Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.     

Influence of gyrA mutation on expression of Erwinia chrysanthemi clb genes cloned in Escherichia coli.

Erwinia chrysanthemi clb genes cloned into Nals Escherichia coli allowed growth on cellobiose, arbutin, or salicin. In contrast, Nalr isogenic strains grew only on cellobiose. It is proposed that expression of cloned E. chrysanthemi clb genes is reduced by the E. coli chromosomal gyrA (Nalr) mutation, resulting in apparent segregation of the Clb and Arb Sal characters.     

DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.     

Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli

Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184. Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively. These two genes showed no homology. The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain. Expression of these genes in E. coli was found not to depend on their orientation in the cloning vector. Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected.     

里氏木霉纤维二糖酶bglII基因的cDNA克隆及其在大肠杆菌中的表达*

将里氏木霉总RNA反转录得到cDNA第一链,并以之为模板进行 RT-PCR合成约1.4kb的纤维二糖酶基因cDNA,将所得的cDNA经测序后克隆到温度敏感型表达载体pJW2中,经PCR和双酶切鉴定筛出阳性重组子,温度诱导后,经酶活测定,bgl II基因在大肠杆菌中得到了表达,酶活为0.75IU/mL。     

里氏木霉纤维二糖酶bgl II基因的cDNA克隆及其在大肠杆菌中的表达

将里氏木霉总RNA反转录得到cDNA第一链,并以之为模板进行 RT-PCR合成约1.4kb的纤维二糖酶基因cDNA,将所得的cDNA经测序后克隆到温度敏感型表达载体pJW2中,经PCR和双酶切鉴定筛出阳性重组子,温度诱导后,经酶活测定,bgl II基因在大肠杆菌中得到了表达,酶活为0.75IU/mL.     

Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase.

The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.     

Functional genes for cellobiose utilization in natural isolates of Escherichia coli.

The genes for utilization of cellobiose are normally cryptic in both laboratory strains and natural isolates of Escherichia coli. A survey of natural isolates of E. coli reveals that functional genes for cellobiose utilization, while rare, are present. The fraction of E. coli that utilized cellobiose ranged from less than 0.01% in human fecal samples to 7% in fecal samples obtained from horses. Samples obtained from sheep, cows, dogs, and pigs contained 0.1 to 0.5% cellobiose-positive E. coli. Neither the previously identified cel genes nor the bgl genes from E. coli K-12 were expressed during growth on cellobiose by any of the 14 naturally occurring Cel+ isolates that were tested. All of the naturally occurring Cel+ isolates possessed a cel operon, but all were deleted for the major portion of the bgl operon. The functional cel+ genes from these natural isolates differed from the mutationally activated cel+ genes obtained in earlier studies in that (i) the mutationally activated cel+ genes were temperature sensitive, while the functional genes were not, and (ii) transport of cellobiose was inducible in the strains carrying functional cel+ genes, while it was expressed constitutively in strains carrying mutationally activated genes.     

Cloning of ten distinct DNA fragments of Clostridium thermocellum coding for cellulases

Abstract Ten distinct Eco RI fragments of Clostridum thermocellum DNA have been cloned in Escherichia coli and shown to express enzymatic activities related to cellulose degradation. Two of the cloned fragments appeared to carry the previously characterized celA and celB genes, which code for the endoglucanases (EG) A and B. Five other cloned fragments code for hitherto unidentified EGs, which can be detected by the Congo red test for hydrolysis of carboxymethylcellulose (CMC). In addition, three separate clones hydrolyzed methylumbelliferyl-β-cellobioside (MUC) but not CMC, hinting that they may express three different cellobiohydrolase genes.     

Physical Mapping of Recombinant Plasmids Containing Broad Bean Chloroplast Ribosomal RNA Genes

Two BamHl fragments containing broad bean chloroplast rRNA genes were cloned using the bacterial plasmid pBR322 as a vector and Escherichia coli HB101 as host bacterial. Physical maps of the two cloned ct DNA BamHI fragments containing rRNA genes were constructed by cleavage with several restriction endonucleases and Southern blot hybridization with E. coli 16S-23S rRNAs. Recombinant plasmids pVFBI6 and pVFB32 contain a 16S rRNA sequence on the 4.70 kb BamHl fragment, a 23S rRNA sequence and 4.5S/5S rRNA sequences on the 5.65 kb BamHl fragment, respectively.     

N-terminal amino acid sequences of the structural proteins of the tick-borne encephalitis virus

DNA-copies of the tick-borne encephalitis RNA fragments have been cloned in plasmid pBR322 in E. coli cells. The sequencing of the cloned DNA-copies revealed clones containing genes coding for viral structural proteins E and C. Strong homology between amino acid sequences of proteins E and C from two TBF strains is found.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.     

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.     

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.