pylori as a signalling molecule

pylori as a signalling molecule selleckchem synthase. Methods Strains and growth culture conditions All strains used in this study

are SB-715992 in vitro listed in Table 1. DH5α was used in the production of proteins needed for AI-2 biosynthesis and cloning [21]. V. harveyi BB170 was used in the bioluminescence bioassay as a reporter strain [22]. E. coli strains were routinely grown in Luria-Bertani (LB) (Bacto) broth or on agar plates at 37°C. V. harveyi was grown in LB or AB medium [23] at 30°C, also under normal atmospheric conditions. H. pylori strains were routinely grown and maintained on Columbia blood agar plates (No.2, with 5% [v/v] horse blood; Oxoid) or grown in Brucella broth (BB) (Bacto) containing 7% (v/v) fetal bovine serum (Gibco). H. pylori J99 was incubated at 37 °C for 24 h to 72 h as required in a MG500 VAIN-cabinet (Don Whitley Scientific) in an atmosphere of 5% CO2, 86% N2, and 6% O2 (all v/v). For motility experiments the method of Wand et al. [24] was used to achieve motile cultures for analysis, see below. Antibiotics were used at the following concentrations: ampicillin at 100 μg/ml, kanamycin at 30 μg/ml. Table 1 Strains FK228 research buy and plasmids used in this study Strains/Plasmids Description Reference Strains     Vibrio harveyi     BB170 luxN :: Tn5 AI-1 sensor negative; AI-2 sensor positive [43] Escherichia coli

    DH5α endA1 recA1 gyrA96 thi-1 hsdR17(rk – mk +) relA1 supE44Δ( lacZYA-argF ) U169 F – Φ80d lacZ Δ M15 deoA phoA λ – [21] DH5α LuxS DH5α containing the plasmid pProEx-luxS EC PAK5 [8] DH5α Pfs DH5α containing the plasmid pProEx HT mtan [8] Helicobacter pylori     J99 (ATCC700824) Wild-type motile strain [44] J99 ΔluxS J99 derivative; ΔluxS :: km; Kmr [15] J99ΔluxS-F J99 derivative; ΔluxS :: km-sacB; Kmr Sucs This study J99 ΔluxS + J99ΔluxS-F derivative; ΔluxS :: km-sacB replaced with original luxS locus; Sucr Kms This study J99 ΔmccA J99 derivative; ΔmccA :: km; Kmr [15] J99 ΔmccB J99 derivative; ΔmccB :: km; Kmr [15] J99 ΔflhB J99 derivative; ΔHP0770 Lys13 to Glu347; Kmr; non-motile

[24] CCUG 17874* Wild-type strain [29] 17874 ΔflaA 17874 derivative; ΔflaA :: cat; Cmr Paul O’Toole 17874 ΔflgE 17874 derivative; ΔflgE :: km; Kmr [30] Plasmids     pGEMT Commercial TA cloning vector; Ampr Promega pGEMTluxSXN396 pGEM-T with inserted 26695 luxS; ΔluxS :: km-sacB; Sucs Kmr [17] pGEMTluxS pGEM-T with inserted full-length luxS fragment This study pProEx-luxS EC pProEX HT containing the luxS gene of E. coli MG1655 [8] pProEx HT mtan PProEX HT containing the pfs gene of E. coli [8] * CCUG 17874 is identical to the type strain NCTC 11637, isolated by B. J. Marshall at Royal Perth Hospital, May 1982 [29]. Molecular biology methods Preparation of plasmid DNA, DNA ligation, gel electrophoresis and transformation of E. coli strains were performed in accordance with standard methods [25]. All PCRs were performed with Taq DNA polymerase (Roche Diagnostics, Lewes, UK). TA cloning was carried out using the pGEM-T vector system (Promega, Madison, WI).

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