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     

An adhesion-defective mutant of Ruminococcus albus SY3 is impaired in its capability to degrade cellulose

An adhesion-defective mutant of Ruminococcus albus SY3 was isolated by a subtractive enrichment procedure, which involved repetitive adsorption of cellobiose-grown cells to cellulose. The growth characteristics of the mutant were compared with those of the wild type. Like the wild-type cells, the mutant was capable of growing on soluble substrates, i.e. cellobiose and xylan. However, in contrast to the wild type strain, the mutant was impaired in its capacity to utilize insoluble substrates, e.g. crystalline cellulose, acid-swollen cellulose or alfalfa cell walls. Scanning electron microscopy revealed protuberance-like surface structures on the wild-type strain which were absent on the mutant. The levels of endoglucanase and xylanase enzymatic activities released into the extracellular culture fluid were higher in the wild type compared to the mutant. However, Avicelase activity was not detected in the extracellular culture fluid of either strains when grown on cellobiose.     

Subcellular distribution of glycanases and related components in Ruminococcus albus SY3 and their role in cell adhesion to cellulose

AIMS: To compare the subcellular distribution of glycanase-related components between wild-type Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes. METHODS AND RESULTS: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions. SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes. The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the 'true cellulase activity' appeared to be entirely confined to the cell membrane fractions. Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R. albus SY3. CONCLUSIONS: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity. A potential cellulosomal scaffoldin candidate was identified in R. albus SY3. SIGNIFICANCE AND IMPACT OF THE STUDY: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R. albus SY3 to cellulose.     

Incorporation of [(15)N] ammonia by the cellulolytic ruminal bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using (15)NH(3). At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH(3)-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH(3). More cell nitrogen was formed from NH(3) during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its (15)N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain

The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albus F-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recombinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40 degrees C, respectively. By Western blot analysis using the antiserum raised against the truncated enzyme, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.     

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     

The isolation and comparison of cellulase genes from two strains of Ruminococcus albus

Endo-1,4-beta-glucanase genes have been cloned from two strains of Ruminococcus albus recently isolated in this laboratory. Although the strains were phenotypically similar, cross-hybridization studies between them showed significant genetic differences, with only 20% of the genome forming DNA heteroduplexes. Heteroduplexes displayed an average dissociation temperature 9 degrees C lower than that of the homoduplex. Consistent with this, restriction maps of the two endoglucanase genes showed no similarity, and hybridization work using the endoglucanase genes as probes revealed that neither gene was present in the genome of the other isolate of R. albus. Comparative enzyme characterization showed differences between the enzymes in their response to temperature, pH and substrate preference.     

Cloning of Ruminococcus albus endo-β-1,4-glucanase and xylanase genes

Buffered solutions of chlorine-releasing agents, sodium hypochlorite (NaOCl), sodium dichloroisocyanurate (NaDCC), and chloramine-T, showed similar activity against vegetative cells of Bacillus subtilis but there was considerable variation in activity against spores, NaOCl showing higher activity than NaDCC, which in turn was more active than chloramine-T. The effect of coat and cortex extraction on sporicidal activity was determined. It was concluded that although spore coats play a role, they do not totally account for chlorine resistance and that the cortex is also involved, probably through its function in maintaining a low water level in the enclosed core. Observed differences in the sporicidal action of NaOCl, NaDCC, and chloramine-T may be related to their differing ability to produce core and cortex degradation.     

Incorporation of [15N]Ammonia by the Cellulolytic Ruminal Bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using ¹⁵NH₃. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH₃-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH₃. More cell nitrogen was formed from NH₃ during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its ¹⁵N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Construction of novel Ruminococcus albus, strains with improved cellulase activity by cloning of Streptomyces rochei endoglucanase gene

The 6.3 kb plasmid pCRB1 containing the eglS gene from Streptomyces rochei, coding for a -1,4 glucanase, was constructed in Bacillus subtilis by using the plasmid vector pIL253 with subsequent activity of the cellulase activity. Strictly anaerobic cellulolytic and xylanolytic strains of Ruminococcus albus, isolated from the rumen of cows and water buffaloes, were used for transformation experiments. The plasmid pCRB1 was introduced by means of electroporation into five freshly isolated strains of R. albus, with frequencies ranging from 10 to 10 /mg of plasmid DNA. Northern analysis demonstrated the expression of the eglS gene in R. albus. All the strains harbouring the heterologous cellulase gene showed an increase of the secreted cellulase activity.     

Albusin B, a bacteriocin from the ruminal bacterium Ruminococcus albus 7 that inhibits growth of Ruminococcus flavefaciens

An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.     

Albusin B, a Bacteriocin from the Ruminal Bacterium Ruminococcus albus 7 That Inhibits Growth of Ruminococcus flavefaciens

An ~32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Formation of Hydrogen and Formate by Ruminococcus albus

Radioisotopic growth studies with specifically labeled (14)C-glucose confirmed that Ruminococcus albus, strain 7, ferments glucose mainly by the Embden-Myerhof-Parnas pathway to acetate, ethanol, formate, CO(2), H(2), and an unidentified product. Cell suspensions and extracts converted pyruvate to acetate, H(2), CO(2), and a small amount of ethanol. Formate was not produced from pyruvate and was not degraded to H(2) and CO(2), indicating that formate was not an intermediate in the production of H(2) and CO(2) from pyruvate. Cell extract and (14)C-glucose growth studies showed that the H(2)-producing pyruvate lyase reaction is the major route of H(2) and CO(2) production. An active pyruvate-(14)CO(2) exchange reaction was demonstrable with cell extracts. The (14)C-glucose growth studies indicated that formate, as well as CO(2), arises from the 3 and 4 carbon positions of glucose. A formate-producing pyruvate lyase system was not demonstrable either by pyruvate-(14)C-formate exchange or by net formate formation from pyruvate. Growth studies with unlabeled glucose and labeled (14)CO(2) or (14)C-formate suggest that formate arises from the 3 and 4 carbon positions of glucose by an irreversible reduction of CO(2). The results of the studies on the time course of formate production showed that formate production is a late function of growth, and the rate of production, as well as the total amount produced, increases as the glucose concentration available to the organism increases.     

Ultrastructure and adhesion properties of Ruminococcus albus.

Morphological studies have shown that cells of the anaerobic rumen bacterium Ruminococcus albus have electron-translucent granules of reserve carbohydrate in their cytoplasm, and that they have a polysaccharide "coat" layer external to their gram-negative cell wall. This coat layer, which stains specifically with ruthenium red, forms a compact mat of fibers adjacent to the cell, and fibrous elements also project as much as 0.6 mum from the cells. These radial fibers are clearly visualized by freeze-etching, and can be seen to extend throughout the extensive intercullular space in centrifuged pellets of these bacteria. Cells of R. albus adhere to cellulose fibers added to the culture medium, and the coat material is seen to mediate this adhesion in addition to its function in the general protection of these cells.     

Nucleotide sequence of the gene encoding the Streptomyces albus G beta-lactamase precursor

A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans [Dehottay et al. (1986) Gene 42, 31-36], was sequenced. The gene codes for a 314-amino-acid precursor, the N-terminal region of which has the characteristics of a signal peptide. The beta-lactamase as excreted by the host strain S. lividans PD6 has a ragged N-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an aminopeptidase. The primary structure (as deduced from the nucleotide sequence) was confirmed by amino acid sequencing of a 16-residue stretch at the amino terminus of the protein, a 12-residue stretch containing the active-site serine [De Meester et al. (1987) Biochem. J. 244, 427-432] and a 23-residue stretch obtained by trypsin digestion of the protein. The beta-lactamase belongs to class A, has three half-cystine residues (one of which occurs on the amino side of the active-site serine) and is inactivated by thiol reagents. Putative ribosome binding site and terminator region were identified.     

Analysis of antibiotic susceptibility and extrachromosomal DNA content of Ruminococcus albus and Ruminococcus flavefaciens

Seventeen Ruminococcus albus and Ruminococcus flavefaciens strains have been screened for naturally occurring antibiotic resistance, as determined by zones of inhibition from antibiotic disks. These strains were also examined for extrachromosomal DNA content. All strains screened are resistant to low levels (10-200 micrograms/mL) of streptomycin. In contrast to the previously reported data, we have found that R. flavefaciens C-94 is now susceptible to both kanamycin and tetracycline. However, R. flavefaciens FD-1 is not susceptible to kanamycin (minimum inhibitory concentration (MIC) = 40 micrograms/mL). Furthermore, R. albus 8 is resistant to tetracycline (MIC = 40 micrograms/mL), and erythromycin (MIC = 100 micrograms/mL). Six freshly isolated strains showed resistance to tetracycline (35-70 micrograms/mL), and all tetracycline-resistant strains also showed resistance to minocycline. None of these Ruminococcus determinants share homology with the streptococcal tetL, tetM, or tetN determinants. All 17 strains were screened for extrachromosomal DNA content. Nine different techniques for the detection and isolation of extrachromosomal DNA were tested. However, owing to difficulties in demonstrating or isolating plasmid DNA, it has not been possible to determine if these antibiotic resistance genes are plasmid borne. Evidence is presented to suggest that the presence of oxygen may affect the quality of the DNA obtained from Ruminococcus.