Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens

AIMS: To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. METHODS AND RESULTS: Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. CONCLUSIONS: A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation of tetracycline-resistant strains of strictly anaerobic bacteria from the rumen

Tetracycline resistant (Tc^R) strains of three of the major species of strictly anaerobic rumen bacteria Megasphaera elsdenii, Selenomonas ruminantium and Butyrivibrio fibrisolvens , were recovered with an isolation medium containing 20 μg/ml tetracycline. Only two of 14 strains of these species from other sources, isolated without antibiotic selection, showed tetracycline resistance. Evidence was found for the presence of plasmids in two tetracycline-resistant strains of M. elsdenii , and in some strains of S. ruminantium.     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis.

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mixed fungal populations and lignocellulosic tissue degradation in the bovine rumen

Anaerobic fungi in ruminal fluid from cows eating Bermuda grass hay plus a grain and minerals supplement were evaluated for diversity in sporangial morphotypes and colony growth patterns and for the degradation of various lignocelluloses. In selective cultures containing streptomycin and penicillin, an active population of ruminal fungi colonized leaf blades and degraded fiber at rates and extents almost equal to that of the total ruminal population. Three major sporangial morphotypes were consistently observed on leaf blades: oval, globose, and fusiform. Fungal colonies representing three distinct growth types consistently developed in anaerobic roll tubes inoculated with strained ruminal fluid. Sporangial morphotypes could not be matched to colony types due to multiple sporangial forms within a colony. Under identical growth conditions, one type exhibited a monocentric growth pattern, while two types exhibited polycentric growth patterns previously unreported in ruminal fungi. Mixed ruminal fungi in selective cultures or in digesta taken directly from the rumen produced a massive clearing of the sclerenchyma. Quantitation of tissue areas in cross sections by light microscopic techniques showed that fungal incubations resulted in significant (P = 0.05) increases in sclerenchyma degradation compared to whole ruminal fluid incubations. The mestome cell wall was at times penetrated and partially degraded by fungi; the colonization was less frequent and to a lesser degree than with the sclerenchyma. Conversely, ruminal bacteria were not observed to degrade the mestome sheath. Phenolic monomers at 1 mM concentrations did not stimulate to a significant (P = 0.05) extent the dry weight loss or fungal colonization of leaf blades; at 10 mM concentrations cinnamic and benzoic acids were toxic to ruminal fungi.     

Use of lectins for a comparative study of cell wall composition of different anaerobic rumen fungal strains

The technique based on fluorescein-linked lectins used to determine the cell wall structure of anaerobic rumen fungi belonging to genera: Neocallimastix, Piromonas and Sphaeromonas, appears to be an interesting tool for distinguishing between strains. Furthermore this technique shows differences of cell wall composition between different parts of the thallus (spores, sporangia, rhizo?ds).     

Enumeration of cellulolytic anaerobic bacteria from the bovine rumen: Comparison of three methods

Three methods—the most probable number technique, a cellulose agar overlay on basal carbohydrate plates, and carboxymethylcellulose in basal carbohydrate plates—were compared for ease of preparation, interpretation of results, and agreement in estimation of size of the cellulolytic bacterial population in digesta samples from the rumen. The most probable number method yielded consistent detection of cellulose hydrolysis in liquid medium but required at least a 10-day incubation, and its mean was associated with wide 95% confidence intervals. The cellulose overlay method was the least consistent, and zones of hydrolysis often were difficult to see. The carboxymethylcellulose method was the easiest method for preparation and required only a 2-day incubation. The three methods estimated the same population size (all within one-half log unit of each other), but the carboxymethylcellulose method had the lowest coefficient of variation.     

Mixed fungal populations and lignocellulosic tissue degradation in the bovine rumen.

Anaerobic fungi in ruminal fluid from cows eating Bermuda grass hay plus a grain and minerals supplement were evaluated for diversity in sporangial morphotypes and colony growth patterns and for the degradation of various lignocelluloses. In selective cultures containing streptomycin and penicillin, an active population of ruminal fungi colonized leaf blades and degraded fiber at rates and extents almost equal to that of the total ruminal population. Three major sporangial morphotypes were consistently observed on leaf blades: oval, globose, and fusiform. Fungal colonies representing three distinct growth types consistently developed in anaerobic roll tubes inoculated with strained ruminal fluid. Sporangial morphotypes could not be matched to colony types due to multiple sporangial forms within a colony. Under identical growth conditions, one type exhibited a monocentric growth pattern, while two types exhibited polycentric growth patterns previously unreported in ruminal fungi. Mixed ruminal fungi in selective cultures or in digesta taken directly from the rumen produced a massive clearing of the sclerenchyma. Quantitation of tissue areas in cross sections by light microscopic techniques showed that fungal incubations resulted in significant (P = 0.05) increases in sclerenchyma degradation compared to whole ruminal fluid incubations. The mestome cell wall was at times penetrated and partially degraded by fungi; the colonization was less frequent and to a lesser degree than with the sclerenchyma. Conversely, ruminal bacteria were not observed to degrade the mestome sheath. Phenolic monomers at 1 mM concentrations did not stimulate to a significant (P = 0.05) extent the dry weight loss or fungal colonization of leaf blades; at 10 mM concentrations cinnamic and benzoic acids were toxic to ruminal fungi.     

Stoichiometry of glucose and starch splitting by strains of amylolytic bacteria from the rumen and anaerobic digester

The stoichiometry of glucose and starch splitting by the amylolytic bacteria Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Eubacterium ruminantium and Clostridium sp. was followed. There were many differences in the ratios of metabolites and in growth yields, as well as in the cell composition, between the growth on glucose and starch. The bacteria employ different nutritional strategies with respect to both energy sources.     

Stoichiometry of glucose and starch splitting by strains of amylolytic bacteria from the rumen and anaerobic digester

The stoichiometry of glucose and starch splitting by the amylolytic bacteria Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Eubacterium ruminantium and Clostridium sp. was followed. There were many differences in the ratios of metabolites and in growth yields, as well as in the cell composition, between the growth on glucose and starch. The bacteria employ different nutritional strategies with respect to both energy sources.     

A simple in situ method for estimating fungal population size in the rumen

Sediment and the overlying water samples were collected from 67 fresh and 38 salt water sites in the UK. Only 13% (five) of the samples taken from salt water sites were negative for magnetotactic bacteria compared with 47·7% (32) of the fresh water ones. Of the seven types of salt water habitats examined, saltmarsh ponds were found to be most suited to magnetotactic bacteria, 96% (25/26) of sites being positive.     

Characterization of two strains of Pelobacter carbinolicus isolated from anaerobic digesters

In anaerobic industrial digesters treating wastewaters from food industry, the sludges showed high capacities to degrade ethanol. The main syntrophic ethanoldegrading organisms were characterized as Pelobacter carbinolicus. During acetoin degradation, butanediol isomers were shown to be transiently produced. In coculture with Methanobrevibacter arboriphilus, the strains oxidized not only primary monoalcohols but also 1,2- and 1,3-diols. Ecological importance as well as metabolic pathways of diol degradation are discussed.     

Isolation and characterization of cellulolytic anaerobic fungi and associated mycoplasmas from the rumen of a steer fed a roughage diet

The isolation of 12 strains of cellulolytic fungi from the rumen of a roughage-fed steer is described. These represented three different genera, including one new genus and species (Orpinomyces bovis). The organisms were indistinguishable on the basis of fermentation products from cellulose, and their fermentation patterns were very similar to those of rumen fungi isolated in other countries. Mycoplasmas were found to be associated with 7 of the 12 isolates. The ecological role of the association of the mycoplasmas with rumen fungi is still unknown.     

PCR-DGGE analysis of bacterial population attached to the bovine rumen wall

We isolated and amplified by PCR 16S rDNA from bacteria attached to the bovine rumen wall and analyzed it by denaturing gradient gel electrophoresis (DGGE) with subsequent sequence analysis. The attached bacterial community differed from the bacteria of rumen content; however, no differences were observed among the five epithelial sampling sites taken from each animal. The DGGE profile of the bacterial population attached to the rumen wall represented a high inter-animal variation.     

Removal of anaerobic fungi from the rumen of sheep by chemical treatment and the effect on feed consumption and in vivo fibre digestion

The population of anaerobic fungi in the rumen of sheep was reduced by the addition of tetronasin (an ionophore antibiotic) to a herbage diet. Fungi were reduced to undetectable levels (< 1 fungal zoospore per ml rumen fluid) by the combined addition of tetronasin and cycloheximide (a protein synthesis inhibitor) and the absence of fungi was maintained with low levels of tetronasin. Sheep with fungi present in the rumen ate 40% more of a straw-based diet (with a fibre digestibility in vivo of 51%) than they ate when without fungi (47% fibre digestibility). Counts of total viable bacteria, cellulolytic bacteria and ciliate protozoa in the rumen were not significantly different when anaerobic fungi were either present or absent.     

Phylogenetic analysis of methanogens from the bovine rumen

Background  

Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified.     

Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen

Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.