Isolation of gene conferring salt tolerance from halophilic bacteria of Lunsu, Himachal Pradesh, India

Background Halophiles offer an attractive source of genes conferring salt tolerance. Halobacillus trueperi SS1 strain of Lunsu, Himachal Pradesh, India, a strict halophile, was exploited to isolate and clone the genes for salt tolerance. The genomic library of BamH1 digest of H. trueperi SS1 was constructed in pUC19, and recombinants were screened for salt tolerance on an LB medium containing ampicillin (100 μg/ml) and NaCl (0 to 1.5 M). Results One recombinant clone named as salt-tolerant clone (STC) conferred salt tolerance to host Escherichia coli/DH5α, which showed growth in the LB medium supplemented with ampicillin and 1.2 M NaCl. Restriction digestion and PCR analysis revealed the presence of an insert of approximately 2000 bp in the STC. DNA sequencing of the 2-kb insert on both strands yielded a sequence of 2301 nucleotides. Protein BLAST analysis of 2301-bp sequence of H. trueperi SS1 present in STC showed 97% identity to multidrug transport ATP binding/permease protein of Halobacillus karajensis. The insert contained in STC was subcloned into pGEX4T2 vector, and the recombinant clone STC/pGEX4T2 conferred salt tolerance to the bacterial host E. coli. Conclusions The present study led to the isolation of salt tolerance gene encoding a putative multidrug transport ATP binding/permease protein from H. trueperi SS1. The salt tolerance gene can be subcloned for transferring salt tolerance traits into agricultural crop plants for cultivation in saline and coastal lands.

Two multi-fragment recombination events resulted in the β-lactam-resistant serotype 11A-ST6521 related to Spain9V-ST156 pneumococcal clone spreading in south-western Europe, 2008 to 2016

Background The successful pneumococcal clone Spain9V-ST156 (PMEN3) is usually associated with vaccine serotypes 9V and 14. Aim Our objective was to analyse the increase of a serotype 11A variant of PMEN3 as cause of invasive pneumococcal disease (IPD) in Spain and its spread in south-western Europe. Methods We conducted a prospective multicentre study of adult IPD in Spain (2008–16). Furthermore, a subset of 61 penicillin-resistant serotype 11A isolates from France, Italy, Portugal and Spain were subjected to whole genome sequencing (WGS) and compared with 238 genomes from the European Nucleotide Archive (ENA). Results Although the incidence of serotype 11A in IPD was stable, a clonal shift was detected from CC62 (penicillin-susceptible) to CC156 (penicillin-resistant). By WGS, three major 11A-CC156 lineages were identified, linked to ST156 (n = 5 isolates; France, Italy and Portugal), ST166 (n = 4 isolates; France and Portugal) and ST838/6521 (n = 52 isolates; France, Portugal and Spain). Acquisition of the 11A capsule allowed to escape vaccine effect. AP200 (11A-ST62) was the donor for ST156 and ST838/6521 but not for ST166. In-depth analysis of ST838/6521 lineage showed two multi-fragment recombination events including four and seven fragments from an 11A-ST62 and an NT-ST344 representative, respectively. Conclusion The increase in penicillin-resistant serotype 11A IPD in Spain was linked to the spread of a vaccine escape PMEN3 recombinant clone. Several recombination events were observed in PMEN3 acquiring an 11A capsule. The most successful 11A-PMEN3 lineage spreading in south-western Europe appeared after two multi-fragment recombination events with representatives of two major pneumococcal clones (11A-ST62 and NT-ST344).

Disruption of hmgA by DNA Duplication is Responsible for Hyperpigmentation in a Vibrio anguillarum Strain

Vibrio anguillarum 531A, isolated from a diseased fish in the Atlantic Ocean, is a mixture composed of about 95 and 5% of highly pigmented cells (strain 531Ad) and cells with normal levels of pigmentation (strain 531Ac), respectively. Analysis of the V. anguillarum 531Ad DNA region encompassing genes involved in the tyrosine metabolism showed a 410-bp duplication within the hmgA gene that results in a frameshift and early termination of translation of the homogentisate 1,2-dioxygenase. We hypothesized that this mutation results in accumulation of homogentisate that is oxidized and polymerized to produce pyomelanin. Introduction in E. coli of recombinant clones carrying the V. anguillarum hppD (4-hydroxyphenylpyruvate-dioxygenase), and a mutated hmgA produced brown colored colonies. Complementation with a recombinant clone harboring hmgA restored the original color to the colonies confirming that in the absence of homogentisate 1,2-dioxygenase the intermediary in tyrosine catabolism homogentisate accumulates and undergoes nonenzymatic oxidation and polymerization resulting in high amounts of the brown pigment. Whole-genome sequence analysis showed that V. anguillarum 531 Ac and 531Ad differ in the hmgA gene mutation and 23 mutations, most of which locate to intergenic regions and insertion sequences.