Novel conjugative transferable multiple drug resistance plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from marine aquaculture environment

Abstract

The emergence of drug-resistant bacteria is a severe problem in aquaculture. The ability of drug resistance genes to transfer from a bacterial cell to another is thought to be responsible for the wide dissemination of these genes in the aquaculture environment; however, little is known about the gene transfer mechanisms in marine bacteria. In this study, we show that a tetracycline-resistant strain of Photobacterium damselae subsp. damselae, isolated from seawater at a coastal aquaculture site in Japan, harbors a novel multiple drug resistance plasmid. This plasmid named pAQU1 can be transferred to Escherichia coli by conjugation. Nucleotide sequencing showed that the plasmid was 204,052 base pairs and contained 235 predicted coding sequences. Annotation showed that pAQU1 did not have known repA, suggesting a new replicon, and contained seven drug resistance genes: bla(CARB-9)-like, floR, mph(A)-like, mef(A)-like, sul2, tet(M) and tet(B). The plasmid has a complete set of genes encoding the apparatus for the type IV secretion system with a unique duplication of traA. Phylogenetic analysis of the deduced amino acid sequence of relaxase encoded by traI in pAQU1 demonstrated that the conjugative transfer system of the plasmid belongs to MOB(H12), a sub-group of the MOB(H) plasmid family, closely related to the IncA/C type of plasmids and SXT/R391 widely distributed among species of Enterobacteriaceae and Vibrionaceae. Our data suggest that conjugative transfer is involved in horizontal gene transfer among marine bacteria and provide useful insights into the molecular basis for the dissemination of drug resistance genes among bacteria in the aquaculture environment.

Fig. 1

Detection of plasmid pAQU1 in the donor and a representative transconjugant using PFGE (A) and Southern hybridization with the tet(M) probe (B). Lanes: M, DNA size standard (lambda ladder); 1, representative transconjugant TJ311W2; 2, E. coli W3110; 3, P. damselae subsp. damselae 04Ya311; and 4, negative control strain of P. damselae subsp. damselae JCM8967. About 20 ng of DNA was loaded in each lane.

Fig. 2

Circular map of pAQU1. CDSs outside and inside of the circle are coded clockwise and counter-clockwise, respectively. Putative functions of the products of the CDSs are indicated in color: red, replication, partition and termination; purple, DNA processing; blue, conjugative transfer; green, transposition or integration; orange, antibiotic resistance; yellow, other functions; and gray, unknown functions. The third circle indicates GC content where purple shows upper GC value above the center line and gray shows lower GC value below the center line.

Fig. 3

DNA sequence of the potential replication origin region. CDSs are boxed. Arrows below the sequences indicate the 16 to18-bp AT-rich direct repeats. Inverted repeats of 15 and 21 bases are indicated by dashed lines.

Fig. 4

Alignment of TraA homologues. Conserved amino acids are indicated by gray boxes.

Fig. 5

Phylogenetic tree of relaxases (TraI) from plasmids and SXT/R391 that belong to the MOB_H family.

Fig. 6

Conserved sequence motifs in relaxases (TraI) grouped in the MOB_H family. Both of the 3H and HD hydrolase motifs indicated by gray boxes are conserved in the relaxase encoded by pAQU1.