Recovery and Analysis of Formyltetrahydrofolate Synthetase Gene Sequences from Natural Populations of Acetogenic Bacteria

Primers for PCR amplification of partial (1,102 of 1,680 bp) formyltetrahydrofolate synthetase (FTHFS) gene sequences were developed and tested. Partial FTHFS sequences were successfully amplified from DNA from pure cultures of known acetogens, from other FTHFS-producing organisms, from the roots of the smooth cordgrass, Spartina alterniflora, and from fresh horse manure. The amplimers recovered were cloned, their nucleotide sequences were determined, and their translated amino acid sequences were used to construct phylogenetic trees. We found that FTHFS sequences from homoacetogens formed a monophyletic cluster that did not contain sequences from nonhomoacetogens and that FTHFS sequences appear to be informative regarding major physiological features of FTHFS-producing organisms.     

Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups

Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.     

Diversity of the Formyltetrahydrofolate Synthetase Gene (fhs), a Key Enzyme for Reductive Acetogenesis,in the Bovine Rumen

A clone library of the partial formyltetrahydrofolate synthetase gene (fhs), a key enzyme in reductive acetogenesis, was constructed from the DNA of bovine rumen contents. Diverse sequences were recovered, the majority of which were clustered with the fhs of authentic acetogens. Low similarity values to known fhs were observed in all sequences, suggesting the presence of unknown acetogens.     

Niche in Pasture-Fed Ruminants for the Large Rumen Bacteria Oscillospira, Lampropedia, and Quin's and Eadie's Ovals

The little-studied large bacteria Oscillospira, Lampropedia, and ovals attach rapidly in large numbers to the cuticular surface of clover and grass leaves in the rumen. The cuticle of green leaves may constitute a specific niche for these bacteria.     

Niche in Pasture-Fed Ruminants for the Large Rumen Bacteria Oscillospira, Lampropedia, and Quin's and Eadie's Ovals

The little-studied large bacteria Oscillospira, Lampropedia, and ovals attach rapidly in large numbers to the cuticular surface of clover and grass leaves in the rumen. The cuticle of green leaves may constitute a specific niche for these bacteria.     

Melatonin-like Immunoreactivity in the Presence of Different Chemicals as Determined by the Radioimmunoassay

To determine the nature of the molecule(s) that is responsible for the melatonin like immunoreactivity (MLI), we measured the effect of pretreatment of plasma samples with detergents, reducing agent, and proteinase K. Nonidet P-40 Triton X-100, and ethylacetate extraction had no effect, while sodium deoxycholate, sodium dodecyl sulfate, β-mercaptoethanol, ether extraction, proteinase K, and temperature increased the MLI. Since the radioimmunoassay (RIA) was sensitive to proteinase K, ionic detergents, and a reducing compound, we hypothesize that a proteinaceous molecule might be responsible for this MLI. We compared our column procedure for RIA of plasma melatonin (1) with procedures involving extraction with either ethylacetate or ether. In our hands preextraction of samples with organic solvents caused a loss of immunoreactivity. We also found that passing samples through the column is more efficient in eliminating interference in the melatonin assay than extracting samples either with ethylacetate or ether.     

Degradation of Dehydrodivanillin by Anaerobic Bacteria from Cow Rumen Fluid

Dehydrodivanillin (DDV; 0.15 g/liter) was biodegradable at 37°C under strictly anaerobic conditions by microflora from cow rumen fluid to the extent of 25% within 2 days in a yeast extract medium. The anaerobes were acclimated on DDV for 2 weeks, leading to DDV-degrading microflora with rates of degradation eight times higher than those initially. Dehydrodivanillic acid and vanillic acid were detected in an ethylacetate extract of a DDV-enriched culture broth by thin-layer, gas, and high-performance liquid chromatographies and by mass spectrometry.     

Molecular Systematics of the Subfamily Caprinae (Artiodactyla, Bovidae) as Determined from Cytochrome b Sequences

We have sequenced the complete mitochondrial DNA cytochrome b gene from 18 species of the subfamily Caprinae and two outgroup taxa. Additional sequences retrieved from the literature were used to constitute a data set of 32 cytochrome b sequences comprising all genera usually included within the Caprinae. Phylogenetic relationships were assessed by PAUP using three new weighting schemes based on homoplasy analyses. Each type of substitution considered at each of the three codon positions was weighted according to its homoplasy level, as measured by the consistency index (CI), the slope of saturation (S), or their product (CIS). These differentially weighted parsimony analyses indicate that (1) the subfamily Caprinae is monophyletic, but only with the exclusion of Saiga from the group; (2) there is no support for monophyly of the four tribes currently recognized (Caprini, Rupicaprini, Ovibovini, and Saigini), suggesting relationships different from those traditionally accepted; (3) the caprine group consists of three major clades corresponding to (a) Budorcas and Ovis, (b) Capricornis, Ovibos, and Naemorhedus, and (c) Capra, Hemitragus, and Pseudois; and (4) the basal branching pattern is very weakly supported by bootstrap or branch support values except for the sister-group relationship of Pantholops with all other caprines, and the phylogenetic positions of Ammotragus, Oreamnos, and Rupicapra remain unclear.     

Substrate Uptake by Uncultured Bacteria from the Genus Achromatium Determined by Microautoradiography

Microautoradiography was used to investigate substrate uptake by natural communities of uncultured bacteria from the genus Achromatium. Studies of the uptake of ¹⁴C-labelled substrates demonstrated that Achromatium cells from freshwater sediments were able to assimilate ¹⁴C from bicarbonate, acetate, and protein hydrolysate; however, ¹⁴C-labelled glucose was not assimilated. The pattern of substrate uptake by Achromatium spp. was therefore similar to those of a number of other freshwater and marine sulfur-oxidizing bacteria. Different patterns of radiolabelled bicarbonate uptake were noted for Achromatium communities from different geographical locations and indicated that one community (Rydal Water) possessed autotrophic potential, while the other (Hell Kettles) did not. Furthermore, the patterns of organic substrate uptake within a single population suggested that physiological diversity existed in natural communities of Achromatium. These observations are consistent with and may relate to the phylogenetic diversity observed in Achromatium communities. Incubation of Achromatium-bearing sediment cores from Rydal Water with ³⁵S-labelled sulfate in the presence and absence of sodium molybdate demonstrated that this bacterial population was capable of oxidizing sulfide to intracellular elemental sulfur. This finding supported the role of Achromatium in the oxidative component of a tightly coupled sulfur cycle in Rydal Water sediment. The oxidation of sulfide to sulfur and ultimately to sulfate by Achromatium cells from Rydal Water sediment is consistent with an ability to conserve energy from sulfide oxidation.     

Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen

Microbes in ruminal contents incorporated (14)C into cells when they were incubated in vitro in the presence of [(14)C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular (14)C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate (14)C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.     

Diversity of the Formyltetrahydrofolate Synthetase (FTHFS) Gene in the Proximal and Mid Ostrich Colon

We analysed fragments of the formyltetrahydrofolate synthetase (FTHFS) gene, which encodes a key enzyme in reductive acetogenesis, from the bacterial flora in the proximal (PC) and mid (MC) colon of three ostriches to assess and compare bacterial diversity in this organ. Two clone libraries of FTHFS fragments were constructed from DNA extracted from digesta of the PC and MC, and a total of 46 cloned sequences were analysed from each library. A wide variety of FTHFS sequences were recovered. The coverage of the PC and MC libraries was 90.0% and 83.3%, respectively. Shannon–Wiener index (H’) and Chao1 of the MC library were higher than those of PC library. The sequences from each library were classified into 15 operational taxonomic units (OTUs) and clusters. Only four OTUs in cluster I were distantly related to known acetogens from human feces and rumen, suggesting the presence of the novel acetogens. Phylogenetic analysis suggests that composition of FTHFS sequences differs for the PC and MC.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

Characterization of Variation in Rumen Methanogenic Communities under Different Dietary and Host Feed Efficiency Conditions,as Determined by PCR-Denaturing Gradient Gel Electrophoresis Analysis

Understanding ruminal methanogens is essential for greenhouse gas mitigation, as well as for improving animal performance in the livestock industry. It has been speculated that ruminal methanogenic diversity affects host feed efficiency and results in differences in methane production. This study examined methanogenic profiles in the rumen using culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for 56 beef cattle which differed in feed efficiency, as well as diet (the cattle were fed a low-energy diet or a high-energy diet). The methanogenic PCR-DGGE profiles detected were greatly affected by diet, and the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 with the low-energy diet to a community containing predominantly Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 with the high-energy diet. For each diet, the methanogenic PCR-DGGE pattern was strongly associated with the feed efficiency of the host. Diet-associated bands for Methanobrevibacter sp. AbM4 and M. smithii SM9 and a feed efficiency-related band for M. smithii PS were identified. The abundance of total methanogens was estimated by determining the numbers of copies of the 16S rRNA genes of methanogens. However, the size of the methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. Thus, the structure of the methanogenic community at the species or strain level may be more important for determining host feed efficiency under different dietary conditions.Ruminal methanogens use methanogenesis pathways to maintain low hydrogen partial pressure and to facilitate fiber digestion in the rumen by converting hydrogen into methane gas (24, 37). However, although it is necessary, this process also has adverse effects because the released methane represents a significant loss of dietary energy for the host animal (14) and it constitutes a large proportion of the agricultural greenhouse gas emitted (4, 10). Many studies to obtain a better understanding of rumen methanogens have been conducted in order to improve the efficiency of ruminal function and to mitigate methane release. Assessments by both cultivation-dependent and cultivation-independent methods have found that members of the genus Methanobrevibacter account for the majority of the methanogens in the rumens of sheep and cattle (11, 18, 21-23, 28, 31, 33, 34). In addition, Methanosphaera stadtmanae, Methanobacterium species, and Methanosarcina barkeri have also been found in some studies (13, 32). Although the phylogenetic positions of the methanogens in the rumen are diverse, these organisms utilize only three major pathways for methanogenesis: the CO₂ reduction pathway, the C₁ compound (e.g., methanol and methylamine) conversion pathway, and the acetate fermentation pathway. Each methanogen species has a substrate preference, and most methanogens can use only one or two substrates (37).Previous studies of rumen methanogens focused primarily on determining the methanogen species composition in different samples and developing strategies to reduce the methane yield from ruminants. Recently, there has been a strong desire to understand the impact of methanogens on host biology. Two primary studies found that feedlot beef cattle with higher feed efficiency (designated “efficient” animals) produced about 20% less methane gas than animals with lower feed efficiency (designated “inefficient” animals) (8, 19). The methanogenic communities of efficient and inefficient animals fed a low-energy diet have been compared, and divergence between the two communities has been reported (36). However, it is not clear how the methanogens in the rumen of cattle change when the animals are fed a different diet.The aims of this study were to describe the methanogenic communities in 56 steers with different feed efficiencies that were fed two distinct diets (a low-energy diet and a high-energy diet) and to understand how methanogenic communities change in response to diet modification using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis. Multivariate analysis was used to analyze the association of PCR-DGGE bands with the daily dry matter intake (DMI), average daily gain (ADG), feed conversion ratio (FCR), and residual feed intake (RFI). Methanogens that were associated with diet and with host feed efficiency were identified. In addition, the methanogen population of each rumen sample was examined by quantitative real-time PCR (qRT-PCR), and the results for different RFI groups and both diets were compared.     

Occurrence of Potentially Pathogenic Bacteria in Epilithic Biofilm Forming Bacteria isolated from Porter Brook River-stones,Sheffield, UK

Biofilms in aquatic ecosystems develop on wet benthic surfaces and are primarily comprised of various allochthonous microorganisms, including bacteria embedded within a self-produced matrix of extracellular polymeric substances (EPS). In such environment, where there is a continuous flow of water, attachment of microbes to surfaces prevents cells being washed out of a suitable habitat with the added benefits of the water flow and the surface itself providing nutrients for growth of attached cells. When watercourses are contaminated with pathogenic bacteria, these can become incorporated into biofilms. This study aimed to isolate and identify the bacterial species within biofilms retrieved from river-stones found in the Porter Brook, Sheffield based on morphological, biochemical characteristics and molecular characteristics, such as 16S rDNA sequence phylogeny analysis. Twenty-two bacterial species were identified. Among these were 10 gram-negative pathogenic bacteria, establishing that potential human pathogens were present within the biofilms. Klebsiella pneumoniae MBB9 isolate showed the greatest ability to form a biofilm using a microtiter plate-based crystal violet assay. Biofilm by K. pneumoniae MBB9 formed rapidly (within 6 h) under static conditions at 37 °C and then increased up to 24 h of incubation before decreasing with further incubation (48 h), whereas the applied shear forces (horizontal orbital shaker; diameter of 25 mm at 150 rpm) had no effect on K. pneumoniae MBB9 biofilm formation.     

Bacterial Content in Samples from Different Sites in the Rumen of Sheep and Cows as Determined in Two Culture Media

Samples of material from the rumen of cattle and of sheep were cultured in both Gall Broth (GB) and Reinforced Clostridium Medium (RCM). Samples were taken, at monthly intervals, from five sites in cattle and two sites in sheep in two series and, at weekly intervals, from two sites in cattle and in sheep in two series. Comparisons were made of the growth of organisms in the media, using the number of fertile tubes per set of six dilutions as a measure of the viable count. RCM gave a greater number of fertile tubes than GB with samples from cattle, but the reverse was the case with samples from sheep. Real differences in the number of fertile tubes from different sites in cattle were found at different times, but there was no consistent pattern. Simultaneous sampling from different sites in the rumen is, therefore, essential to provide an estimate of sampling error within animals. Although no site to site differences were detectable with rumen material from sheep, it is advisable to take more than one sample at a given time to assess sampling error.     

Identification of a Novel Toluene-Degrading Bacterium from the Candidate Phylum TM7, as Determined by DNA Stable Isotope Probing

The dominant bacterium responsible for carbon uptake from toluene in an agricultural soil was identified by stable isotope probing. Samples were amended with unlabeled toluene or labeled [ring-¹³C₆]toluene, and DNA was extracted over time. Sequencing indicated that the organism involved belongs to the candidate phylum TM7. Microorganisms in this candidate phylum are of particular interest because although they have been found in a variety of habitats, no stable culture of any species exists, so their general metabolic capabilities are largely unknown.The application of PCR technology has uncovered the impressive diversity of the microbial world. It has been estimated that less than half of the recognized bacterial phyla include cultured representatives (14). Here, stable isotope probing (SIP) (a method that links function to identity in mixed microbial samples) was used to identify dominant toluene degraders in an environment previously unexposed to the contaminant but likely containing a diverse microbial community of previously undiscovered toluene degraders.Soil samples were collected from a field in Michigan previously under corn production. This field received biosolids from a wastewater treatment plant 2 to 3 years before sample collection. Following collection, soils were homogenized, sieved (4-mm screen), and stored at 4°C until use (<1 year). The microcosms consisted of phosphate-buffered mineral medium (20 ml) (10) and soil (6 g [wet weight]) in serum bottles (150 ml). The bottles were sealed with rubber stoppers and an aluminum seal. The treatment groups included no-toluene controls, autoclaved controls, and samples amended with unlabeled (1 μl, 99%; Chem Service) or labeled (1 μl, ring-¹³C₆, 99%; Cambridge Isotope Laboratories) toluene. Eight samples for each treatment (two for each time point) were incubated at room temperature (∼20°C) with reciprocal shaking. The concentrations of toluene in headspace samples (200 μl) after toluene addition (∼2 h) and at each time point were determined with a gas chromatograph (Perkin Elmer) equipped with a flame ionization detector and a capillary column (DB-624 [diameter, 0.53 mm]; J&W Scientific). The injector and detector temperatures were set at 200°C, and the column temperature was 80°C.At four time points (3, 5, 6, and 8 days after toluene addition), two samples from the labeled and unlabeled treatment groups were sacrificed for soil DNA extraction using a Powersoil DNA extraction kit (MO BIO Laboratories, Inc., Carlsbad, CA). At each time point, DNAs from two microcosms were pooled, and SIP involved terminal restriction fragment length polymorphism (TRFLP) analysis of all fractions (labeled and unlabeled). Approximately 10 μg DNA (quantified with an ND-1000 spectrometer; Nanodrop) was added to Quick-Seal polyallomer tubes (13 by 51 mm, 5.1 ml; Beckman Coulter), along with a Tris-EDTA (pH 8.0)-CsCl solution. Before the tubes were sealed (Quick-Seal tube topper; Beckman Coulter), buoyant density (BD) was determined (∼1.77 g ml⁻¹) with a model AR200 digital refractometer (Leica Microsystems, Inc.) and adjusted by adding CsCl solution or Tris-EDTA buffer. The tubes were centrifuged at 178,000 × g (20°C) for 48 h in a Stepsaver 70 V6 vertical titanium rotor (eight tubes, 5.1-ml capacity each) within a Sorvall WX 80 Ultra Series centrifuge (Thermo Scientific). Following centrifugation, a fraction recovery system (Beckman Coulter) was used for fraction (150 μl) collection. The BD of each fraction was measured, and CsCl was removed by glycogen-assisted ethanol precipitation.The fractions were PCR amplified using 27F-FAM (5′-AGAGTTTGATCMTGGCTCAG [5′ end labeled with carboxyfluorescein]) and 1492R (5′-GGTTACCTTGTTACGACTT) (Operon Biotechnologies) as previously described (4). Briefly, this involved the following conditions: 94°C (5 min); 30 cycles at 94°C (30 s), 55°C (30 s), and 72°C (1.5 min); and 72°C (5 min). The presence of PCR products was confirmed by gel electrophoresis. PCR products were purified with a QIAquick PCR purification kit (Qiagen, Inc.) (∼150 ng) and digested with HaeIII (New England Biolabs). In addition, three other enzymes (MspI, MseI, and HincII; New England Biolabs) were used for digestion of a number of heavy fractions. DNA fragments were separated by capillary electrophoresis (ABI Prism 3100 genetic analyzer; Applied Biosystems) at the Research Technology Support Facility at Michigan State University. Data were analyzed with GeneScan software (Applied Biosystems), and the percent abundance of each fragment was determined (18). Heavy-fraction ¹³C-labeled DNAs (day 8 fractions with BD values of 1.744 g ml⁻¹) were amplified, as described above, with unlabeled primers and cloned into Escherichia coli TOP10 by using a TOPO TA cloning kit (Invitrogen Corporation). E. coli clones were grown on Luria-Bertani medium solidified with 15 g agar liter⁻¹ in the presence of 50 μg ampicillin liter⁻¹ for 16 h at 37°C. Colonies with inserts were verified by PCR with primers M13 forward (5′-TGTAAAACGACGGCCAGT-3′) and M13 reverse (5′-AACAGCTATGACCATG-3′), plasmids were extracted from the positive clones with a QIAprep miniprep system (Qiagen, Inc.), and the insertions were sequenced (using primers M13 forward and M13 reverse) at the Research Technology Support Facility at Michigan State University. The partial 16S rRNA gene sequences obtained were aligned and edited with Chromas Pro (Technelysium, Pty. Ltd.). The Ribosomal Database Project (Center for Microbial Ecology, Michigan State University) analysis tool “classifier” (16) was utilized to assign taxonomic identity. In addition, the Ribosomal Database Project classifier checked the deposited sequence for chimeras.Toluene removal occurred rapidly, starting after 3 days and reaching completion after 8 days (Table ​(Table1).1). The low percentage of recoveries was likely caused by toluene sorption to the soil; however, the difference between the controls and samples clearly illustrates a biological removal mechanism. DNAs extracted over time (on days 3, 5, 6, and 8) from both labeled and unlabeled samples were subjected to ultracentrifugation, fractionation, and TRFLP on every fraction. TRFLP analyses indicated that one fragment (394 bp) was highly enriched in the heavy fractions (>1.732 g ml⁻¹) obtained from [¹³C]toluene microcosms but not in fractions with similar BD values obtained from the unlabeled controls, and further, the level of enrichment increased with time (Fig. ​(Fig.1).1). At day 3, the fragment was present in only one fraction TRFLP profile, indicating a low natural relative abundance of this organism. The peak TRFLP relative abundance values for the 394-bp fragment were 11.3%, 41.5%, and 62.1% on days 5, 6, and 8, respectively (Fig. ​(Fig.2).2). Consistent with this trend, the BD of this fragment also increased with time, with the peak relative abundances being found at BD values of 1.732 g ml⁻¹, 1.740 g ml⁻¹, and 1.744 g ml⁻¹ on days 5, 6, and 8, respectively (Fig. ​(Fig.2).2). These trends indicate that the organism represented by this fragment is directly involved in toluene transformation. A number of other TRFLP peaks were found in the heavy ¹³C fractions; however, because they were also present in the heavy ¹²C fractions, they were excluded from further analysis.Open in a separate windowFIG. 1.Dominance of the 394-bp fragment over time in TRFLP profiles from heavy fractions of soil samples amended with unlabeled (¹²C) or labeled (¹³C) toluene. The pattern is representative of other heavy-fraction samples at these time points. The BD values are given in the lower-right corners of the profiles.Open in a separate windowFIG. 2.Relative abundances of the dominant 394-bp fragment over a range of BD values from DNA extracted after 5 days (a), 6 days (b), or 8 days (c) from soil amended with either labeled (¹³C) or unlabeled (¹²C) toluene.

TABLE 1.

Ranges for percentages of toluene remaining in soil-liquid slurries over time