Recipient competence in F''lac matings of Escherichia coli K-12.

We studied recipient mating ability in the presence of excess F'lac donors. Ninety-five percent of recipients were able to receive F'lac in 30-min matings. Competition between an F'-lac donor and an F'lac traI donor, which mobilized a ColE1 derivative (pML2), showed that each recipient mated with an average of two to three donors in 30 min. Experiments in which the competing donor was added at different times showed that some competition occurred throughout the 30-min mating period, which suggested that aggregate formation was spread over this time.     

A plausible mechanism for large-scale chromosomal DNA amplification in streptomycetes

Recent advances in our understanding of the structure of highly amplified DNA sequences in Streptomyces fradiae and lividans have enabled us to formulate a possible mechanism by which amplification may occur. An essential feature of the model is the generation of an amplification precursor, which comprises a circularised copy of the DNA to be amplified, attached to one arm of the chromosome by a replication fork. Multiple copies of the amplifiable DNA are generated by rolling circle replication. The model adequately accounts for many features of gene amplification in these two species, including the tendency for deletions to occur to one side, but not the other, of the amplified DNA.     

Characterization of multilayer-enhanced surface-enhanced raman scattering (SERS) substrates and their potential for SERS nanoimaging

Multilayer gold surface-enhanced Raman scattering (SERS) substrates, which consist of continuous gold films that are separated by self-assembled monolayers (SAMs) and cast over 430-nm diameter silica nanospheres on a glass slide, have been evaluated as a means of further enhancing the SERS signals produced from conventional metal film over nanostructure substrates. Evaluation of the effect of various SAMs, with different terminal functional groups, on the SERS enhancement factor were measured and compared to conventional single-layer gold film over nanostructure substrates, revealing relative enhancements as great as 22.4-fold in the case of 2-mercapto-ethanol spacer layers. In addition to evaluation of the effect of different terminal functionalities, the effect of spacer length was also investigated, revealing that the shorter chain length alcohols provided the greatest signals. Employing the optimal SERS multilayer geometry, SERS nanoimaging probes were fabricated and the SERS enhancement factor and variability in enhancement factor were measured over the SERS active imaging area, providing absolute enhancements similar to previous silver-based SERS nanoimaging probes (i.e., 1.2 × 10⁸). Varying the size of the multilayer gold islands that were deposited on the tip of the SERS active nanoimaging probe, it is possible to tune the optimal SERS excitation wavelength accurately and predictably over the range of approximately 450 to 600 nm, without coating the entire surface of the probe and significantly reducing the transmission and resulting signal-to-noise ratio of the images obtained.     

Rapid evolution of escape ability in Trinidadian guppies (Poecilia reticulata)

Predators are widely assumed to create selection that shapes the evolution of prey escape abilities. However, this assumption is difficult to test directly due to the challenge of recording both predation and its evolutionary consequences in the wild. We examined these events by studying natural and experimental populations of Trinidadian guppies, Poecilia reticulata, which occur in distinct high-predation and low-predation environments within streams. Importantly, in the last two decades several populations of guppies have been experimentally introduced from one type of predatory environment into the other, allowing measurements of the consequences of change. We used this system to test two hypotheses: First, that changes in predatory environments create phenotypic selection favoring changes in escape ability of guppies, and second, that this selection can result in rapid evolution. For the first test we compared escape ability of wild caught guppies from high- versus low-predation environments by measuring survival rates during staged encounters with a major predator, the pike cichlid Crenicichla alta. We used guppies from three streams, comparing two within-stream pairs of natural populations and three within-stream pairs of an introduced population versus its natural source population. In every comparison, guppies from the high-predation population showed higher survival. These multiple, parallel divergences in guppy survival phenotype suggest that predatory environment does create selection of escape ability. We tested our second hypothesis by rearing guppies in common garden conditions in the laboratory, then repeating the earlier experiments using the F2 generation. As before, each comparison resulted in higher survival of guppies descended from the high-predation populations, demonstrating that population differences in escape ability have a genetic basis. These results also show that escape ability can evolve very rapidly in nature, that is, within 26-36 generations in the introduced populations. Interestingly, we found rapid evolutionary loss of escape ability in populations introduced into low-predation environments, suggesting that steep fitness trade-offs may influence the evolution of escape traits.     

Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell

We report here the application of an antibody-based nanoprobe for in situ measurements of a single cell. The nanoprobe employs antibody-based receptors targeted to a fluorescent analyte, benzopyrene tetrol (BPT), a metabolite of the carcinogen benzo[a]pyrene (BaP) and of the BaP-DNA adduct. Detection of BPT is of great biomedical interest, since this species can serve as a biomarker for monitoring DNA damage due to BaP exposure and for possible precancer diagnosis. The measurements were performed on the rat liver epithelial clone 9 cell line, which was used as the model cell system. Before making measurements, the cells were treated with BPT. Nanoprobes were inserted into individual cells, incubated 5 min to allow antigen-antibody binding, and then removed for fluorescence detection. We determined a concentration of 9.6+/-0.2x10(-11) M for BPT in the individual cells investigated. The results demonstrate the possibility of in situ measurements inside a single cell using an antibody-based nanoprobe.     

An experimental test of the thermoregulatory hypothesis for the evolution of endothermy

The thermoregulatory hypothesis proposes that endothermy in mammals and birds evolved as a thermoregulatory mechanism per se and that natural selection operated directly to increase body temperature and thermal stability through increments in resting metabolic rate. We experimentally tested this hypothesis by measuring the thermoregulatory consequences of increased metabolic rate in resting lizards (Varanus exanthematicus). A large metabolic increment was induced by feeding the animals and consequent changes in metabolic rate and body temperature were monitored. Although metabolic rate tripled at 32 degrees C and quadrupled at 35 degrees C, body temperature rose only about 0.5 degrees C. The rate of decline of body temperature in a colder environment did not decrease as metabolic rate increased. Thus, increasing the visceral metabolic rate of this ectothermic lizard established neither consequential endothermy nor homeothermy. These results are inconsistent with a thermoregulatory explanation for the evolution of endothermy.     

Isolation and cloning of Streptomyces terminal fragments

Streptomyces species have a linear chromosome of approximately 8 Mb in size. Many strains also carry linear plasmids. Most of these linear elements contain terminal proteins covalently bound to the 5 ends of the DNA. Using a method for the visualisation of terminal DNA fragments in agarose gels, it was possible to see three fragments in S. rimosus and five fragments in S. avermitilis. The method was also used to clone the 298 bp BamHI fragment carrying the left end of plasmid SLP2. Analysis of the sequence showed that the end resembled other Streptomyces chromosome and plasmid ends, but there were eight palindromes (instead of seven) and a tandem duplication of a 14 bp sequence.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).