Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles

Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of Mycobacterium tuberculosis isolates from KwaZulu-Natal.

CAPRISA, 2015.Abstract available in pdf

Evolution of extensively drug-resistant tuberculosis over four decades revealed by whole genome sequencing of Mycobacterium tuberculosis from KwaZulu-Natal, South Africa

The largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) was identified in Tugela Ferry, KwaZulu-Natal (KZN), South Africa in 2005. The antecedents and timing of the emergence of drug resistance in this fatal epidemic XDR outbreak are unknown, and it is unclear whether drug resistance in this region continues to be driven by clonal spread or by the development of de novo resistance. A whole genome sequencing and drug susceptibility testing (DST) was performed on 337 clinical isolates of Mycobacterium tuberculosis (M.tb) collected in KZN from 2008 to 2013, in addition to three historical isolates, one of which was isolated during the Tugela Ferry outbreak. Using a variety of whole genome comparative approaches, 11 drug-resistant clones of M.tb circulating from 2008 to 2013 were identified, including a 50-member clone of XDR M.tb that was highly related to the Tugela Ferry XDR outbreak strain. It was calculated that the evolutionary trajectory from first-line drug resistance to XDR in this clone spanned more than four decades and began at the start of the antibiotic era. It was also observed that frequent de novo evolution of MDR and XDR was present, with 56 and 9 independent evolutions, respectively. Thus, ongoing amplification of drug-resistance in KwaZulu-Natal is driven by both clonal spread and de novo acquisition of resistance. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics that assess only rifampicin resistance

Isoniazid resistance is the first step towards drug resistance.

<p>Acquisition of <i>katG</i> S315 mutations precedes all other resistance mutations, including rifampicin, in all instances in which the order of acquisition can be disambiguated. For the 214 strains with genotypic resistance to two or more MDR or XDR defining drugs, and in which the order of acquisition of these mutations could be disambiguated, we quantified the number of evolutions in which resistance to one drug was gained before resistance to a second drug. Isoniazid resistance was divided into mutations conferred by the <i>katG</i>S315 codon versus “Other INH” mutations (defined as loss-of-function mutations in <i>katG</i> that do not involve codon 315 or mutations in the <i>inhA</i> promoter). Reported numbers represent the number of independent evolutionary events (not the number of strains) in which the drug resistance indicated by the row labeled “first resistance” was acquired before the drug resistance indicated by the column labeled “second resistance.” The background color is shaded to indicate the fraction of unambiguous evolutionary events in which the “first resistance” was acquired before the “second resistance” for that given drug pair.</p

Demographic characteristics of participants and phenotypic drug susceptibility of strains.

<p>* HIV-positive and HIV-negative individuals were compared using Fisher’s exact test for categorical variables and nonparametric Mann-Whitney test for continuous variables.</p><p>^† Data in this row are from the XDR data, not the total dataset.</p><p>Results having a <i>p</i>-value < 0.05 were considered statistically significant.</p><p>Data are <i>n</i> (%) or mean ± standard deviation (SD).</p

Diverse strains contribute to drug resistance in KwaZulu-Natal.

<p>(A) Midpoint rooted maximum-likelihood phylogeny of 340 <i>M</i>. <i>tuberculosis</i> isolates. Four of the seven known <i>M</i>. <i>tuberculosis</i> lineages were identified: CAS (<i>Lin1</i>), Beijing (<i>Lin2</i>), EAI (<i>Lin 3</i>), and Euro-American (<i>Lin4</i>). Digital spoligotyping identified 17 unique spoligotypes in the dataset; spoligotypes are shown on this figure if they are represented by three or more strains. Corresponding spoligotypes and phenotypes are reported for all strains in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001880#pmed.1001880.s009" target="_blank">S4 Table</a>. Phenotypic XDR, MDR, poly- and monodrug resistance (labeled “Drug-resistant other”), and pansusceptible strains are indicated by colored tick marks at the tip of each leaf node. (B) Histogram of pairwise SNP distances between strains. The number of pairs within each SNP distance range is plotted. The peaks correspond to the distance between major lineages. The peak at the far left of the figure corresponds to the distance between pairs of strains within a clone.</p

Molecular evolution and dating of drug resistance emergence within the Tugela Ferry XDR Clone.

<p>Midpoint rooted maximum-likelihood phylogeny of 107 <i>M</i>. <i>tuberculosis</i> isolates of the LAM4 spoligotype. The gray shaded box identifies the Tugela Ferry XDR Clone. KZN605, the historical XDR strain collected in Tugela Ferry during the outbreak, is a member of this clone. Two additional historical isolates, KZN1435 and KZN4207, are not members of the Tugela Ferry XDR Clone. Each evolutionary gain of a drug resistance mutation was assigned to its position on the phylogenetic tree by parsimony (colored circles). A–E traces the stepwise order of drug resistance acquisition in the Tugela Ferry XDR Clone and estimates the year when each mutation was gained. Gray bars indicate the 95% highest posterior density (HPD) intervals. (A) <i>katG</i> S315T (isoniazid); <i>gidB</i> 130 bp deletion (streptomycin); 1957 (95% HPD: 1937–1971); (B) <i>inhA</i> promoter -8 (isoniazid and ethionamide); 1964 (95% HPD: 1948–1976); (C) <i>embB</i> M306V (ethambutol); 1967 (95% HPD: 1950–1978); (D) <i>rpoB</i> L452P (rifampicin); <i>pncA</i> 1bp insertion (pyrazinamide); 1984 (95% HPD: 1974–1992); and (E) <i>rpoB</i> D435G (rifampicin); <i>rrs</i> 1400 (kanamycin); <i>gyrA</i> A90V (ofloxacin); 1995 (95% HPD: 1988–1999). The accumulation of individual drug-resistant mutations within a strain is denoted to the right of the phylogenetic tree. The dates of drug discovery are displayed at the bottom of the figure [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001880#pmed.1001880.ref053" target="_blank">53</a>]. Four additional LAM4 strains on a distant branch were not included in this figure because of size constraints. Bootstrap values are provided for lettered nodes, and bootstrap values for all nodes are shown in S5 Fig.</p