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P L Bergquist

Publications and source records attributed to P L Bergquist.

At least 19 recordsLinked to original sources
PubMed1999

Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. strain Rt69B.1.

Three family 10 xylanase genes (xynA, xynB, and xynC) and a single family 11 xylanase gene (xynD) were identified from the extreme thermophile Caldicellulosiruptor strain Rt69B.1 through the use of consensus PCR in conjunction with sequencing and polyacrylamide gel electrophoresis. These genes appear to comprise the complete endoxylanase system of Rt69B.1. The xynA gene was found to be homologous to the xynA gene of the closely related Caldicellulosiruptor strain Rt8B.4, and primers designed previously to amplify the Rt8B.4 xynA gene could amplify homologous full-length xynA gene fragments from Rt69B.1. The complete nucleotide sequences of the Rt69B.1 xynB, xynC, and xynD genes were obtained using genomic walking PCR. The full-length xynB and xynC genes are more than 5 kb in length and encode highly modular enzymes that are the largest xylanases reported to date. XynB has an architecture similar to the family 10 xylanases from Thermoanaerobacterium saccharolyticum (XynA) and Clostridium thermocellum (XynX) and may be cell wall associated, while XynC is a bifunctional enzyme with an architecture similar to the bifunctional beta-glycanases from Caldicellulosiroptor saccharolyticus. The xynD gene encodes a two-domain family 11 xylanase that is identical in architecture to the XynB family 11 xylanase from the unrelated extreme thermophile Dictyoglomus thermophilum strain Rt46B.1. The sequence similarities between the Rt69B.1 xylanases with respect to their evolution are discussed.

Bacteria, Anaerobic↗
PubMed1998

Expression and secretion of a xylanase from the extreme thermophile, thermotoga strain FjSS3B.1, in Kluyveromyces lactis.

The yeast Kluyveromyces lactis has been developed as a host for extracellular production of thermophilic hemicellulases by employing expression vectors based on the 2 mu-like plasmid pKD1 of Kluyveromyces drosophilarium. A beta-1,4-xylanase gene (xynA) from the extreme thermophile Thermotoga sp. strain FjSS3B.1 was fused inframe with a synthetic secretion signal derived from the K. lactis killer toxin and expressed under control of the K. lactis LAC4 (beta-galactosidase) promoter. Correctly processed xylanase enzyme with full biological activity on oat spelts xylan was secreted during shake-flask cultivation of K. lactis transformants. The transcriptional activity of the LAC4 promoter dramatically affected mitotic stability of the expression vector under nonselective conditions. However, one combination of host strain and expression plasmid showed higher stability and good yield and has been employed for scaled-up production of XynA and other thermostable hemicellulases in chemostat culture. XynA secreted by K. lactis is as thermostable as the native enzyme, having a half-life of 48 h at 90 degrees C.

Amino Acid Sequence↗
PubMed1998

Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp.

A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from Dictyoglomus thermophilum Rt46B.1. Family 11 xylanase consensus fragments (GXCFs) were amplified from Rt46B.1 genomic DNA by using different sets of consensus PCR primers that exhibited broad specificity for conserved motifs within fungal and/or bacterial family 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (xynB) was identified. The entire gene sequence was obtained in the second step by using genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstream and downstream of the xynB GXCF region. The putative XynB peptide (M(r), 39,800) encoded by the Rt46B.1 xynB open reading frame was a multidomain enzyme comprising an N-terminal catalytic domain (M(r), 22,000) and a possible C-terminal substrate-binding domain (M(r), 13,000) that were separated by a short serine-glycine-rich 23-amino-acid linker peptide. Seven xylanases which differed at their N and C termini were produced from different xynB expression plasmids. All seven xylanases exhibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85 degrees C. Pretreatment of Pinus radiata and eucalypt kraft-oxygen pulps with XynB resulted in moderate xylan solubilization and a substantial improvement in the bleachability of these pulps.

Amino Acid Sequence↗
PubMed1997

Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus.

A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95,732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5'-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.

Amino Acid Sequence↗
PubMed1997

Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis.

The xynA structural gene from the extremely thermophilic anaerobe Dictyoglomus thermophilum Rt46B.1 was fused in frame with the secretion signal of the Kluyveromyces lactis killer toxin in episomal expression vectors based on the Kluyveromyces plasmid pKD1. XynA was secreted predominantly as an unglycosylated 35-kDa protein which comprised up to 90% of the total extracellular proteins and reached a concentration of 130 micrograms/ml in shake-flask cultures grown under selective conditions.

Archaea↗
PubMed1996

Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation.

The gene for a highly thermostable neutral proteinase (Npr) was isolated from Bacillus sp. strain EA1 by the polymerase chain reaction using consensus primers based on the sequences of npr genes from related species. The gene was sequenced and shown to be closely related to a neutral proteinase gene from Bacillus caldolyticus strain YP-T; the mature form of the enzyme differing by only a single amino acid. Enzyme samples were prepared from both the native organisms and also from recombinant Escherichia coli expressing the two npr genes. The proteinase from strain EA1 was shown to be significantly more thermostable than that from B. caldolyticus and that this difference is the result of a single amino acid substitution which is situated proximal to a region of the enzyme known to be crucial to conferring thermal stability. The phylogenetic relationship of EA1 to other Bacilli is also described.

Amino Acid Sequence↗
PubMed1996

Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4.

A gene encoding a beta-mannanase (manA) has been cloned from an obligately anaerobic extreme thermophile, Caldicellulosiruptor strain Rt8B.4, which is most closely related to Caldicellulosiruptor saccharolyticus (formerly Caldocellum saccharolyticum). The gene codes for a multidomain enzyme with a C-terminal beta-mannanase domain which was amplified by the polymerase chain reaction and cloned into a temperature-inducible expression vector in Escherichia coli. Sequence comparisons have shown that the Man domain of Rt8B.4 ManA is related to a thermophilic Dictyoglomus mannanase and a mesophilic mannanase from a Bacillus species. It appears to be unrelated to the beta-mannanase domain of C. saccharolyticus, implying acquisition of the genes from unrelated sources by the two bacteria.

Amino Acid Sequence↗
PubMed1996

Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor.

A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in lambda ZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70 degrees C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.

Amino Acid Sequence↗
PubMed1995

A single emm gene-specific oligonucleotide probe does not recognise all members of the Streptococcus pyogenes M type 1.

Serological typing of the streptococcal M protein has recently been challenged by a number of unique molecular methodologies based on oligonucleotide recognition of allelic variations within the M protein (emm) gene. In these methods, stringent hybridization of an oligonucleotide probe to a polymerase chain reaction amplified emm gene is used as confirmation of specific M type identity. A sample of 17 isolates from 7 previously defined distinct genotypes were tested using a single M1 oligonucleotide probe. Isolates from only three of the genotypes hybridized with the probe. The results demonstrate that a single emm-specific oligonucleotide probe can not identify all members of M type 1, as defined by conventional serotyping using polyclonal antisera.

Amino Acid Sequence↗
PubMed1995

A gene encoding a thermophilic alkaline serine proteinase from Thermus sp. strain Rt41A and its expression in Escherichia coli.

The extreme thermophile Thermus sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus. The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.

Amino Acid Sequence↗
PubMed1995

Sequence and expression of a xylanase gene from the hyperthermophile Thermotoga sp. strain FjSS3-B.1 and characterization of the recombinant enzyme and its activity on kraft pulp.

A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp.

Amino Acid Sequence↗
PubMed1995

Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate.

A genomic library of the Dictyoglomus sp. strain Rt46B.1 was constructed in the phage vector lambda ZapII and screened for xylanase activity. A plaque expressing xylanase activity, designated B6-77, was isolated and shown to contain a genomic insert of 5.3 kb. Subcloning revealed that the xylanase activity was restricted to a internal 1,507-bp PstI-HindIII fragment which was subsequently sequenced and shown to contain a single complete open reading frame coding for a single-domain xylanase, XynA, with a putative length of 352 amino acids. Homology comparisons show that XynA is related to the family F group of xylanases. The temperature and pH optima of the recombinant enzyme were determined to be 85 degrees C and pH 6.5, respectively. However, the enzyme was active across a broad pH range, with over 50% activity between pH 5.5 and 9.5. XynA was shown to be a true endo-acting xylanase, being capable of hydrolyzing xylan to xylotriose and xylobiose, but it could not hydrolyze xylobiose to monomeric xylose. XynA was also shown to hydrolyze xylan present in Pinus radiata kraft pulp, indicating that it may be of use as an aid in pulp bleaching. The equivalent xylanase gene was also isolated from the related bacterium Dictyoglomus thermophilum, and DNA sequencing showed these genes to be identical, which, together with the 16S small-subunit rRNA gene sequencing data, indicates that Rt46B.1 and D. thermophilum are very closely related.

Amino Acid Sequence↗
PubMed1995

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.

The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.

Bacterial Proteins↗
PubMed1994

Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium.

A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor, as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.

Bacteria, Anaerobic↗
PubMed1994

Secondary structure model for an unusual SSU rRNA from the extremely thermophilic bacterium strain AZ3 B.1.

The SSU rRNA gene of the extremely thermophilic bacterium strain AZ3 B.1, encodes an rRNA containing four large inserts. A secondary structure model has been constructed which predicts that the inserts form large stem loop structures with a common sequence motif at the base of the helices. To date, these structures have only been detected in related, thermophilic organisms.

Bacteria, Anaerobic↗
PubMed1994

Phylogeny and lipid composition of Thermonema lapsum, a thermophilic gliding bacterium.

1,490 nucleotides of the 16S rRNA gene of a Gram-negative, thermophilic and gliding bacterium, Thermonema lapsum, have been sequenced. Phylogenetic analysis indicates that T. lapsum is related to cytophaga-flavobacteria-bacteroides (CFB) and is confirmed by the identification signature nucleotides that define this group. Further phylogenetic analysis indicates that T. lapsum forms the deepest branch in the CFB group; this observation was confirmed by the identification of unique nucleotide and nucleotide pairs which separate T. lapsum from all other members of this group. The phospholipid fatty acid (PLFA) profile also confirmed that T. lapsum is related to the cytophaga-flavobacteria-bacteroides group and also to selected members of the genus Flexibacter; the PLFA profile is unique to T. lapsum.

Bacteria, Aerobic↗
PubMed1994

Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.

The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.

Amino Acid Sequence↗