A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

The differential roles of D-Pax2 variants in regulating Drosophila eye and bristle development

The ability to appropriately interact with the environment is crucial to an organism’s survival. The establishment of functional sensory systems, such as the bristles and eyes in Drosophila, is a critical event during the development of the organism. The transcription factor D Pax2 is involved in the differentiation of the shaft and glial cells in the developing bristle (Kavaler et al., Dev, 126:2261-2272, 1999) and of the cone and primary pigment cells in the developing eye (Fu and Noll, Genes Dev, 11:389-405, 1997). How D-Pax2 contributes to distinct differentiative pathways in different cell types is not known. Recent work by Anna Czechowski and Katherine Harmon (personal communication) identified a mutation in the D-Pax2 gene that introduced a stop codon at the end of exon 9, effectively truncating the protein. This mutation affects bristle, but not eye, development. We thus suspected regions after exon 9 are required for D-Pax2 function only in the bristles and may also be associated with alternative splicing of the D Pax2 transcript. We plan to assess the role of the carboxy terminal region of the protein by establishing transgenic lines bearing rescue constructs of D-Pax2 with either the complete coding sequence or with deletions of specific exons. To date, we have generated the first rescue construct bearing the complete coding region of the gene driven by a 3 KB upstream regulatory region of D-Pax2 and are currently generating transgenic fly lines with this construct.