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.     

Circularity of the Bacillus subtilis chromosome and further studies on its bidirectional replication

Replicating, circular chromosomes of Bacillus subtilis have been displayed by autoradiography of samples obtained from thymine-requiring spores germinating in the presence of [³H]thymine and proceeding into the second round of replication. The observed chromosomes have the expected Cairns-type configuration, and the measured length of a completed (non-replicating) chromosome is 900 to 1100 μm.The extent to which bidirectional replication can proceed following initiation of a round has been investigated further. Thymine-requiring spores germinating in the presence of [³H]thymidine were diluted into higher specific activity medium during the first round of replication and autoradiographs prepared from samples obtained shortly afterwards. From the pattern of labelling within individual loops that become visible, it is clear that replication can proceed in a bidirectional manner up to a stage that would account for replication of at least 50% of the whole chromosome. And, until this stage, the replication rates in both directions are approximately equal.     

GEOGRAPHIC VARIATION IN ALLOZYMES IN A “RING SPECIES,” THE PLETHODONTID SALAMANDER ENSATINA ESCHSCHOLTZII OF WESTERN NORTH AMERICA

The ring species Ensatina eschscholtzii (a plethodontid salamander) of western North America has a circle of subspecies surrounding the Central Valley of California which come into contact and are sympatric in southern California. We examined 26 proteins in 19 populations (maximum of 10 specimens per population) collected throughout the range in order to gain an understanding of the degree of differentiation in the group. Allozymic differentiation is profound, with genetic distances in excess of 0.5 (Rogers or Nei) between populations. Naturally hybridizing populations differ by genetic distances greater than 0.4. Two general classes of color morphs, blotched and unblotched, are segregated geographically, but they do not form discrete genetic units. Both are deeply differentiated, and genetic distances among populations of either class exceed those measured between the classes where they are sympatric in southern California. This study disclosed little evidence of gene exchange around the ring of populations and sampling of many additional populations in regions between populations sampled thus far will be required to determine whether smooth intergradation occurs. Although genetic distances measured exceed those between some co-occurring species of plethodontid salamanders, we find no evidence of borders between cryptic species.     

‘In-Format’ screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules

Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an ‘in-format’ screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC₅₀) when assessed in-format were identified. In contrast, the corresponding dAb monomers had ～1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening naïve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time.