High-throughput comparison of gene fitness among related bacteria

ABSTRACT: BACKGROUND: The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains to reveal differences not apparent at the gene sequence level. RESULTS: A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 strain (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of Tn insertions or had very few. For three of these nine genes, part of the annotated gene lacked Tn integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498.L, STM14_2872, STM14_3360.RJ, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential. CONCLUSIONS: Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene fitness among syntenic homologous genes. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Water cytotoxicity and dioxins bioaccumulation in an Egyptian delta wetland ecosystem

Manzala Lake, as one of the main Egyptian wetland ecosystems, is facing risks of pollution. An in vitro cytotoxicity test using a mammalian cell line was employed to determine the toxicity of multiple pollutants in the water and Tilapia zillii fish sampled from the lake. The concentrations of seven polychlorinated dibenzo-p-dioxins and ten polychlorinated dibenzofurans were investigated in water and muscle of the fish in 2014. Cytotoxicity testing showed that the percentage inhibition of cell viability in the studied sites ranged between 56.16% and 83.22%. Dioxin analysis indicated that the average concentrations of 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzofuran and 1,2,3,4,6,7,8,9-octachlorodibenzofuran were higher than the toxic equivalence quotients (TEQs) set by the World Health Organization (WHO) in all water and fish muscle samples; however, the average concentration of 2,3,7,8-tetrachlorodibenzofuran was higher only in fish muscle samples. The bioaccumulation factor (BAF) ranged dramatically between 2 and 58.5 for the detected dioxins. Adverse human health effects through the consumption of fish are not expected, because dioxin levels in fish muscle are deemed safe for human consumption. Implementation of a strategic multidisciplinary action plan is strongly recommended to sustain this delta wetland ecosystem.     

Construction of a gene library from the nitrogen-fixing aerobe Azotobacter vinelandii

We have cloned the DNA of Azotobacter vinelandii in the cosmid pHC79. Recombinant cosmids that can transform Escherichia coli leuB- to a Leu+ phenotype, as well as those having sequence homology to the nitrogenase structural genes of Klebsiella pneumoniae have been selected from this library.