 mL -1 ) was observed using Mc'Ilvaine buffer at pH 7.0.This low activity was also observed using purified Bgl.The B. subtilis BglH also showed very low activity against non-phosphorylated β-glucosides.The enzyme activity against aryl-phospho--β-D-glucosides was not measured because there was no commercial supplier for this substrate.

Figure 1 -
Figure 1 -Nucleotide sequence of the entire insert (2484 bp) and the deduced protein sequence of the bglH gene from Bacillus pumilus.Start and stop codons and the ribosome-bounding site (RBS) are underlined.Partial nucleotide sequence of the bglP gene is in italic font.

Figure 3 -
Figure 3 -Over-expression of the BglH protein in Escherichia coli BL21 CodonPlus(DE3) was analyzed by 12% SDS-PAGE.Proteins were stained with Coomassie Blue R-250.a) Lane 1, lysate from untransformed cells not induced with IPTG; Lane 2, lysate from untransformed cells induced with IPTG; Lane 3, lysate from transformed cells with pAB1 not induced with IPTG; Lane 4, lysate from transformed cells with pAB1 induced with IPTG.b) Lane 1, lysate from transformed cells with pAB1 induced with IPTG; Lane 2, Purified protein obtained from transformed cells with pAB1.Molecular weight markers (MW) are indicated in kDa.Arrow indicates the BglH protein.