Contrary to expectations, the DNA binding activity of PriB shows little preference for specific DNA structures (Figure 2). The apparent dissociation constants range from 628 ± 95 nM (3′ Overhang) to 690 ± 51 nM (Fork 2), and the observed differences among apparent dissociation constants for the various DNA structures are insignificant given the experimental uncertainty of the

measurements (Table 2). This observation, together with the low affinity with which N. gonorrhoeae PriB binds DNA relative to E. coli PriB, suggests that the surface of this PriB homolog might have been adapted for a purpose other than binding ssDNA. Furthermore, it raises the important question of whether N. gonorrhoeae PriB can stimulate its cognate PriA’s helicase

LY2835219 order activity, since in E. coli this stimulatory effect depends on PriB’s strong ssDNA-binding activity [7]. Figure 2 DNA binding activity of N. gonorrhoeae PriB. PriB was serially diluted and incubated with 1 nM fluorescein-labeled ssDNA (squares), dsDNA (closed diamonds), 3′ Overhang (circles), or Fork 2 (triangles). Measurements are reported in triplicate and error bars represent one standard deviation of the mean. PriA helicase activity is limited to short stretches of duplex DNA To test the functional consequences of N. gonorrhoeae PriB’s weak DNA binding activity, we first had to examine N. gonorrhoeae PriA’s helicase activity. We used the partial duplex and forked DNA structures shown in Table 1 as substrates based on extensive www.selleckchem.com/products/GDC-0449.html studies of substrate preference

and helicase activity of E. coli PriA [22, 28, 29]. For each of these substrates, the fluorescein-labeled check details strand represents the nascent lagging strand arm, and the degree of duplex DNA unwinding of the fluorescein-labeled strand was determined using fluorescence polarization spectroscopy. For these experiments, the DNA substrates were incubated with PriA and ATP for 10 min at 37°C, the reactions were terminated by addition of SDS, and the fluorescence polarization of Cediranib (AZD2171) the samples was measured. The degree of unwinding was determined by comparing the fluorescence polarization of the samples to that of the DNA substrate incubated in buffer alone (fully intact DNA substrate) and to the samples heated briefly to 95°C and fast-cooled back to 25°C (fully denatured DNA substrate). This allowed us to measure the fraction of each DNA substrate that is unwound by various concentrations of PriA. Of the DNA substrates examined, PriA shows greatest unwinding activity on forked DNA substrates with relatively short duplex lagging strand arms. Levels of maximal unwinding are approximately 83% for Fork 1 (15 bp lagging strand arm), 70% for Fork 2 (25 bp lagging strand arm), and 42% for Fork 3 (40 bp lagging strand arm) (Figure 3).

The microarray data have been deposited in the NCBI Gene Expression Ommibus (http://​www.​ncbi.​nlm.​nih.​gov/​gds/​) and the accession number is GSE43026. Quantitative real-time RT-PCR A quantitative real-time RT-PCR (qRT-PCR) was used to confirm the expression levels of representative genes that were identified as differentially expressed by the microarray. Briefly, reactions were performed using the iQTM SYBRR Green Super Mix (Bio-Rad,

Hercules, CA) and MyiQTM instrument (Bio-Rad). Primers were designed by Primer 3 software (http://​frodo.​wi.​mit.​edu/​) and are listed in Table 6. The 16S rRNA transcript was used to normalize target gene expression. Amplification efficiency and relative transcript abundance (R) were calculated as previously described [37]. R values were log2 transformed to meet

assumptions of normality and variance; statistical significance was determined by the two #this website randurls[1|1|,|CHEM1|]# tailed Student’s t-test under the null hypothesis of R = 0. Construction and complementation of insertional mutants Isogenic C. jejuni NCTC 11168 mutant strains with a disrupted copy of cj0309c-cj0310c, cj0423-cj0425, cj1169c-cj1170c, or cj1173-cj1174 genes were constructed by insertional mutagenesis with antibiotic resistance cassettes. The strategies are shown in Figure 1. Primers used in the construction and complementation of mutants are listed in Table 6. The chloramphenicol (cat) and kanamycin (aphA-3) resistance cassettes were PCR amplified using however Ex-Taq (Takara see more Bio Inc.) from plasmids pUOA18 and pMW10 with cat and aphA3 primers, respectively, as described in a previous study [38]. PCR products were digested with the appropriate restriction enzymes (Table 6, Figure 1). The PCR products and a resistance cassette

were ligated by T4 DNA ligase (Promega, Madison, WI), cloned into suicide vector pUC19 (Invitrogen, Carlsbad, CA), and transformed into competent E. coli DH5α (Invitrogen). Recombinant clones with the intended mutation were confirmed by PCR. Plasmids were extracted from DH5α and used to transform wild-type NCTC 11168 by the standard biphasic method for natural transformation [39]. Transformants were colony purified on MH plates with supplemented antibiotics. Single colonies were selected and confirmed by PCR. Mutations were complemented by inserting the entire set of the wild-type copy of genes between the structural genes of the ribosomal gene cluster in the corresponding mutant strains as described previously [37, 40]. PCR amplification and sequencing were performed on positive clones to confirm no mutations occurred in the cloned sequences. All strains were stored at −80°C for later use. Oxidative stress tests To determine if the mutated genes affected the susceptibility of C. jejuni to oxidative stress, wild-type NCTC 11168 and mutant strains (KO39Q、KO73Q、KO425Q、KOp50Q and DKO01Q) were compared using two oxidative stress tests.

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