Transposon mutagenesis of marine Vibrio spp

Abstract

MATERIALS AND METHODSReagents, enzymes, and DNA analysis. Antibiotics were purchased from Sigma Chemical Co., St. Louis, Mo., and were used at the following concentrations: tetracycline, 20 ,ug/ml for Escherichia coli and 10 ,ug/ml for Vibrio spp.; ampicillin, 80 ,ug/ml; chloramphenicol,20 ,ug/ml; kanamycin, 80 ,ug/ml. Restriction endonucleases were purchased from New England BioLabs, Inc., Boston, Mass. T4 DNA ligase was purchased from Bethesda Research Laboratories, Rockville, Md. Conditions for restriction and ligation of DNA followed the instructions of the manufacturer. ONPG (oto the arms of TnS-132 (pAl3105) was obtained from Alan Boyd, University of California, San Diego.Mutagenesis with Tn5. Transposon TnS-132 inserted into the genome of transducing phage P1 (P1 clrlOOCM) was obtained as a lysogen of E. coli C600 from D. Berg, Washington University. This transposon was constructed to encode tetracycline resistance in place of the wild-type kanamycin resistance (4, 16). Wild-type TnS could not be used, since Vibrio strains show considerable natural resistance to kanamycin. P1 lysates were prepared by temperature induction (5). The recipient bacterium, V. harveyi BB7 (from R. Belas, Agouron Institute), was grown in LM broth (10 g of tryptone [Difco], 5 g of yeast extract [Difco], and 20 g of NaCl per liter) to 5 x 108 cells per ml. CaCl2 was added to 25 mM, and P1 clrlOOCM::TnS-132 was added to a multiplicity of infection of 0.1. Cells were incubated at 30°C without shaking for 30 min to allow phage adsorption. The cell suspension was then diluted with 5 volumes of LM broth and incubated for 2 h at 30°C with shaking to permit expression of the tetracycline resistance phenotype. To select tetracy-on August 4, 2020 by guest

Behaviour of IncP-1 plasmids and a miniMu transposon in a marine Vibrio sp.: isolation of starvation inducible lac operon fusions

“…Twenty/zl of 1 mM sterile CaCI 2 solution and 100 t~l chloroform was added and after vortexing for 1 min the lysate was centrifuged at 8000 × g for 20 rain. The supernatant containing a portion of PlclrlOOCM transducing particles packaged with mini-Mu (Tet r) DNA [12], was transferred to a new tube and stored at 4"C.…”

“…Mu phage mutagenesis has successfully been used to obtain lac fusion mutants of E. coil and S. typhimurium and bacterial gene regulation studies have benefited greatly from analysis of such mutants ~7, [9][10][11]. Belas et al [12] previously reported the use of the PlclrlOOCM phage for mobilization of their newly constructed mini-Mu (Tet r) transposon into the genome of Fibrio parahaemolyticus. This transfer system was tested with Fibr/o sp.…”

“…In this paper we describe the use of a genetic transfer system, based on IncP-1 broad host range plasmids. An E. coli donor strain was constructed that was capable of conjugatively transferring the mini-Mu (Tet r) transposon [12] to Fibrio sp. S14 and other Gram-negative bacterial species.…”

Strain and dose infectivity ofVibrio parahaemolyticus: the causative agent of early mortality syndrome in shrimp

“…Three V. parahaemolyticus strains were used as follows: EMS 13-028/A3 (EMS) (Han, Tang, Pantoja, et al 2015;Han, Tang, Tran et al 2015), RIMD2210633 (RIMD) (Makino et al 2003) and LM5312 (LM) (Belas et al 1984;McCarter 1998). The bacteria were grown at 30°C in trypticase soy broth or on 1.5% trypticase soy agar (Becton Dickinson and Company, Spark, MD, USA) supplemented with an additional 2% NaCl (TSB2+) to make the overall salinity 25 parts per thousand (ppt) matching the salinity of the water used for housing the shrimp.…”

“…RIMD2210633 (RIMD) is a human clinical isolate obtained in 1996 from a patient with 'travellers' diarrhoea' at the Kansai International Airport, Japan (Makino, Oshima, Kurokawa, Yokoyama, Uda, Tagomori, Iijima, Najima, Nakano, Yamashita, Kubota, Kimura, Yasunaga, Honda, Shinagawa, Hattori & Iida 2003). LM5312 (LM), also known as BB22OP, is an environmental strain that was obtained from Bangladesh in the early 1980s (Belas, Mileham, Simon & Silverman 1984;McCarter 1998). In addition to the comparative strain analysis, pathogenicity of the EMS strain of V. parahaemolyticus to shrimp at different inoculation levels was examined to establish a disease challenge assay.…”