Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.     

The cytoplasmic membrane as the site of the antimicrobial action of N-octylethanolamine

On agar spread plates, N-octylethanolamine was biocidal at comparable minimum inhibitory concentration (MIC) values (3–4mm) against Pseudomonas aeruginosa (two strains), Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida tropicalis, and Acremonium sp. which had been grown on a number of different media. The inhibition was greater at higher pH values. In liquid culture, growth inhibition by 3mm N-octylethanolamine was accompanied by cell lysis. Both effects could be prevented by the presence of 1mm spermine or spermidine, but only in bacteria, and not at high pH values. These effects of the polyamines were shown to be non-specific, being shared by other polycations and Mg²⁺ ions. N-Octylethanolamine at concentrations above its MIC caused total inhibition of the oxidation of 1mm glucose by Ps. aeruginosa (CAS1 and PAO1), E. coli, or C. tropic an effect that was partially reversible by Mg²⁺ ions. At concentrations below the MIC, there was little inhibit ion of glucose oxidation but a potent inhibition of the extrusion of ethidium bromide from intact cells of E. coli, suggesting that at such concentrations N-octylethanolamine is uncoupling oxidative phosphorylation. The data presented confirm the view that the biocidal effects are due to action on the cytoplasmic membrane.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

m-Octopamine: Normal Occurrence with p-Octopamine in Mammalian Sympathetic Nerves

The development of a radiochemical enzyme assay for p-octopamine in 1969 led to its identification in a large number of invertebrate nerve systems and in mammalian sympathetic nerves. The original method by which p-octopamine was measured has now been found to be nonspecific; however, modifications of this procedure can determine both m- and p-octopamine. We recently developed a new specific method for the unequivocal identification and quantitative determination in tissue of the six octopamine and synephrine isomers. With this method--negative chemical ionization gas chromatography-mass spectrometry--the more physiologically active m-octopamine has been found in association with p-octopamine in 10 organs of the rat. m-Octopamine is present in concentrations equal to those of p-octopamine in heart, spleen, and liver and in concentrations from 30 to 60% of p-octopamine in adrenals, vas deferens, brain, kidney, large intestine, bladder, and lungs. In vivo inhibition of monoamine oxidase markedly increased the concentrations of both m- and p-octopamine in all organs examined. Both amines were virtually absent from all organs except the adrenals following chemical sympathectomy with 6-hydroxydopamine, thereby establishing that m- and p-octopamine are localized within sympathetic nerve endings.     

Intraspecific trait variation influences physiological performance and fitness in the South Africa shrub genus Protea (Proteaceae)

Background and AimsGlobal plant trait datasets commonly identify trait relationships that are interpreted to reflect fundamental trade-offs associated with plant strategies, but often these trait relationships are not identified when evaluating them at smaller taxonomic and spatial scales. In this study we evaluate trait relationships measured on individual plants for five widespread Protea species in South Africa to determine whether broad-scale patterns of structural trait (e.g. leaf area) and physiological trait (e.g. photosynthetic rates) relationships can be detected within natural populations, and if these traits are themselves related to plant fitness.MethodsWe evaluated the variance structure (i.e. the proportional intraspecific trait variation relative to among-species variation) for nine structural traits and six physiological traits measured in wild populations. We used a multivariate path model to evaluate the relationships between structural traits and physiological traits, and the relationship between these traits and plant size and reproductive effort.Key ResultsWhile intraspecific trait variation is relatively low for structural traits, it accounts for between 50 and 100 % of the variation in physiological traits. Furthermore, we identified few trait associations between any one structural trait and physiological trait, but multivariate regressions revealed clear associations between combinations of structural traits and physiological performance (R² = 0.37–0.64), and almost all traits had detectable associations with plant fitness.ConclusionsIntraspecific variation in structural traits leads to predictable differences in individual-level physiological performance in a multivariate framework, even though the relationship of any particular structural trait to physiological performance may be weak or undetectable. Furthermore, intraspecific variation in both structural and physiological traits leads to differences in plant size and fitness. These results demonstrate the importance of considering measurements of multivariate phenotypes on individual plants when evaluating trait relationships and how trait variation influences predictions of ecological and evolutionary outcomes.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

CAM variations in the leaf-succulent Delosperma tradescantioides (Mesembryanthemaceae), native to southern Africa

Drought responses of diurnal gas exchange, malic acid accumulation and water status were examined in Delosperma tradescantioides , a succulent that grows in drought-prone microenvironments in summer rainfall and all-year rainfall regions of southern Africa. When well-watered, this species exhibited Crassulacean acid metabolism (CAM)-cycling, but its carbon fixation pattern changed during the development of drought, shifting to either low-level CAM or to CAM-idling. The rate and pattern of this change depended on environmental conditions, duration of water stress and leaf age. At the onset of drought, diurnal malate fluctuation increased, but was strongly depressed (by ca 70%) as drought continued, and when leaf water content and water potential were low (ca 35 and 50% of the initial levels, respectively). When rewatered, rates of growth and photosynthesis, gas exchange and water status recovered fully to pre-stressed values within two days. Whole-shoot carbon uptake rates suggested that leaf growth had continued unabated during a short-term (∼ one week) drought. This emphasises that CAM-idling allows the maintenance of active metabolism with negligible gas exchange when soil water is limiting. It is possible that old or senescent leaves may provide water for the expansion of developing leaves during initial periods of drought. Regardless of the water regime and environmental conditions, leaf nocturnal malate accumulation and water content were positively correlated and increased with leaf age. Thus the gradual loss of water from older mature leaves may induce CAM-idling, which reduces water loss. An important ecological consequence of this combination of CAM modes is the potential to switch rapidly between fast growth via C₃ gas exchanges when well-watered to water-conserving CAM-idling during drought.     

An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques

From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0.35+/-0.02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA-RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0.45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70-75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0.23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0.23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RC(rel) mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RC(rel) strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.     

Fine-grain pattern in Southern Cape plateau forests

Abstract. Regeneration levels, size class distributions and a nearest neighbour technique were used to describe apreliminary dynamics frameworkfortheplateauforests. Taken over a large area, most of the canopy dominants have a negative exponential distribution of stems > 10 cm dbh per size class. In small (0.04 ha) plots, if present as more than one stem, most species are present as both canopy (> 25 cm dbh) and understorey (10–15 cm dbh) individuals. The canopy dominants maintain their rank in the bank of advance regeneration (> 5 cm height < 10 cm dbh). However, relative numbers of all regeneration of most canopy individuals are not strongly correlated with canopy closure or local abundance of conspecific adults. The mean area of nearest neighbour polygons of canopy individuals around saplings of the more common species are small. In conclusion, most species appearto be shade tolerant and locally persistent conferring a fine grain on this forest. We support recent questioning of universality of the gaps/non gaps paradigm.