Activity of Key Enzymes Involved in Carbohydrate Metabolism in Phaseolus vulgaris Cell Suspension Cultures

Activities of some key enzymes of glycolysis and sucrose metabolismwere investigated in relation to the physiological growth stagein bean cell suspension cultures. Activities of sucrose synthase,pyrophosphate:fructose-6-phosphate phosphotransferase, ATP:fructose-6-phosphatephosphotransferase, UDP glucose pyrophosphorylase, acid andalkaline invertase were detected. Both pyrophosphate:fructose-6-phosphatephosphotransferase and sucrose synthase activities increasedduring the active phase of cell division. Thereafter activitiesbegan to decline when sugar in the medium was depleted. Theincrease in enzyme activities coincided with a sharp decreasein the endogenous sucrose, glucose and fructose levels. Thelargest change occurred in the activity of sucrose synthase,which was more than seven fold higher in logarithmic phase cellsthan in lag hase cells. Transfer of mid-logarithmic phase cellsto fresh medium, containing 93 mmol dm^–3 sucrose, or additionof sucrose to existing medium, resulted in a further increasein PPjifructose- 6-phosphate phosphotransferase and sucrosesynthase activities. ²Present address: Plant Biotechnology Research Centre, PrivateBag X293, Pretoria 0001, Republic of South Africa.     

The impact of commercial harvesting on Warburgia salutaris (‘pepper-bark tree’) in Mpumalanga, South Africa

Commercialisation often increases the difficulty in managing harvested plant populations sustainably. The bark of the popular medicinal species, Warburgia salutaris (Bertol.f.) Chiov. (Canellaceae) (pepper-bark tree), is widely traded throughout southern Africa. The impact of commercial harvesting on this Red Data species was assessed by comparing commercially harvested populations with populations growing on private land or in protected areas (termed protected populations) in Mpumalanga and Limpopo Province, South Africa. The basal diameters and heights of stems in commercially harvested populations were significantly lower than those of the protected populations. The density of young/small plants was low in all populations. W. salutaris is usually resilient to high levels of bark harvesting. In this study, 75% of heavily harvested stems (>10% of the stem below 2 m) coppiced (resprouted). However, individuals that had been affected by regular fires, or repeatedly harvested, appeared prone to a fungal disease and had high percentage mortality. The populations occurring on private land appeared the most vigorous. Habitat in one protected area had been reduced through the construction of a dam. In another, small W. salutaris populations exhibited a shrubby growth form, probably due to frequent fires. Our current knowledge for this species supports a global IUCN status of EN A4acd. Plant conservation needs to become a higher priority both within and outside protected areas. Commercially harvested populations should be better managed through improved harvesting techniques and monitoring. Cultivation levels urgently need to be increased. Further research should be conducted on factors limiting regeneration, including the most appropriate fire regime.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and IB comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.     

Spatial phylogenetics of the native woody plant species in Hainan,China

To better identify biodiversity hotspots for conservation on Hainan Island, a tropical island in southern China, we assessed spatial variation in phylogenetic diversity and species richness using 18,976 georeferenced specimen records and a newly reconstructed molecular phylogeny of 957 native woody plants. Within this framework, we delineated bioregions based on vegetation composition and mapped areas of neoendemism and paleoendemism to identify areas of priority for conservation. Our results reveal that the southwest of Hainan is the most important hot spot for endemism and plant diversity followed by the southeast area. The distribution of endemic species showed a scattered, rather than clustered, pattern on the island. Based on phylogenetic range‐weighted turnover metrics, we delineated three major vegetational zones in Hainan. These largely correspond to natural secondary growth and managed forests (e.g., rubber and timber forests) in central Hainan, old‐growth forests and natural secondary growth forest at the margins of Hainan, and nature reserves on the island (e.g., Jianfeng and Diaoluo National Nature Reserves). Our study helps to elucidate potential botanical conservation priorities for Hainan within an evolutionary, phylogenetic framework.     

A network-based approach to classify the three domains of life

Background

Chromosomal orthologs can reveal the shared ancestral gene set and their evolutionary trends. Additionally, physico-chemical properties of encoded proteins could provide information about functional adaptation and ecological niche requirements.

Results

We analyzed 7080 genes (five groups of 1416 orthologs each) from Rhizobiales species (S. meliloti, R. etli, and M. loti, plant symbionts; A. tumefaciens, a plant pathogen; and B. melitensis, an animal pathogen). We evaluated their phylogenetic relationships and observed three main topologies. The first, with closer association of R. etli to A. tumefaciens; the second with R. etli closer to S. meliloti; and the third with A. tumefaciens and S. meliloti as the closest pair. This was not unusual, given the close relatedness of these three species. We calculated the synonymous (dS) and nonsynonymous (dN) substitution rates of these orthologs, and found that informational and metabolic functions showed relatively low dN rates; in contrast, genes from hypothetical functions and cellular processes showed high dN rates. An alternative measure of sequence variability, percentage of changes by species, was used to evaluate the most specific proportion of amino acid residues from alignments. When dN was compared with that measure a high correlation was obtained, revealing that much of evolutive information was extracted with the percentage of changes by species at the amino acid level. By analyzing the sequence variability of orthologs with a set of five properties (polarity, electrostatic charge, formation of secondary structures, molecular volume, and amino acid composition), we found that physico-chemical characteristics of proteins correlated with specific functional roles, and association of species did not follow their typical phylogeny, probably reflecting more adaptation to their life styles and niche preferences. In addition, orthologs with low dN rates had residues with more positive values of polarity, volume and electrostatic charge.

Conclusions

These findings revealed that even when orthologs perform the same function in each genomic background, their sequences reveal important evolutionary tendencies and differences related to adaptation. This article was reviewed by: Dr. Purificación López-García, Prof. Jeffrey Townsend (nominated by Dr. J. Peter Gogarten), and Ms. Olga Kamneva.     

Molecular analyses of Pythium irregulare isolates from grapevines in South Africa suggest a single variable species

The Pythium irregulare species complex is the most common and widespread Pythium spp. associated with grapevines in South Africa. This species complex has been subdivided into several morphological and phylogenetic species that are all highly similar at the sequence level [internal transcribed spacer (ITS) and cytochrome c oxidase (cox) regions]. The complex includes Pythium regulare and Pythium cylindrosporum, which are morphologically distinct, and P. irregulare sensu stricto (s.s.) and Pythium cryptoirregulare, which are morphologically similar. The aim of the current study was to determine whether 50 South African grapevine P. irregulare isolates represented more than one phylogenetically distinct species. The isolates were characterised using nuclear (ITS and β-tubulin) and mitochondrial (cox1 and cox2) gene region phylogenies and two isozyme loci [glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase (Mdh-1)]. Some of the gene sequence data were difficult to interpret phylogenetically, since some isolates contained two or more polymorphic ITS copies within the same isolate (intra-isolate variation) that clustered into different ITS sub-clades, i.e. the P. irregulare s.s. and P. cryptoirregulare sub-clades. The molecular data furthermore only revealed the presence of one phylogenetic species, P. irregulare. Morphological analyses of a subset of the isolates confirmed that the isolates were P. irregulare, and further showed that the P. cylindrosporum ex-type strain formed typical P. irregulare oogonia, and not the previously reported distinct elongated oogonia. Some of the molecular analyses suggested the occurrence of outcrossing events and possibly the formation of aneuploids or polyploids since (i) the nuclear and mitochondrial gene data sets were incongruent, (ii) polymorphic ITS copies were present within the same isolate, (iii) heterozygosities were observed in the β-tubulin gene and Gpi and Mdh-1 loci in some isolates and (iv) more than two β-tubulin alleles were detected in some isolates. Altogether, the data suggest that P. irregulare, P. cryptoirregulare, P. cylindrosporum, and possibly P. regulare should be synonimised under the name P. irregulare.