TABLE 2.

Sequences of the primers used in this study

Primer name	Sequence (restriction enzyme)a	Purpose
enoup5	5′-TTGCGGCCGCTCAAGCACGGCGGTCTG-3′ (NotI)	PCR amplification of the upstream flanking DNA fragment of the enolase gene from A. hydrophila
enoup3	5′-CCAGATCTATGTGTATTTCCTCAGGT-3′ (BglII)
enodn5	5′-GTAGATCTATCGTCGCCGGTTCTCTTG-3′ (BglII)	PCR amplification of the downstream flanking DNA fragment of the enolase gene from A. hydrophila
enodn3	5′-TTTCTAGAGGATCCTCGGATCGGCGG-3′ XbaI
eno5	5′-GTAGTACTATGTCCAAGATCGTTAAAGTG-3′ (ScaI)	PCR amplification of the DNA fragment encoding the enolase gene from A. hydrophila for cloning into plasmid pBR322
eno3	5′-TCCTGCAGTTAAGCCTGGTTCTTCACTTC-3′ (PstI)
Sm5	5′-ATGCGCTCACGCAACTGGTC-3′	Identification of the deletion of the enolase gene on the chromosome of A. hydrophila
Sm3	5′-TTATTTGCCGACTACCTTGG-3′
ENT5	5′-CCTACAAGTCCGTCAACGAG-3′
ENT3	5′-ACGTGCAGCGCATTGAGCAC-3′
enoF	5′-CGCGGATCCATGTCCAAGATCGTTAAAGTGATCGG-3′ (BamHI)	Cloning of the native eno gene into pALTER-1 vector
enoR	5′-CCCAAGCTTTAAGCCTGGTTCTTCACTTCTTTCAG-3′ (HindIII)
enoK-Q343	5′-GCCAACTCCATCCTGATC(A)CAGTTCAACCAGATCGG-3′	Site-directed mutagenesis reactions
enoK-M394	5′-CCGCTGCTGGCCAGATCA(A)TGACCGGTTCCATGAGC-3′
enoK-L420	5′-AAGCCCTGGGTGCC(AA)TTGGCTCCGTTCCGCGGTCTG 3′
enoK-N427	5′-GGGTGCCAAGGCTCCGTTCCGCGGTCTGAA(A)TGAAG-3′
enoK-R430	5′-TCCGCGGTCTGAAAGAAGTGA(A)GGAACCAGGCTTAA-3′
enoN	5′-GGGTTTCATATGTCCAAGATCGTTAAAGTGATCGGTCGTG 3′ (NdeI)	Cloning of the enolase gene (native or mutated) into pET-30a(+) vector for hyperexpression
enoC	5′-CCCCTCGAGAGCCTGGTTCTTCACTTCTTTCAGACCGCGG 3′ (XhoI)
enoC427	5′-CCCCTCGAGAGCCTGGTTCTTCACTTC(T)ATTCAGACCGCGG-3′ (XhoI)
enoC430	5′-CCCCTCGAGAGCCTGGTTC(T)CTCACTTCTTTCAGACCGCGG-3′ (XhoI)

^aThe underlining indicates the restriction endonuclease site. Boldface indicates mutated nucleotide(s) in the enolase gene of A. hydrophila; the original nucleotide is shown in parentheses. All primers were developed for this study.