TABLE 2.
Bacterial strains and plasmids
| Plasmid or strain | Descriptiona | Source or reference |
|---|---|---|
| Plasmids | ||
| pJRD215 | Template for PCR amplification (primers 1 and 2) of kan (the aph-3 gene for Kmr without its transcriptional terminator) | 8 |
| EZ-TN5<DHFR-1> | Template for PCR amplification of dhfr (primers 3 and 4) (the gene for Tmr without its transcriptional terminator) | Epicentre |
| pDONR/Zeo | Cloning vector; Zeor | Invitrogen |
| p1FimQ | pDONR/Zeo containing a PCR-amplified (primers 5 and 6) region extending from upstream of strain T14V fimQ to the middle of fimQ | This study |
| p2FimQ | p1FimQ with an XbaI site created 317 bp upstream of fimQ using a QuikChange site-directed mutagenesis kitb and primers 7 and 8 | This study |
| p3FimQ | p2FimQ with kan in the XbaI site | This study |
| p4FimQ | pDONR/Zeo containing a PCR-amplified (primers 5 and 9) region extending from upstream of strain T14V fimQ to the ATG starting codon of fimQ | This study |
| p5FimQ | p4FimQ with an XbaI site created at the same position as in p2FimQ by site-directed mutagenesis (primers 7 and 8) | This study |
| p6FimQ | p5FimQ with kan in the XbaI site | This study |
| p7FimQ | pDONR/Zeo containing a PCR-amplified (primers 10 and 11) region of strain T14V fimQ corresponding to nucleotides 1066 to 2348 of the complete gene | This study |
| p8FimQ | p7FimQ with the NdeI-HpaI fragment replaced with an NdeI-HpaI fragment from p6FimQ, resulting in a 1,063-bp deletion of fimQ (from ATG starting codon) | This study |
| p1Srt | pDONR/Zeo containing a PCR-amplified (primers 12 and 13) srtC1 region of strain T14V | This study |
| p2Srt | p1Srt with an XbaI site created 42 bp upstream of the 3′ end of srtC1 by site-directed mutagenesis (primers 14 and 15) | This study |
| p3Srt | p2Srt with kan in the XbaI site | This study |
| p4Srt | p2Srt with srtC1 deleted (all coding sequence except the 42 bp at the 3′ end) by inverse PCR (primers 16 and 17); the resultant plasmid contains an XbaI site at the same position as in p2Srt | This study |
| p5Srt | p4Srt with kan in the XbaI site | This study |
| p6Srt | p2Srt with dhfr in the XbaI site | This study |
| p1OrfC | pDONR/Zeo containing a PCR-amplified (primers 18 and 19) orfC region of A. naeslundii T14V | This study |
| p2OrfC | p1OrfC with an XbaI site created 6 bp downstream of the 3′ end of orfC by site-directed mutagenesis (primers 20 and 21) | This study |
| p3OrfC | p2OrfC with kan in the XbaI site | This study |
| p4OrfC | p2OrfC with orfC deleted (all coding sequence except the first 12 bp from the 5′ end) by inverse PCR (primers 22 and 23); the resultant plasmid contains an XbaI site at the same position as in p2OrfC | This study |
| p5OrfC | p4OrfC with kan in the XbaI site | This study |
| A. naeslundii strains | ||
| T14V | Wild-type strain; Kms Smr | 5 |
| FimQ-Km | Kmr transformant of T14V obtained with p3FimQ | This study |
| ΔFimQ | Kmr transformant of T14V obtained with p8FimQ | This study |
| SrtC1-Km | Kmr transformant of T14V obtained with p3Srt | This study |
| ΔSrtC1 | Kmr transformant of T14V obtained with p5Srt | This study |
| SrtC1-DHFR | Tmr transformant of ΔSrtC1 obtained with p6Srt | This study |
| OrfC-Km | Kmr transformant of T14V obtained with p3OrfC | This study |
| ΔOrfC | Kmr transformant of T14V obtained with p5OrfC | This study |
a
The sequences of all primers used in the present study are listed in Table S1 in the supplemental material. Antibiotic abbreviations: Km, kanamycin; Sm, streptomycin; Tm, trimethoprim; Zeo, zeocin.
b
From Stratagene, La Jolla, CA.