Cloning and sequencing of a Bacteroides ruminicola B(1)4 endoglucanase gene.

Bacteroides ruminicola B(1)4, a noncellulolytic rumen bacterium, produces an endoglucanase (carboxymethylcellulase [CMCase]) that is excreted into the culture supernatant. Cultures grown on glucose, fructose, maltose, mannose, and cellobiose had high specific activities of CMCase (greater than 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. rumincola did not grow on either ball-milled or acid-swollen cellulose even though the CMCase could hydrolyze swollen cellulose. The CMCase gene was cloned into Escherichia coli, and its nucleotide sequence contained a single open reading frame coding for a protein of 40,481 daltons. The enzyme was overproduced in E. coli under the control of the tac promoter and purified to homogeneity. The N-terminal sequence, amino acid composition, and molecular weight of the purified enzyme were similar to the values predicted from the open reading frame of the DNA sequence. However, the CMCase present in B. ruminicola was found to have a monomer molecular weight of 88,000 by Western immunoblotting. This discrepancy appeared to have resulted from our having cloned only part of the CMCase gene into E. coli. The amino acid sequence of the CMCase showed homology to sequences of beta-glucanases from Ruminococcus albus and Clostridium thermocellum.     

The cellular location of Prevotella ruminicola beta-1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.     

Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing

We have characterized hygromycin B and apramycin resistance genes from an E. coli plasmid. We have localized the coding and control regions of these genes by deletion of DNA fragments from plasmids containing the genes. It was found that polypeptides with apparent molecular weights of 33,000 and 31,500 daltons are encoded by the apramycin resistance gene and polypeptides with apparent molecular weights of 42,500 and 41,500 daltons are encoded by the hygromycin B resistance gene. DNA sequence analysis identified a typical promoter sequence upstream of the genes. Deletion of this promoter eliminated both resistance phenotypes, and hygromycin B resistance could be restored by substitution of a promoter from a foreign gene. The region known to be necessary for hygromycin B resistance contained an open reading frame large enough to encode the hygromycin B resistance gene product. This open reading frame was fused with the amino terminus of beta-galactosidase. This hybrid gene conferred hygromycin resistance to E. coli, and expression of resistance was under IPTG control.     

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage.

A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular level. Nucleotide sequence determination revealed the presence of two open reading frames. Both open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.     

Cloning and sequencing of an Escherichia coli gene, nlp, highly homologous to the ner genes of bacteriophages Mu and D108.

An nlp (Ner-like protein) gene was isolated from Escherichia coli. The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed. It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide. The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108. The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region. The nlp gene was located at 69.3 min on the E. coli genetic map.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.     

Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.     

Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4.

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1)4 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW). pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients. B. uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cmr, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC). The xylanase promoter allowed the B. uniformis transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells. Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid-swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. Under these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B(1)4 (pTC-XRCMC) transconjugates expressed Tcr and had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was the carbon source. On the basis of these results, it appears that not all of the promoters recognized by B. uniformis and P. ruminicola 23 are functional in P. ruminicola B(1)4. However, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstructed CMCase in P. ruminicola B(1)4.     

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.     

Organization and nucleotide sequence of the glutamine synthetase (glnA) gene from Lactobacillus delbrueckii subsp. bulgaricus.

A 3.3-kb BamHI fragment of Lactobacillus delbrueckii subsp. bulgaricus DNA was cloned and sequenced. It complements an Escherichia coli glnA deletion strain and hybridizes strongly to a DNA containing the Bacillus subtilis glnA gene. DNA sequence analysis of the L. delbrueckii subsp. bulgaricus DNA showed it to contain the glnA gene encoding class I glutamine synthetase, as judged by extensive homology with other prokaryotic glnA genes. The sequence suggests that the enzyme encoded in this gene is not controlled by adenylylation. Based on a comparison of glutamine synthetase sequences, L. delbrueckii subsp. bulgaricus is much closer to gram-positive eubacteria, especially Clostridium acetobutylicum, than to gram-negative eubacteria and archaebacteria. The fragment contains another open reading frame encoding a protein of unknown function consisting of 306 amino acids (ORF306), which is also present upstream of glnA of Bacillus cereus. In B. cereus, a repressor gene, glnR, is found between the open reading frame and glnA. Two proteins encoded by the L. delbrueckii subsp. bulgaricus gene were identified by the maxicell method; the sizes of these proteins are consistent with those of the open reading frames of ORF306 and glnA. The lack of a glnR gene in the L. delbrueckii subsp. bulgaricus DNA in this position may indicate a gene rearrangement or a different mechanism of glnA gene expression.     

Cloning and molecular analysis of cDNA encoding a carboxymethylcellulase of the yeast Cryptococcus flavus.

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.     

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.     

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen

Abstract The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyne disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum .     

Nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes.

2,3-Dihydroxybiphenyl dioxygenase, which catalyzes ring metacleavage of 2,3-dihydroxybiphenyl, is encoded by the bphC gene of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and T. Miyazaki, J. Bacteriol. 166:392-398, 1986). We determined the nucleotide sequence of a DNA fragment of 2,040 base pairs which included the bphC gene. The fragment included one open reading frame of 912 base pairs to accommodate the enzyme. The predicted processed amino acid sequence of the enzyme subunit consisted of 302 residues, and its 12 NH2-terminal residues were in perfect agreement with those determined for the enzyme. Approximately 10 base pairs upstream from the initiation codon for 2,3-dihydroxybiphenyl dioxygenase, there was a base sequence complementary to the 3' end of the 16S rRNA from Pseudomonas aeruginosa. There was no promoterlike sequence in the region upstream of the bphC gene, but another long open reading frame was present. A putative bphD gene encoding a metacleavage compound-hydrolyzing enzyme was suggested in the region downstream of the bphC gene.     

Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus

Bacillus cereus secretes phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5-kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin-hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the phosphatidylcholine-hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster. As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli. The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20-30-fold in the presence of MgCl2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCl2.     

The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

Sequencing and expression of the gene encoding the Clostridium stercorarium beta-xylosidase Xyl43B in Escherichia coli

The Clostridium stercorarium F-9 xyl43B gene encoding the beta-xylosidase Xyl43B consists of an open reading frame of 1,491 nucleotides that encodes a putative protein, classified in family 43, of 497 amino acids with a predicted molecular weight of 56,355. The deduced amino acid sequence of Xyl43B has sequence similarity with beta-xylosidases from Bacteriodes thetaiotaomicron (57% sequence identity), Prevotella ruminicola (45%), Streptomyces coelicolor (40%), and Clostridium acetobutylicum (36%), all of which have been classified in family 43 of the glycoside hydrolases. Xyl43B was purified from a recombinant Escherichia coli and characterized. The optimum pH of the purified enzyme was 3.5 and it was stable over pH from 3.0 to 8.0. Its optimum temperature was 80 degrees C and it showed thermostability in the temperature range from 50 to 70 degrees C. Xyl43B had a K(m) of 6.2 mM and a V(max) of 15 micromol min(-1) mg(-1) for p-nitrophenyl-beta-D-xylopyranoside.