Figure 2 Resistance phenotypes determined by the CZC and MER modules.
MICs of cobalt, zinc and mercury ions for wild-type strains (dark gray) and strains carrying pBBR-ZM3CZCMER (the plasmid contains CZC and MER resistance modules) (light gray) of Pseudomonas sp. LM7R, Pseudomonas sp. LM12R, A. tumefaciens LBA288 and E. coli TG1. This analysis revealed that introduction of pBBR-ZM3CZCMER into strain LM7R resulted in a significant increase in the MICs of cobalt (6-fold) and zinc (3-fold), which indicates resistance. In contrast, the level of tolerance to mercury was not changed (Figure 2). Different results were obtained with the transconjugants of strains LM12R and LBA288, which exhibited resistance to mercury (MIC increases of 1.5- and 3-fold, respectively), but not SB525334 Cyclosporin A nmr to cobalt or zinc. Interestingly, none of the tested
strains was resistant to cadmium. Introduction of the plasmid pBBR-ZM3CZCMER into E. coli TG1 did not result in cobalt or mercury resistance; however, an unexpected increase in sensitivity to zinc was observed (Figure 2). Besides the CZC and MER modules, plasmid pBBR-ZM3CZCMER also carries orf15 encoding a protein related to metallo-beta-lactamases, many of which confer resistance to beta-lactam antibiotics, e.g. . Therefore, we tested whether the CP-868596 datasheet pBBR-ZM3CZCMER-containing strains (LM7R, LM12R, LBA288, TG1) acquired resistance to antibiotics representing three classes of beta-lactams: (i) ampicillin (penicillins), (ii) ceftazidime (cefalosporins) and (iii) meropenem (carbapenems). The MICs, determined by Epsilometer tests, revealed no resistance phenotype, indicating that Orf15 protein does not exhibit beta-lactamase Megestrol Acetate activity in these strains. Identification and characterization of transposable elements (TEs) For the identification of functional TEs of Halomonas sp. ZM3 we employed the mobilizable BHR trap plasmid pMAT1, carrying the sacB cassette, which enables positive selection of transposition events . A pool of putative transposition mutants was collected and analyzed as described in Methods.
From this set of mutants, two classes of pMAT1 derivatives were identified, containing inserted elements of respective sizes 1 kb and 1.5 kb, which is typical for the majority of insertion sequences (ISs). DNA sequencing and comparison of the obtained nucleotide sequences (NCBI and ISfinder databases) revealed that the identified elements were novel insertion sequences, designated ISHsp1 and ISHsp2. ISHsp1 carries identical terminal inverted repeat sequences (IRs) of 15 bp at both ends (Figure 3). Transposition of the element into the sacB cassette of pMAT1 resulted in duplication of a short (6 bp) target sequence (5′-TACTTA-3′) to form direct repeats (DRs) (Figure 3). Within the 1518-bp-long sequence of ISHsp1 (G+C content – 56.7%) only one ORF was identified (nt position 113–1495), encoding a putative protein (460 aa; 52.