TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this work
| Strain, plasmid, or oligonucleotide | Relevant characteristics | Source or reference |
|---|---|---|
| Strains | ||
| Cp1015 | Wild type; str1 hexA | 12 |
| Cp8056 | str1 hexA nox K71−→stop codon | 1 |
| Plasmids | ||
| pAM239 | PBR322 derivative; ColE1 origin; lacZα selection | 8 |
| pLDH1 | PstI/XbaI fragment of pTSS7 (GenBank accession no. AF000336, positions 434 to 1103) cloned into pAM239 | This work |
| pLDH2 | LDH11-LDH12a PCR fragment of ldh cloned into pAM239 (PstI/XbaI) | This work |
| pTSS7 | Mutated PCR fragment (241G→T and 243T→A) of the ldh gene (GenBank accession no. AF000336, positions 233 to 1103) cloned into pBluescript (12a) | This work |
| pTSS13 | PCR fragment of the spxB gene (GenBank accession no. L39074, positions 23 to 1983) cloned into pBluescript (12a) | This work |
| Probes | ||
| nox | A 1,124-bp BamHI-EcoRI nox fragment from pNOX2 (1) (GenBank accession no. AF014458, positions 10 to 1133) | This work |
| ldh | An 847-bp HpaI-SpeI ldh fragment from pTSS7 (GenBank accession no. AF000336, positions 267 to 1103) | This work |
| spxB | A 1,206-bp HincII-HincII spxB fragment from pTSS13 (GenBank accession no. L39074, positions 475 to 1680) | This work |
| S16 | A 650-bp fragment from pP16 (GenBank accession no. X58312, positions 166 to 816) | 5 |
| gap | A 699-bp GAPD1-GAPD2 PCR fragmentb | |
| Oligonucleotide primers | ||
| GAPD1 | 5′-CCTGAGAAGCTTGCGTCAAACAATGAACCGTA | This work |
| HindIII | ||
| GAPD2 | 5′-CCACTGCTGCAGGGTCGTCTTGCTTTCCGTCG | This work |
| PstI | ||
| FPAM | 5′-CGAGCTCGGTACCCGGGG | This work |
| LDH11 | 5′-CCACACCTGCAGGGGTGAACACGGTGACTCTG | This work |
| PstI | ||
| LDH12 | 5′-CCACACTCTAGATGTGGGAAGATGTATAATGG | This work |
| XbaI |
The LDH11 primer was designed from the GenBank (accession number AF000336) sequence (positions 720 to 739), whereas the LDH12 sequence was retrieved from the 3836 contig sequence of the TIGR unfinished genome sequences database (positions 149 to 168 upstream of the stop codon of the ldh gene).
The gap gene was identified on the basis of its homology to the gapC gene of Streptococcus equisimilis. The gapC gene sequence from S. equisimilis (GenBank accession number Y12602) was used to search for homolog sequences into the TIGR unfinished genome sequences database. A 1,005-nucleotide sequence that showed 87% identity with the Streptococcus equisimilis gapC gene was retrieved in contig 3836.