Purification and properties of ferredoxin and rubredoxin from Butyribacterium methylotrophicum.

A ferredoxin and a rubredoxin from Butyribacterium methylotrophicum, which displays a carbonyl-dependent acetyl-coenzyme A synthesis, were purified to electrophoretic homogeneity. The two electron carriers showed absorption spectra similar to those in Clostridium species. The ferredoxin displayed absorption peaks at 280 and 391 nm, while rubredoxin displayed absorption peaks at 279, 382, and 482 nm. Minimum molecular weights calculated from the respective amino acid compositions were 5,727 for ferredoxin and 5,488 for rubredoxin, excluding iron and inorganic sulfur atoms. Both electron carriers were isolated as monomers, according to gel-filtration data. Electron spin resonance analysis revealed that the ferredoxin was a 2[4Fe-4S]-type and that both clusters had a midpoint redox potential value of -410 mV, whereas rubredoxin contained one acid-stable iron and had a redox value of -40 mV. The coupling of these electron carriers to hydrogenase and carbon monoxide dehydrogenase activities was investigated. Rubredoxin showed higher activity towards carbon monoxide dehydrogenase, whereas ferredoxin showed higher activity towards hydrogenase.     

Biochemical route and control of butyrate synthesis in Butyribacterium methylotrophicum

 Butyribacterium methylotrophicum produced more butyrate when grown on lactate than when grown on glucose, and only acetate was detected during growth on pyruvate. Higher levels of NADH were found in butyrate-producing than in acetate-producing cells. The addition of neutral red, an electron-flow modulator, to cells growing on pyruvate altered the carbon and electron flow from acetate plus H₂ synthesis to butyrate synthesis. Enzymatic analysis suggested that pyruvate was produced from glucose via an Embden-Meyerhof-Parnas pathway. Pyruvate was further metabolized to butyryl-CoA via, β-hydroxybutyryl-CoA and butyryl-CoA dehydrogenases. Lactate dehydrogenase, unlike butyryl-CoA dehydrogenase, was inducible and detected only in lactate-grown cells. Both of these dehydrogenases utilized 2,6-dichloroindophenol and other artificial electron acceptors but not NAD(P). Ferredoxin–NAD oxidoreductase levels were highest in lactate and lowest in pyruvate-grown cells. Cells contained both a ferredoxin–neutral-red reductase activity and a neutral-red–NAD reductase activity that coupled electron flow to butyrate synthesis. These results showed that butyrate synthesis by B. methylotrophicum was regulated by the carbon source and was dependent on the cellular NADH/NAD ratios, and the levels and direction of ferredoxin- and NAD-linked oxidoreductases. Received: 3 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

 Exogenous H₂/CO₂ and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO₂ reduction to acetate was coupled to H₂ consumption. The addition of either H₂ or CO₂ to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H₂-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N₂ was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO₂ was the feed gas as compared to H₂ or N₂. The increase in H₂-consuming activity and decrease in H₂-producing activity was correlated with an increase in butyrate synthesis. H₂-consuming and ferredoxin (Fd)–NAD reductase activities increased while H₂-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H₂ did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H₂ production. Received: 22 August 1995/Received revision: 18 December 1995/Accepted: 22 January 1996     

Selection of sulphur sources for the growth of Butyribacterium methylotrophicum and Acetobacterium woodii

Sulphide and cysteine inhibited growth of batch cultures of Butyribacterium methylotrophicum at moderate concentrations (above 0.5 mM) during growth on glucose (10 mM). The ability of several sulphur sources to replace sulphide was tested in cultures of B. methylotrophicum or Acetobacterium woodii. With sulphite (1 mM), thiosulphate (0.5 mM), elemental sulphur, and dithionite (1 mM), but not sulphate (1 mM), cultures of both organisms grew and produced some sulphide. With elemental sulphur as the sulphur source, toxic levels of sulphide accumulated. Optimal levels for the cultivation of B. methylotrophicum with sulphite were 0.5–2.0 mM, but at higher concentrations the growth rate decreased rapidly, while with dithionite up to 4.0 mM the growth rate was relatively unaffected. In chemostat cultures of B. methylotrophicum with dithionite (1 mM) as the sulphur source and glucose as the limiting substrate, dilution rates up to 0.40 h^–1 were obtained. Thiosulphate could only be used in batch cultures in combination with the reductant titanium(III)nitriloacetate, but in continuous cultures the addition of the reductant to the reservoir was not necessary, because once growth had started enough sulphide was produced to keep the fermentor reduced. The maximum growth rate of B. methylotrophicum with thiosulphate in batch and continuous culture was 0.26 h^–1. Both thiosulphate and dithionite are more convenient sulphur sources than sulphide, but dithionite is more versatile because of its reductive properties and the faster growth it allows.Offprint requests to: T. A. Hansen     

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.     

Metabolism of H2-CO2, methanol, and glucose by Butyribacterium methylotrophicum.

The fermentative metabolism of Butyribacterium methylotrophicum grown on either H2-CO2, methanol, glucose, or CO is described. The following reaction stoichiometries were obtained: 1.00 H2 + 0.52 CO2 leads to 0.22 acetate + 0.06 cell C; 1 methanol + 0.18 CO2 + 0.01 acetate leads to 0.24 butyrate + 0.29 cell C; and 1.00 glucose leads to 0.31 CO2 + 1.59 acetate + 0.21 butyrate + 0.13 H2 + 1.58 cell C. Cell yields of 1.7 g (dry weight) per mol of H2, 8.2 g (dry weight) per mol of methanol, 42.7 g (dry weight) per mol of glucose, and 3.0 g (dry weight) per mol of CO were obtained from linear plots of cell synthesis and substrate consumption. Doubling times of 9.0, 9.0, and 3 to 4 h were observed during batch growth on H2-CO2, methanol, and glucose, respectively. Indicative of a growth factor limitation, glucose fermentation in defined medium displayed a lower cell synthesis efficiency than when yeast extract (0.05%) was present. B. methylotrophicum fermentation displayed atypically high substrate/cell carbon synthesis conversion ratios for an anaerobe, as greater than 24% of the carbon was assimilated into cells during growth on methanol or glucose. The data indicate that B. methylotrophicum conserves carbon-bound electrons during growth on single-carbon or multicarbon substrates.     

Stability of characters and construction of phylogenetic trees.

Parsimony methods infer phylogenetic trees by minimizing number of character changes required to explain observed character states. From the perspective of applicability of parsimony methods, it is important to assess whether the characters used to infer phylogeny are likely to provide a correct tree. We introduce a graph theoretical characterization that helps to assess whether given set of characters is appropriate to use with parsimony methods. Given a set of characters and a set of taxa, we construct a network called character overlap graph. We show that the character overlap graph for characters that are appropriate to use in parsimony methods is characterized by significant under-representation of subnetworks known as holes, and provide a validation for this observation. This characterization explains success in constructing evolutionary trees using parsimony method for some characters (e.g., protein domains) and lack of such success for other characters (e.g., introns). In the latter case, the understanding of obstacles to applying parsimony methods in a direct way has lead us to a new approach for detecting inconsistent and/or noisy data. Namely, we introduce the concept of stable characters which is similar but less restrictive than the well known concept of pairwise compatible characters. Application of this approach to introns produces the evolutionary tree consistent with the Coelomata hypothesis.     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

Student construction of phylogenetic trees in an introductory biology course

Background

Phylogenetic trees have become increasingly essential across biology disciplines. Consequently, learning about phylogenetic trees has become an important component of biology education and an area of interest for biology education research. Construction tasks, in which students generate phylogenetic trees from some type of data, are often used for instruction. However, the impact of these exercises on student learning is uncertain, in part due to our fragmented knowledge of what students construct during the tasks. The goal of this project was to develop a more robust method for describing student-generated phylogenetic trees, which will support future investigations that attempt to link construction tasks with student learning.

Results

Through iterative examination of data from an introductory biology course, we developed a method for describing student-generated phylogenetic trees in terms of style, conventionality, and accuracy. Students used the diagonal style more often than the bracket style for construction tasks. The majority of phylogenetic trees were constructed conventionally, and variable orientation of branches was the most common unconventional feature. In addition, the majority of phylogenetic trees were generated correctly (no errors) or adequately (minor errors only) in terms of accuracy. Suggesting extant taxa are descended from other extant taxa was the most common major error, while empty branches and extra nodes were very common minor errors.

Conclusions

The method we developed to describe student-constructed phylogenetic trees uncovered several trends that warrant further investigation. For example, while diagonal and bracket phylogenetic trees contain equivalent information, student preference for using the diagonal style could impact comprehension. In addition, despite a lack of explicit instruction, students generated phylogenetic trees that were largely conventional and accurate. Surprisingly, accuracy and conventionality were also dependent on each other. Our method for describing phylogenetic trees constructed by students is based on data from one introductory biology course at one institution, and the results are likely limited. We encourage researchers to use our method as a baseline for developing a more generalizable tool, which will support future investigations that attempt to link construction tasks with student learning.

     

BIMLR: A method for constructing rooted phylogenetic networks from rooted phylogenetic trees

Rooted phylogenetic trees constructed from different datasets (e.g. from different genes) are often conflicting with one another, i.e. they cannot be integrated into a single phylogenetic tree. Phylogenetic networks have become an important tool in molecular evolution, and rooted phylogenetic networks are able to represent conflicting rooted phylogenetic trees. Hence, the development of appropriate methods to compute rooted phylogenetic networks from rooted phylogenetic trees has attracted considerable research interest of late. The C_ASS algorithm proposed by van Iersel et al. is able to construct much simpler networks than other available methods, but it is extremely slow, and the networks it constructs are dependent on the order of the input data. Here, we introduce an improved C_ASS algorithm, BIMLR. We show that BIMLR is faster than C_ASS and less dependent on the input data order. Moreover, BIMLR is able to construct much simpler networks than almost all other methods. BIMLR is available at http://nclab.hit.edu.cn/wangjuan/BIMLR/.     

VOSTORG: a package of microcomputer programs for sequence analysis and construction of phylogenetic trees

VOSTORG is a new, versatile package of programs for the inference and presentation of phylogenetic trees, as well as an efficient tool for nucleotide (nt) and amino acid (aa) sequence analysis (sequence input, verification, alignment, construction of consensus, etc.). On appropriately equipped systems, these data can be displayed on a video monitor or printed as required. They are implemented on IBM PC/XT/AT/PS-2 or compatible computers and hardware graphic support is recommended. The package is designed to be easily handled by occasional computer users and yet it is powerful enough for experienced professionals.     

Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees

Background  

In several studies, secondary structures of ribosomal genes have been used to improve the quality of phylogenetic reconstructions. An extensive evaluation of the benefits of secondary structure, however, is lacking.     

Accuracy of estimated phylogenetic trees from molecular data

The accuracies and efficiencies of three different methods of making phylogenetic trees from gene frequency data were examined by using computer simulation. The methods examined are UPGMA, Farris' (1972) method, and Tateno et al.'s (1982) modified Farris method. In the computer simulation eight species (or populations) were assumed to evolve according to a given model tree, and the evolutionary changes of allele frequencies were followed by using the infinite-allele model. At the end of the simulated evolution five genetic distance measures (Nei's standard and minimum distances, Rogers' distance, Cavalli-Sforza's f theta, and the modified Cavalli-Sforza distance) were computed for all pairs of species, and the distance matrix obtained for each distance measure was used for reconstructing a phylogenetic tree. The phylogenetic tree obtained was then compared with the model tree. The results obtained indicate that in all tree-making methods examined the accuracies of both the topology and branch lengths of a reconstructed tree (rooted tree) are very low when the number of loci used is less than 20 but gradually increase with increasing number of loci. When the expected number of gene substitutions (M) for the shortest branch is 0.1 or more per locus and 30 or more loci are used, the topological error as measured by the distortion index (dT) is not great, but the probability of obtaining the correct topology (P) is less than 0.5 even with 60 loci. When M is as small as 0.004, P is substantially lower. In obtaining a good topology (small dT and high P) UPGMA and the modified Farris method generally show a better performance than the Farris method. The poor performance of the Farris method is observed even when Rogers' distance which obeys the triangle inequality is used. The main reason for this seems to be that the Farris method often gives overestimates of branch lengths. For estimating the expected branch lengths of the true tree UPGMA shows the best performance. For this purpose Nei's standard distance gives a better result than the others because of its linear relationship with the number of gene substitutions. Rogers' or Cavalli-Sforza's distance gives a phylogenetic tree in which the parts near the root are condensed and the other parts are elongated. It is recommended that more than 30 loci, including both polymorphic and monomorphic loci, be used for making phylogenetic trees. The conclusions from this study seem to apply also to data on nucleotide differences obtained by the restriction enzyme techniques.     

Accuracy of estimated phylogenetic trees from molecular data

Summary The accuracies and efficiencies of four different methods for constructing phylogenetic trees from molecular data were examined by using computer simulation. The methods examined are UPGMA, Fitch and Margoliash's (1967) (F/M) method, Farris' (1972) method, and the modified Farris method (Tateno, Nei, and Tajima, this paper). In the computer simulation, eight OTUs (32 OTUs in one case) were assumed to evolve according to a given model tree, and the evolutionary change of a sequence of 300 nucleotides was followed. The nucleotide substitution in this sequence was assumed to occur following the Poisson distribution, negative binomial distribution or a model of temporally varying rate. Estimates of nucleotide substitutions (genetic distances) were then computed for all pairs of the nucleotide sequences that were generated at the end of the evolution considered, and from these estimates a phylogenetic tree was reconstructed and compared with the true model tree. The results of this comparison indicate that when the coefficient of variation of branch length is large the Farris and modified Farris methods tend to be better than UPGMA and the F/M method for obtaining a good topology. For estimating the number of nucleotide substitutions for each branch of the tree, however, the modified Farris method shows a better performance than the Farris method. When the coefficient of variation of branch length is small, however, UPGMA shows the best performance among the four methods examined. Nevertheless, any tree-making method is likely to make errors in obtaining the correct topology with a high probability, unless all branch lengths of the true tree are sufficiently long. It is also shown that the agreement between patristic and observed genetic distances is not a good indicator of the goodness of the tree obtained.     

Predicting rates of interspecific interaction from phylogenetic trees

Integrating phylogenetic information can potentially improve our ability to explain species' traits, patterns of community assembly, the network structure of communities, and ecosystem function. In this study, we use mathematical models to explore the ecological and evolutionary factors that modulate the explanatory power of phylogenetic information for communities of species that interact within a single trophic level. We find that phylogenetic relationships among species can influence trait evolution and rates of interaction among species, but only under particular models of species interaction. For example, when interactions within communities are mediated by a mechanism of phenotype matching, phylogenetic trees make specific predictions about trait evolution and rates of interaction. In contrast, if interactions within a community depend on a mechanism of phenotype differences, phylogenetic information has little, if any, predictive power for trait evolution and interaction rate. Together, these results make clear and testable predictions for when and how evolutionary history is expected to influence contemporary rates of species interaction.     

Single-carbon catabolism in acetogens: analysis of carbon flow in Acetobacterium woodii and Butyribacterium methylotrophicum by fermentation and 13C nuclear magnetic resonance measurement.

The catabolism of methanol, formate, or carbon monoxide to acetate or butyrate or both was examined in two acetogenic bacteria. Butyribacterium methylotrophicum simultaneously transformed methanol and formate mainly to butyrate with concomitant H2 and CO2 production and consumption. In contrast, methanol plus CO was primarily converted to acetate, and only slight amounts of CO2 were produced. In vivo 13C nuclear magnetic resonance analysis of [13C]methanol transformation by B. methylotrophicum indicated that methanol was predominantly incorporated into the methyl of acetate. 13CO2 was produced and then consumed, and butyrate was formed from the condensation of two acetate precursors. The analysis of the position of acetate labeled by a given 13C single-carbon substrate when B. methylotrophicum or Acetobacterium woodii was grown in the presence of a second one-carbon substrate indicated two trends: when methanol was consumed, CO, CO2, or formate predominantly labeled the acetate carboxyl; when CO was consumed, CO2 and formate were principally funneled into the acetate methyl group, and CO remained a better carboxyl precursor. These data suggest a model of acetate synthesis via the combined operation of two readily reversible single-carbon pathways which are linked by CO2.     

fastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood

We have developed a new tool, called fastDNAml, for constructingphylogenetic trees from DNA sequences. The program can be runon a wide variety of computers ranging from Unix workstationsto massively parallel systems, and is available from the RibosomalDatabase Project (RDP) by anonymous FTP. Our program uses amaximum likelihood approach and is based on version 3.3 of Felsenstein'sdnaml program. Several enhancements, including algorithmic changes,significantly improve performance and reduce memory usage, makingit feasible to construct even very large trees. Trees containing40–100 taxa have been easily generated, and phylogeneticestimates are possible even when hundreds of sequences exist.We are currently using the tool to construct a phylogenetictree based on 473 small subunit rRNA sequences from prokaryotes.     

MRBAYES: Bayesian inference of phylogenetic trees

SUMMARY: The program MRBAYES performs Bayesian inference of phylogeny using a variant of Markov chain Monte Carlo. AVAILABILITY: MRBAYES, including the source code, documentation, sample data files, and an executable, is available at http://brahms.biology.rochester.edu/software.html.     

ProfDist: a tool for the construction of large phylogenetic trees based on profile distances

SUMMARY: ProfDist is a user-friendly software package using the profile-neighbor-joining method (PNJ) in inferring phylogenies based on profile distances on DNA or RNA sequences. It is a tool for reconstructing and visualizing large phylogenetic trees providing new and standard features with a special focus on time efficency, robustness and accuracy. AVAILABILITY: A Windows version of ProfDist comes with a graphical user interface and is freely available at http://profdist.bioapps.biozentrum.uni-wuerzburg.de     

Secator: a program for inferring protein subfamilies from phylogenetic trees

With the huge increase of protein data, an important problem is to estimate, within a large protein family, the number of sensible subsets for subsequent in-depth structural, functional, and evolutionary analyses. To tackle this problem, we developed a new program, Secator, which implements the principle of an ascending hierarchical method using a distance matrix based on a multiple alignment of protein sequences. Dissimilarity values assigned to the nodes of a deduced phylogenetic tree are partitioned by a new stopping rule introduced to automatically determine the significant dissimilarity values. The quality of the clusters obtained by Secator is verified by a separate Jackknife study. The method is demonstrated on 24 large protein families covering a wide spectrum of structural and sequence conservation and its usefulness and accuracy with real biological data is illustrated on two well-studied protein families (the Sm proteins and the nuclear receptors).