5 M sucrose+10 mM potassium phosphate, and (5) 272 mM sucrose+7 mM sodium phosphate+1 mM MgCl2. Electroporation was performed using a Bio-Rad Gene Pulser with field strength settings from 5 to 20 kV cm−1 and a Bio-Rad Pulse Controller Plus with resistance settings of 200–400Ω. A 2.1-kb fragment containing the rpsL gene was generated by PCR with primers rpsLup-F and rpsLdn-R (Table 2 and Fig. 1), using genomic DNA of the spontaneous streoptomycin resistance mutant (SR1) as template. The PCR amplicon was cloned into the pGEM-T easy

vector (Promega) to generate pSR1-rpsL. To introduce the silent CYC202 mouse point mutations used to identify true transformants, plasmid pSR1-rpsL was used as template for inverse PCR using the phosphorylated primers

rpsL-WM-F (containing the silent point mutations) and rpsL-WM-R (Table 2 and Fig. 1). Then the PCR reaction was purified and digested with DpnI to eliminate the template plasmid pSR1-rpsL. The PCR fragment, which actually was a linearized plasmid, was then self-ligated and transformed into E. coli. The plasmid containing the expected silent point mutations was confirmed by sequencing and designated as pWM-rpsL. Using the resulting plasmid as template, the 2.1-kb fragment with the introduced point mutations was generated with primer INCB024360 datasheet pair rpsLup-F/rpsLdn-R (Table 2 and Fig. 1). It has been reported in other bacteria that spontaneous mutations in the rpsL gene can confer streptomycin resistance (Shima et al., 1996; Bjorkman et al., 1998; Barnard et al., 2010). To generate a selective marker for testing the transformability of V. parvula PK1910, we isolated spontaneous streptomycin-resistant mutants and sequenced the rpsL gene of these mutants. From the Isotretinoin eight

clones randomly selected for sequencing, all carried a single point mutation at codon 43 of the rpsL gene, among which five had a change from AAG to AAC (named SR1) while three from AAG to AAT (named SR2). These mutations resulted in exactly the same substitution of the wild-type lysine (K) by asparagine (N) at codon 43. This indicates that it is the K43N mutation in RpsL that confers streptomycin resistance in V. parvula. Analysis of the draft sequence of V. parvula PK1910 revealed a type I restriction system, suggesting a potential transformation barrier for foreign DNA. Thus, to avoid complications with the restriction system, we chose to use the chromosomal DNA from the isogenic rpsL mutant strain as transforming DNA to optimize transformation conditions. Several factors have been reported to affect the efficiency of electroporation-mediated transformation, including cultivation conditions, composition of the electroporation buffer, and electroporation conditions.