Genome sequence of the ruminal bacterium Megasphaera elsdenii

Megasphaera elsdenii is a Gram-negative ruminal bacterium. It is being investigated as a probiotic supplement for ruminants as it may provide benefits for energy balance and animal productivity. Furthermore, it is of biotechnological interest due to its capability of producing various volatile fatty acids. Here we report the complete genome sequence of M. elsdenii DSM 20460, the type strain for the species.     

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.     

The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid

AIMS: To isolate predominant ruminal bacteria that produce trans-10, cis-12 conjugated linoleic acid (CLA) from linoleic acid (LA). METHODS AND RESULTS: Mixed bacteria from ruminal contents of a cow fed grain were enriched with DL-lactate and trypticase. They produced more trans-10, cis-12 CLA than those that were not enriched (7 vs 2 microg mg protein(-1), P < 0.05). Enrichments had an abundance of large cocci that produced trans-10, cis-12 CLA from LA. Strain YJ-4 produced the most trans-10, cis-12 CLA (approx. 7 microg mg protein(-1)) and 16S rDNA sequencing indicated that YJ-4 was a strain of Megasphaera elsdenii. Megasphaera elsdenii T81 produced approx. 4 microg trans-10, cis-12 CLA mg protein(-1) while strains B159, AW106 and JL1 produced < 0.5 microg mg protein(-1). The trans-10, cis-12 CLA production of YJ-4 was first order with respect to cell concentration (0-800 microg protein ml(-1)), but kinetics were not first order with respect to substrate concentration. CONCLUSIONS: Some M. elsdenii strains produce significant amounts of trans-10, cis-12 CLA. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-10, cis-12 CLA appears to cause milk fat depression in cattle fed diets supplemented with grain and polyunsaturated fatty acids, but predominant ruminal bacteria that produced trans-10, cis-12 CLA from LA had not previously been isolated.     

Role of Megasphaera elsdenii in the Fermentation of dl-[2-C]lactate in the Rumen of Dairy Cattle

Since Megasphaera elsdenii ferments a variable part of dl-lactate to butyrate, measurement of the percentage of dl-lactate fermented to propionate via the acrylate pathway in rumen contents will underestimate the participation of M. elsdenii in the dl-lactate fermentation. The percentage of dl-[2-C]lactate fermented via the acrylate pathway and the percentage of dl-lactate fermented to butyrate can be measured with C-FT (Fourier transform)-nuclear magnetic resonance. On the average, the contribution of M. elsdenii to dl-lactate fermentation in the rumen of dairy cattle was found to be 74% (standard deviation, 13%), but differed with animal or diet. After feeding a cow readily fermentable carbohydrates, the contribution of M. elsdenii to the fermentation of dl-lactate increased as a consequence of catabolite repression in other dl-lactate-fermenting bacteria.     

GATC-specific restriction-modification systems in ruminal bacteria

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.     

Interactions between rumen amylolytic and lactate-utilizing bacteria in growth on starch

The growth and metabolism of the rumen amylolytic bacteria Streptococcus bovis, Butyrivibrio fibrisolvens and Bacteroides ruminicola, growing in pure cultures and co-cultures with the rumen lactilytic bacteria Megasphaera elsdenii and Veillonella alcalescens were followed. The interaction of amylolytic bacteria with V. alcalescens represents a simple food chain. The interaction with M. elsdenii is more complex, since there is a simultaneous competition for products of the starch degradation.     

Limited genetic variability inMegasphaera elsdenii strains

Levels of phenotypic and genotypic diversity among seven Megasphaera elsdenii strains recovered from rumen contents of cattle, sheep and lambs were determined by a combination of antibiotic-resistance analysis and PCR fingerprint techniques targeted both to the ribosomal RNA operon (ARDRA, RISA) and the whole genome (ERIC-PCR, RAPD-PCR). Despite exhibiting different antibiotic resistance profiles, the tested strains represent genetically nearly identical isolates. Close genetic relatedness was found among M. elsdenii isolates that originated from vastly different habitats worldwide, as revealed by the comparison of 16S rDNA sequences.     

Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria

Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested. While restriction endonucleases isolated from M. elsdenii strains were found to be sensitive to Dam methylation, enzymes from M. multiacida cleaved DNA irrespective of Dam methylation. The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M. elsdenii and M. multiacida strains. R-M systems are believed to represent the main defense tool against phage infection. Based on the results of our experiments it could be assumed that M. elsdenii and M. multiacida use the different strategy for bacteriophage protection compared to S. ruminantium.     

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

AIMS: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. METHODS AND RESULTS: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 10(10)R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. CONCLUSIONS: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. SIGNIFICANCE AND IMPACT OF THE STUDY: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.     

Cell Envelope Morphology of Rumen Bacteria

The cell walls of three species of rumen bacteria (Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii) were studied by a variety of morphological methods. Although all the cells studied were gram-negative and had typical cytoplasmic membranes and outer membranes, great variation was observed in the thickness of their peptidoglycan layers. Megasphaera elsdenii evidenced a phenomenally thick peptidoglycan layer whose participation in septum formation was very clearly seen. All species studied have cell wall "coats" external to the outer membrane. The coat of Bacteroides ruminicola is composed of large (approximately 20 nm) globules that resemble the protein coats of other organisms, whereas the coat of Bacteroides succinogenes is a thin and irregular carbohydrate coat structure. Megasphaera elsdenii displays a very thick fibrillar carbohydrate coat that varies in thickness with the age of the cells. Because of the universality of extracellular coats among rumen bacteria we conclude that the production of these structures is a protective adaptation to life in this particular, highly competitive, environment.     

Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine

Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.     

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.     

Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets

Abstract Samples of rumen ingesta from two rumen-fistulated dairy cows fed grass silage-based diets were examined for numbers and types of bacteria that developed colonies on rumen fluid-agar media designated to support the growth of (a) a wide range of species, (b) cellulolytic bacteria, (c) lactate-fermenting bacteria, (d) non-fermentative bacteria. The most numerous species was Bacteroides ruminicola followed by Butyrivibrio fibrisolvens . The most abundant cellulolytic species were Eubacterium cellulosolvens and Ruminococcus flavefaciens. Megasphaera elsdenii and Selenomonas ruminantium were important lactate fermenters but an unidentified bacterium that grew poorly on maintenance medium was by far the most numerous among bacteria isolated from lactate-containing medium. One strain remained sufficiently viable to show that it fermented lactate to propionate and acetate.     

Increased amount of Bifidobacterium thermacidophilum and Megasphaera elsdenii in the colonic microbiota of pigs fed a swine dysentery preventive diet containing chicory roots and sweet lupine

AIMS: To investigate which specific bacterial species that were stimulated or inhibited in the proximal colon of pigs when a fructan-rich diet was compared with a diet that contained resistant carbohydrates. The study focussed especially on Bifidobacterial species by using a noncultureable approach. METHODS AND RESULTS: Terminal restriction fragment length polymorphism (T-RFLP) was used to describe differences in the total colonic microbiota as well as in the populations of Bifidobacterium spp. in pigs fed with a fructan-rich diet and a diet containing resistant carbohydrates. The fructan-rich diet has previously been shown to prevent swine dysentery caused by Brachyspira hyodysenteriae. The T-RFLP profiling, 16S rRNA gene cloning and in situ hybridization showed that the pigs fed with the fructan-rich diet had a higher proportion of Bifidobacterium thermacidophilum subsp. porcinum and Megasphaera elsdenii. CONCLUSIONS: These findings suggested that the bacterial fructan fermentation occurring in the porcine colon might be cross-feeding of lactate produced by B. thermacidophilum and used by M. elsdenii. SIGNIFICANCE AND IMPACT OF THE STUDY: B. thermacidophilum and M. elsdenii may be the course of the inhibition of the pathogenic bacteria Brach. hyodysenteriae in colon of pigs when they are fed fructan-rich diets.     

A lack of predatory interaction between rumen ciliate protozoa and Shiga-toxin producing Escherichia coli

AIMS: To investigate interactions between rumen protozoa and Shiga toxin-producing Escherichia coli (STEC) and to ascertain whether it is likely that rumen protozoa act as ruminant hosts for STEC. METHODS AND RESULTS: The presence of stx genes in different microbial fractions recovered from cattle and sheep rumen contents and faeces was examined using PCR. In animals shedding faecal STEC, stx genes were not detected in the rumen bacterial or rumen protozoal fractions. Direct interactions between ruminal protozoa and STEC were investigated by in vitro co-incubation. Rumen protozoa did not appear to ingest STEC, a STEC lysogen or non-STEC E. coli populations when co-incubated. CONCLUSIONS: The ruminal environment is unlikely to be a preferred habitat for STEC. Bacterial grazing by rumen protozoa appears to have little, if any, effect on STEC populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that ruminal protozoa are unlikely to be a major factor in the survival of STEC in ruminants. They appear as neither a host that protects STEC from the ruminal environment nor a predator that might reduce STEC numbers.     

Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum . increased the extent of microcrystalline cellulose degradation by 10·12% and 7·96%, respectively. Biomass yield in co-cultures was increased by 89·9% and 59·4% for M. elsdenii and E. limosum . Y_cellulose for fungus alone was 52·29 g dry matter mol^-1 glucose. These values were 64·93 and 55·92 g mol^-1 glucose unit in co-culture with M. elsdenii and E. limosum , respectively.     

Studies on some characteristics of hydrogen production by cell-free extracts of rumen anaerobic bacteria

Hydrogen production was studied in the following rumen anaerobes: Bacteroides clostridiiformis, Butyrivibrio fibrisolvens, Enbacterium limosum, Fusobacterium necrophorum, Megasphaera elsdenii, Ruminococcus albus, and Ruminococcus flavefaciens. Clostridium pasteurianum and Escherichia coli were included for comparative purposes. Hydrogen production from dithionite, dithionite-reduced methyl viologen, pyruvate, and formate was determined. All species tested produced hydrogen from dithionite-reduce methyl viologen, but only C. pasteurianum, B. clostridiiformis, E. limosum, and M. elsdenii produced hydrogen from dithionite. All species except E. coli produced hydrogen from pyruvate, but activity was low or absent in extracts of E. limosum, F. necrophorum, R. albus, and R. flavefaciens unless methyl viologen was added. Hydrogen was produced from formate only by E. coli, B. clostridiiformis, E. limosum, F. necrophorum, and R. flavefaciens. Extracts were subjected to ultracentrifugation in an effort to determine the solubility of hydrogenase. The hydrogenase of all species except E. coli appeared to be soluble, although variable amounts of hydrogenase activity were detected in the pellet. Treatment of extracts of the rumen microbial species with DEAE-cellulose resulted in loss ofhydrogen production from pyruvate. Activity was restored by the addition of methyl viologen. It is concluded that hydrogen production in these rumen microorganisms is similar to that in the saccharolytic clostridia.     

Characterization of the gene encoding the [Fe]-hydrogenase from Megasphaera elsdenii

The gene encoding the [Fe]-hydrogenase from the anaerobic bacterium Megasphaera elsdenii has been cloned and sequenced. The gene is monocistronic, in keeping with the protein being a monomer. The translated protein sequence (484 residues, M(r)=53 kDa) comprises a small 2[4Fe-4S] ferredoxin-like domain and a large domain containing the catalytic site. Comparisons with other [Fe]-hydrogenase sequences, including two of which the crystal structures are known, show that the M. elsdenii protein is among the smallest of these enzymes and provide useful indications regarding the basic structural core common to all [Fe]-hydrogenases. It is, nevertheless, to be noted that the genome of Thermotoga maritima encodes a putative [Fe]-hydrogenase that would consist of only 301 residues.     

Correlation between composition of the bacterial community and concentration of volatile fatty acids in the rumen during the transition period and ketosis in dairy cows

The transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity of Veillonellaceae organisms was reduced and that of Streptococcaceae organisms was increased in rumen samples from the transition period and ketosis groups, with the number of Lactobacillaceae organisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes, Megasphaera elsdenii and Selenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium, Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number of Lactobacillus sp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers of Selenomonas ruminantium and Megasphaera elsdenii organisms in the rumen.     

Restriction and modification systems of ruminal bacteria

A high frequency of type II restriction endonuclease activities was detected inSelenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated fromS. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected inS. ruminantium strains as well as in several other species belonging to theSporomusa subbranch of low G+C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).