One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.     

Prevalence of bacterial pathogens in biofilms of drinking water distribution systems

Water for human consumption is required to be free from any bacteria that might pose a health risk. The presence of biofilms in the drinking water distribution system may play a role in the presence of potential pathogens in the drinking water supply. Ninety-five biofilm samples from various parts of South Africa were tested for the presence of Escherichia coli, Aeromonas, Pseudomonas, Salmonella, Shigella and Vibrio spp. Members of these genera were quantified by the three-tube most probable number (MPN) approach using enrichment broths and plating on selective agars. The heterotrophic culturable counts were determined for both the planktonic and biofilm phases of the samples. Biofilm density varied between 10 and 1.9 x 10(9) colony forming units cm(-2). The 16S rRNA identity of the putative pathogenic isolates revealed that high numbers of Aeromonas, Pseudomonas, Klebsiella and Enterobacter were present, but no putative Salmonella and Shigella could be confirmed. None of the Pseudomonas isolates belonged to the pathogenic Pseudomonas aeruginosa or Pseudomonas mendocina while the Aeromonas isolates showed relatedness to known pathogenic members of this group.     

MALDI-TOF MS meets WGS in a VRE outbreak investigation

The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization–time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology–oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.     

Genotypic characterization and comparison of full-length envelope glycoproteins from South African HIV type 1 subtype C primary isolates that utilize CCR5 and/or CXCR4

CCR5 has preferentially been used by all circulating HIV-1 subtype C viruses for cell entry. Recently, we reported the highest proportion of CXCR4-utilizing primary isolates among a cohort of 20 South African AIDS patients. This study describes and compares the Env genotypic characteristics from these 20 HIV-1 subtype C (and unique CD recombinant) primary isolates. Fourteen primary isolates utilized CCR5, four (including the CD recombinant) used CXCR4, and two were dual tropic. Extensive analysis and comparison of important structural motifs such as the N-linked glycosylation sites, signal sequences, CD4-binding sites, variable loops, cleavage sites, known neutralizing antibody and small molecule inhibitor binding sites confirmed that other than the expected differences in the V3 loop, no sequence motifs distinguished between R5 and X4 tropism. Further correlation of the env genotype to functionally relevant motifs is necessary to elucidate the relationship between biologically and immunologically relevant sites and aid vaccine and novel drug design.     

Affinity isolation of cultured tumor cells by means of drugs and hormones covalently bound to glass and Sepharose beads.

Isoproterenol, corticotropin (ACTH), and triodothyronine immobilized on glass and Sepharose beads by diazotization procedures have been shown to interact with cultured tumor cells of "target tissue" origin. Cells used were rat glioma cells (C6), rat adrenal tumor cells (Y-1), and rat pituitary tumor cells (GH3). The rat glioma cells bound principally to immobilized isoproterenol, whereas the rat adrenal tumor cells bound to immobilized corticotropin, and rat pituitary tumor cells bound to immobilized triiodothyronine. Binding was inhibited by preincubation of the cells in soluble drug or hormone. With C6 cells there was a positive correlation between adenylate cyclase [ATP pyrophosphate-lyase (cyclizing, EC 4.6.1.1] stimulation and the degree of binding to the immobilized isoproterenol. Norepinephrine, bound through the ethanolamine side chain via an amide linkage, did not bind cells, demonstrating specific structural requirements for drug-cell interactions. HeLa cells were shown to bind tightly to diphtheria toxin coupled to Sepharose beads via an amide bond. This binding was inhibited by prior incubation of the Sepharose toxin with purified antitoxin. Toxin bound to Sepharose via an azo bond did not bind cells. These data suggest that the cell affinities are due to cell surface receptors interacting with the immobilized drugs and hormones, and that the observed affinities possibly reflect the relative receptor complement of these cells.