Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1

Summary The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.Abbreviations CMCase carboxymethylcellulase - celA gene coding for CeIA - CelA cellodextrinase - ORF open reading frame - phoA gene encoding alkaline phosphatase - pNPC p-nitrophenyl--d-cellobioside     

Analysis of a cellodextrinase cloned from Ruminococcus flavefaciens FD-1

Abstract A cellulase gene from Ruminococcus flavefaciens FD-1 had previously been cloned in Escherichia coli . The product of this gene, CelA, was purified from E. coli and characterised. This 39 kDa cellulase is antigenically related, and of similar mass, to a protein in R. flavefaciens . The enzyme has cellodextrinase activity with predominantly exo-type action. CelA activity was optimal at pH 6.5 and 41°C, and was inhibited in the presence of divalent metal cations. The K _m and V _max were determined as 0.68 mM and 1.89 μmol min⁻¹ mg⁻¹ of CelA, respectively. Cellobiose was the major end product of cellodextrin hydrolysis, and our results suggest that celluboise is competitive inhibitor of CelA.     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.     

Organisation and Variable Incidence of Genes Concerned with the Utilization of Xylans in the Rumen Cellulolytic Bacterium Ruminococcus flavefaciens

Strains of the rumen cellulolytic bacterium Ruminococcus flavefaciens vary in their ability to utilise isolated plant xylans for growth. Here an 11.5 kb fragment of genomic DNA from the xylan-utilizing R. flavefaciens strain 17 that contains the xynD gene, which encodes an extracellular xylanase/β -(1,3-1,4)-glucanase, was analysed. Sequencing revealed five consecutive open reading frames downstream from xynD on the same strand, preceded by the divergently transcribed ORF3. These include the following genes likely to be involved in utilisation of xylan breakdown products: xylA, encoding a β -(1,4)-xylosidase, xsi, encoding a xylose isomerase and ORF8 encoding part of an ABC-type sugar transporter. The products of ORF3 and of a partial ORF1 found upstream of xynD, show significant sequence similarity to AraC-type regulatory proteins while ORF4 and ORF7 show no close relationship to other known proteins. Homologues of the xylA and xsi genes, and inducible β -xylosidase activity, were readily detectable in three xylan-utilizing R. flavefaciens strains 17, B1a and C94 but not in two xylan non-utilizing strains, C1a and B34b, suggesting that this cluster may be absent from xylan non-utilizing strains.     

Nucleotide sequence of the Ruminococcus albus SY3 endoglucanase genes ce1A and ce1B

Summary The complete nucleotide sequences of Ruminococcus albus genes celA and celB coding for endoglucanase A (EGA) and endoglucanase B (EGB), respectively, have been determined. The celA structural gene consists of an open reading frame of 1095 bp. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified EGA. The celB structural gene consists of an open reading frame of 1227 bp; 7 by upstream of the translational start codnn of celB is a typical gram-positive Shine-Dalgarno sequence. The deduced N-terminal region of EGB conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete celB gene, cloned into pUC vectors, caused lethality in Escherichia coli. In contrast, celA cloned in pUC18, under the control of lacZp, directed high-level synthesis of EGA in E. coli JM83. EGA in cell-free extract, purified to near homogeneity by ionexchange chromatography, had a M_r of 44.5 kDa. Gene deletion and subcloning studies with celA revealed that EGA hydrolysed both CMC and xylan, and did not contain discrete functional domains. EGA and EGB showed considerable homology with each other, in addition to exhibiting similarity with Egl (R. albus), EGE (Clostridium thermocellum) and End (Butyrivibrio fibrisolvens).Abbreviations CMC carboxymethylcellulose - CMCase carboxymethylcellulase - celA gene coding for EGA - EGA endoglucanase A - celB gene coding for EGB - EGB endoglucanase B - S-D Shine-Dalgarno     

Nucleotide sequence and regulated expression of the Salmonella fljA gene encoding a repressor of the phase 1 flagellin gene

Summary The nucleotide sequence of Salmonella abortus-equi fljA, which together with the phase 2 flagellin gene constitutes the fljBA operon and encodes the repressor for the phase 1 flagellin gene fliC, was determined. The repressor was predicted to be a basic protein consisting of 179 amino acid residues (M_r = 20419 Da) encoded by ORFII. This was confirmed by the fact that host fliC is repressed by plasmid-encoded ORFII, which indeed expresses a 20 kDa product as determined by urea SDS-polyacrylamide gel electrophoresis. An amino acid sequence capable of forming a helix-turn-helix type of structure was predicted in the C-terminal region of FljA. A rho-independent intercistronic terminator was detected between fljB and ftjA. Chloramphenicol acetyltransferase (CAT) assays of fusions indicated that the terminator is capable of reducing expression of fljA to the level of a few percent, relative to fljB in broth cultures and to 1 % in M9 glycerol cultures.     

Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli

Summary The nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl--d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent M _r of approximately 61000. The calculated M _r of the ced1 gene product was 61023.Abbreviations Ap ampicillin - ced1 gene coding for Ced1 - Ced1 cellodextrinase from B. fibrisolvens - CMC carboxymethylcellulose - LB Luria Bertani - ORF open reading frame - pNPC p-nitrophenyl--d-cellobioside - PC phosphate citrate - HCA hydrophobic cluster analysis     

Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase

The nucleotide sequence of 1400 bp from R-plasmid R538-1 containing the streptomycin/spectinomycin adenyltransferase gene (aadA) was determined, and the location of the aadA gene was identified by a combination of insertion and deletion mutants. Its gene product, aminoglycoside 3"-adenylyltransferase (AAD(3")(9), has a Mr of 31,600.     

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell. Received: 15 July 1996 / Accepted: 24 October 1996     

Valorisation of wastepaper using the fibrolytic/hydrogen producing bacterium Ruminococcus albus

The present study aimed to investigate the biotransformation of different kinds of wastepaper to hydrogen by the fibrotylic bacterium Ruminococcus albus. Five different types i.e. paper tissue, office paper, illustrated magazine paper, paperboard and newspaper, were selected as representatives of the most common types of wastepaper found in municipal solid wastes. The percentage of total carbohydrates measured as glucose equivalents, ranged from 50% to 100% (w/w), whereas the bioconversion by R. albus ranged from 18% to 100% of their initial weigh. The only metabolic products detected in all cases were acetate, ethanol, formate, hydrogen and carbon dioxide. The hydrogen yields ranged from 46 to 280 L H₂/kg paper, indicating that wastepaper could be a promising candidate for second generation biohydrogen production. Subsequently, hydrolysis was investigated for paper tissue and paperboard. It was shown that in both cases the degradation process could be satisfactory described by zero order kinetics and it was identified to be the rate limiting step for the whole process, controlling biomass growth and metabolites generation rate.     

Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens

A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl--d-glucosaminide, isolated and shown to encode an endo--N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.     

Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin-like antibiotics

The nucleotide sequence of a 1883 bp fragment isolated from a resistance plasmid harbored by a Staphylococcus aureus clinical isolate and carrying the gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin family has been determined. The sequence contains one open reading frame which extends from the ATG codon at nt 641 to a TGA codon at nt 1537 and which potentially codes for a protein of 33.035 Da. This value is in agreement with the apparent size (33 kDa) of the protein observed, in minicell extracts. Inactivation of the B components of the virginiamycin antibiotics as well as resistance to these antibiotics were expressed in a virginiamycin sensitive mutant of Escherichia coli recipient containing the gene on a high copy number plasmid.