Succinogenes Strains Research Articles

Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures.

Microbes present one of the most diverse sources of biochemistry in nature, and mRNA sequencing provides a comprehensive view of this biological activity by quantitatively measuring microbial transcriptomes. However, efficient mRNA capture for sequencing presents significant challenges in prokaryotes as mRNAs are not poly-adenylated and typically make up less than 5% of total RNA compared with rRNAs that exceed 80%. Recently developed methods for sequencing bacterial mRNA typically rely on depleting rRNA by tiling large probe sets against rRNAs; however, such approaches are expensive, time-consuming, and challenging to scale to varied bacterial species and complex microbial communities. Therefore, we developed EMBR-seq+, a method that requires fewer than 10 short oligonucleotides per rRNA to achieve up to 99% rRNA depletion in diverse bacterial species. Finally, EMBR-seq+ resulted in a deeper view of the transcriptome, enabling systematic quantification of how microbial interactions result in altering the transcriptional state of bacteria within co-cultures.

A global analysis of gene expression in Fibrobacter succinogenes S85 grown on cellulose and soluble sugars at different growth rates

BackgroundCellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. Fibrobacter succinogenes strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of F. succinogenes S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium.ResultsSeveral patterns of gene expression were observed for the major cellulases produced by F. succinogenes S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose.ConclusionsThis work emphasizes the complexity of the fiber-degrading system utilized by F. succinogenes S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.

Optimization of succinic acid fermentation with Actinobacillus succinogenes by response surface methodology (RSM)

Succinic acid is considered as an important platform chemical. Succinic acid fermentation with Actinobacillus succinogenes strain BE-1 was optimized by central composite design (CCD) using a response surface methodology (RSM). The optimized production of succinic acid was predicted and the interactive effects between glucose, yeast extract, and magnesium carbonate were investigated. As a result, a model for predicting the concentration of succinic acid production was developed. The accuracy of the model was confirmed by the analysis of variance (ANOVA), and the validity was further proved by verification experiments showing that percentage errors between actual and predicted values varied from 3.02% to 6.38%. In addition, it was observed that the interactive effect between yeast extract and magnesium carbonate was statistically significant. In conclusion, RSM is an effective and useful method for optimizing the medium components and investigating the interactive effects, and can provide valuable information for succinic acid scale-up fermentation using A. succinogenes strain BE-1.

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes. Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes. DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii, B. fibrisolvens, Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes-like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.

Fibrolytic capabilities of ruminal bacteriumFibrobacter succinogenesin relation to its phylogenetic grouping

To characterize the fibrolytic function of Fibrobacter succinogenes strains in relation to their phylogenetic grouping, 32 strains were newly isolated from the rumen of sheep. All new strains were classified into phylogenetic groups 1 or 2 including a novel subgroup of group 2. Importantly, the majority of the strains belonging to group 1 were isolated from ruminally incubated hay. Although almost complete degradation of Avicel was observed among all strains, significantly lower digestibility of three different forages was recorded for strain HM2 of group 3 than for the strains of groups 1 and 2. In a comparison of all strains, two group 1 strains showed significantly higher digestibility of alfalfa and orchard grass hays, while two strains of the novel subgroup of group 2 had lower digestibility of orchard grass hay. Adhesion ability of each strain did not necessarily associate with the extent of digestibility. Maximum growth on Avicel was higher in group 1 than in group 2 strains, and two group 1 strains showed a shorter lag time. The results suggest that the ecological prominence of group 1 is due to a mixture of strains that are diverse in their fibrolytic capability making this group highly adaptable to any forage.

Criopreservación de aislados nativos de la bacteria ruminal Fibrobacter succinogenes

En el presente trabajo se estandarizó una técnica de criopreservación en nitrógeno líquido, usando como crioprotector dimetil sulfóxido al 5%, para la preservación de aislados nativos bacterianos de Fibrobacter succinogenes. El respectivo protocolo de criopreservación se evaluó determinando la viabilidad bacteriana por el transcurso de un año. La escala de MacFarland, ampliamente conocida en la estimación de poblaciones bacterianas aerobias, fue validada como técnica de estimación de bacterias anaerobias, usando la técnica de conteo bacteriano por roll-tube (R2 = 0.95). Se utilizaron dos aislados nativos, C7 y C9, de Fibrobacter succinogenes, obtenidos del rumen de bovinos en pastoreo de Brachiaria decumbens, en los Llanos Orientales de Colombia. Quince días después de la exposición de los cultivos al proceso de criopreservación, se apreció una reducción en el número de bacterias viables con relación a la población inicial de cada uno de los aislados (C7 = 9.25 x 108 UFCml-1 vs. 6.15 x 108 UFCml-1 y C9 = 12.51 x 108 UFCml-1 vs. 9.26 x 108 UFCml-1). Sin embargo, no se presentaron diferencias en la población de bacterias viables en los muestreos posteriores (C7 = 6.13 – 6.16 x 108; C9 = 9.26 – 9.35 x 108 UFCml-1). Esta técnica permite mantener la viabilidad bacteriana y puede considerarse como un procedimiento eficiente y de fácil aplicación para la preservación de bacterias ruminales. Además, constituye una herramienta fundamental para el establecimiento de bancos de germoplasma de microorganismos anaerobios ruminales en Colombia. El uso conjunto de las técnicas de criopreservación y la escala de MacFarland, posee ventajas sobre los métodos de preservación y estimación de poblaciones por cultivos activos, por su confiabilidad, eficiencia y bajo costo.

Fiber-degrading systems of different strains of the genus Fibrobacter.

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.

Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes

To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae– Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes– E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn 9 chloramphenicol and the Tn 10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.

Construction of a Fibrobacter succinogenes genomic map and demonstration of diversity at the genomic level.

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies.

Molecular cloning, expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and35S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley β-glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)-β-d-glucanase.

Digestion of cell-wall monosaccharides of ryegrass and alfalfa hays by the ruminal bacteria Fibrobacter succinogenes and Butyrivibrio fibrisolvens.

The ruminal bacteria Fibrobacter succinogenes strains S85 and BL2 were grown in monocultures or in coculture with strain D1 of Butyrivibrio fibrisolvens, and the solubilization of ryegrass and alfalfa cell walls (CW) and digestion of CW monosaccharides were measured. Fibrobacter succinogenes monocultures and cocultures with B. fibrisolvens D1 degraded 58-69% of ryegrass CW, solubilizing 67-78% of CW glucose, 65-71% of CW xylose, 69-75% of hemicellulose, and 68-77% of total CW monosaccharides. When grown on alfalfa CW, those cultures degraded 28-39% of alfalfa CW, solubilizing 42-58% of CW glucose, 30-36% of CW xylose, and 37-45% of hemicellulose. With respect to both substrates, F. succinogenes strains solubilized CW carbohydrates better than did B. fibrisolvens D1. Complementary interaction between B. fibrisolvens D1 and the F. succinogenes strains was identified with respect to the utilization of some solubilized carbohydrates, but not with respect to the extent of CW solubilization, which was determined mainly by the F. succinogenes strains. For both substrates, utilization of cellulose by F. succinogenes monocultures was high (96-98%), whereas that of hemicellulose was lower (24-26% in ryegrass and 49-50% in alfalfa). Under scanning electron microscopy, F. succinogenes bacterial cells attached to and colonized on CW particles were characterized by the appearance of protuberant surface structures that we have identified as "polycellulosome complexes."

Digestion of structural polysaccharides of Panicum and vetch hays by the rumen bacterial strains Fibrobacter succinogenes BL2 and Butyrivibrio fibrisolvens D1

The rumen bacterial strains Fibrobacter succinogenes BL2 and Butyrivibrio fibrisolvens D1, were grown in monocultures and pair combination on cell walls (CW) of two tropical hays: Panicum (grass) and vetch (legume), and their ability to solubilize and utilize CW structural carbohydrate was determined. With respect to both substrates, F. succinogenes BL2 was a better solubilizer of CW carbohydrate than B. fibrisolvens D1. However, the solubilization of Panicum constituents by any bacterial monoculture and co-culture was higher than that of vetch. Complementary interaction between B. fibrisolvens D1 and F. succinogenes BL2 was identified only with respect to carbohydrate utilization, but not with the extent of CW solubilization, which was determined mainly by the F. succinogenes strain. In both substrates, utilization of solubilized cellulose by BL2 monocultures was high (86.4–97.5%), whereas that of solubilized xylan and hemicellulose was much lower (35.2–41.6%). Under scanning electron microscopy visualization, the BL2 bacterial cell mass attached to and colonized on CW particles was characterized by the appearance of protuberant structures known as “polycellulosome complexes” on their surface topology.

The degradation and utilization of structural polysaccharides of sorghum straw by defined ruminal bacteria

Cell wall (CW) preparations of untreated sorghum straw (SORG) and sulfur dioxide-treated SORG (T-SORG) were used as substrates for the solubilization and utilization of CW carbohydrates by pure cultures or pair-combinations of defined rumen bacterial strains. Ruminococcus flavefaciens FD1 and R. albus 7 monocultures and cocultures with Butyrivibrio fibrisolvens D1 had very little solubilizing effect on SORG and even less on T-SORG CW. Fibrobacter succinogenes S85 was the best solubilizer of SORG CW components. The T-SORG CW was composed of fewer hemicellulose components and more cellulose and total CW polysaccharides than the SORG CW. Consequently, F. succinogenes S85 and BL2 monocultures and their cocultures with D1 degraded T-SORG CW polysaccharides by 21–33% units better than those of SORG CW, solubilizing 61–66% of T-SORG CW-glucose, 58–62% of CW-xylose, 58–63% of hemicellulose and 61–65% of total T-SORG structural polysaccharides. Complementary action between B. fibrisolvens D1 and the F. succinogenes strains was identified with respect to coculture growth and carbohydrate utilization, but not with respect to the extent of CW solubilization which was determined mainly by the F. succinogenes strains during 120 h of coculture incubation. In both CW substrates, utilization of solubilized cellulose by F. succinogenes S85 and BL2 monocultures was higher than that of CW-xylose and hemicellulose, being 97–98%, 53–69% and 54–75%, respectively. Under scanning electron microscopic visualization SORG CW particles were less colonized by attached bacterial cells than T-SORG. In both substrates attached F. succinogenes cells were characterized by the appearance of protuberant structures on their surface topology.

Untreated and delignified cotton stalks as model substrates for degradation and utilization of cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Pure cultures of the defined ruminal bacterial strains Fibrobacter succinogenes S85 and BL2, Ruminococcus flavefaciens FD1, Ruminococcus albus 7 and Butyrivibrio fibrisolvens D1, and the pair combinations of strains S85 + D1, BL2 + D1, FD1 + D1 and 7 + D1, were grown on isolated cell walls (CW) of untreated cotton stalks (CS) or ozone-treated CS (OCS) as the sole added carbohydrate substrate. F. succinogenes S85 was the best CW hydrolyzer of CS, solubilizing 27·9% of CW-glucose polymers, 21·9% of xylan, 24·8% of hemicellulose and 26·9% of total CS-CW polysaccharides. The Ruminococci monocultures and cocultures had very little hydrolytic effect on CS but somewhat better hydrolytic effect on OCS-CW. Since OCS-CW contained less lignin and hemicellulose components and more cellulose and total CW polysaccharides than CS-CW all bacterial cultures and cocultures degraded OCS-CW components better than CS-CW polysaccharides. S85 was the best hydrolyzer of OCS residual CW, solubilizing 52·8% of CW-glucose, 51·2% xylan, 51·5% hemicellulose and 52·6% of total OCS-CW monosaccharides. There was complementary action between B. fibrosolvens D1 and the F. succinogenes strains S85 and BL2 with respect to coculture growth and carbohydrate utilization, but not to the extent of CW hydrolysis, which was determined mainly by the F. succinogenes strains during the 120 h of coculture incubations. Utilization of solubilized cellulose, xylan, hemicellulose sugars and total carbohydrates by S85 monoculture was 94·4%, 80·3%, 61·4% and 84·1%, respectively, with respect to CS components, and 96·7%, 64·8%, 62·1% and 89·6% with respect to OCS-CW carbohydrates. Under scanning electron microscopy (SEM) visualization, S85 formed the most dense layer of bacterial cell mass attached to, and colonized on, OCS-CW stems. CW particles of CS were less crowded with attached bacteria. The cell-surface topology of S85 cells attached to CW particles was specified by a coat of characteristic protuberant structures.

The degradation and utilization of wheat-straw cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Cell walls (CW) of untreated wheat straw and sulphur-dioxide (SO2)-treated wheat straw were used as model substrates for the hydrolysis and utilization of CW carbohydrates by pure cultures or pair-combinations of defined rumen bacterial strains. Fibrobacter succinogenes S85 and BL2 strains and their co-cultures with D1 were the best degraders of CW among ruminal cultures, solubilizing 37.2–39.6% of CW carbohydrates of untreated straw and 62.2–74.5% of SO2-treated straw. Complementary action between Butyrivibrio fibrisolvens D1 and the F. succinogenes strains was identified with respect to co-culture growth and carbohydrate utilization. However, the extent of CW solubilization was determined mainly by the F. succinogenes strains. In both substrates, utilization of solubilized cellulose by F. succinogenes S85 and BL2 monocultures was higher than that of xylan and hemicellulose: 96.5–98.3%, 34.4–40.5% and 33.5–36.2%, respectively. Under scanning electron microscopy visualization, S85 and BL2 cells of the co-cultures comprised the most dense layer of bacterial cell mass attached to and colonized on straw stems and leaves, whereas D1 cells were always nearby. Stems and leaves of the untreated straw were less crowded by attached bacteria than that of the SO2-treated straw. In both materials, the cell surface topography of S85 and BL2 bacteria attached to CW particles was specified by a coat of characteristic protuberant structures, “polycellulosome complexes”.

Revision of Campylobacter, Helicobacter, and Wolinella Taxonomy: Emendation of Generic Descriptions and Proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and "Campylobacter upsaliensis." Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus "Flexispira," with "Flexispira rappini" as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.

Transfer of Bacteroides succinogenes (Hungate) to Fibrobacter gen. nov. as Fibrobacter succinogenes comb. nov. and Description of Fibrobacter intestinalis sp. nov.

A comparison of 16S ribosomal ribonucleic acid sequences showed that strains classified as Bacteroides succinogenes are not closely related to other species of Bacteroides, including the type species, Bacteroides fragilis. Flavobacterium heparinum, for example, is more closely related to B. fragilis than B. succinogenes is. Therefore, we propose that B. succinogenes strains be renamed as members of a new genus, Fibrobacter. An analysis of 16S ribosomal ribonucleic acid sequence data revealed that there are two species within this new genus. Isolates from rumina are placed in Fibrobacter succinogenes; the type strain is strain S85 (= ATCC 19169). Isolates from ceca of nonruminant animals are placed in Fibrobacter intestinalis; the type strain is strain NR9 (= ATCC 43854). Members of Fibrobacter succinogenes can be differentiated from Fibrobacter intestinalis by their requirement for biotin; the site of isolation may not be diagnostic. Fibrobacter succinogenes consists of two subspecies; the cells of Fibrobacter succinogenes subsp. succinogenes strains are broad rods that are often pleomorphic and coccoid (type strain, S85), whereas cells of Fibrobacter succinogenes subsp. elongata are slender rods (type strain, HM2 [= ATCC 43856]).

Enumeration ofBacteroides succinogenesin the rumen of sheep fed maize-straw diets

Estimates of Bacteroides succinogenes in the rumen of two sheep fed a maize-straw diet exceeded 108 per g of rumen ingesta and were even higher when the straw was supplemented with pelleted maize grain. A number of B. succinogenes strains were characterized in some detail and very few were found to ferment starch. Cells of freshly isolated strains lost viability more rapidly when grown on agar medium than did cells of strains which had been subjected to more transfers.

Enumeration of Bacteroides succinogenes in the rumen of sheep fed maize-straw diets

Estimates of Bacteroides succinogenes in the rumen of two sheep fed a maize-straw diet exceeded 10 8 per g of rumen ingesta and were even higher when the straw was supplemented with pelleted maize grain. A number of B. succinogenes strains were characterized in some detail and very few were found to ferment starch. Cells of freshly isolated strains lost viability more rapidly when grown on agar medium than did cells of strains which had been subjected to more transfers.

Alpha-ketoglutarate metabolism by cytochrome-containing anaerobes.

During growth in the presence of tracer amounts of exogenously supplied alpha-keto[1-14C]glutarate (AKG) or alpha-keto [5-14C]glutarate, cytochrome-containing Bacteroides fragilis strain 2044 and Bacteroides vulgatus strain 8482 incorporated extremely small amounts of radioactivity into cell macromolecules and protoheme. Under identical conditions, Bacteroides "l" strain 7CM and Bacteroides buccae strain J1 incorporated substantial label from [5-14C]AKG, but not [1-14C]AKG, into cellular macromolecules and protoheme. Bacteroides succinogenes strain S85 incorporated radioactivity from both [1-14C]AKG and [5-14C]AKG into cell macromolecules, but only label from [5-14C]AKG appeared in protoheme. Selenomonas ruminantium strain HD1 and Butyrivibrio fibrisolvens strain D1, both of which are devoid of cytochromes, incorporated substantial label from both [1-14C]AKG and [5-14C]AKG into cell macromolecules, but failed to incorporate label from either position into protoheme. Bacteroides ruminicola sp. brevis strain GA33 incorporated label from both [1-14C]AKG and [5-14C]AKG into both cell macromolecules and protoheme. A substantial portion of the heme synthesized by this organism may be formed by the "plant" pathway involving the intact use of the AKG carbon skeleton. Major differences exist in the manner and extent of AKG utilization among cytochrome-containing anaerobes and between these organisms and bacteria devoid of cytochromes obtained from similar environments.