(A) Southern hybridization analysis of EcoRI-restricted genomic DNA of Tn5-mob insertion mutants probed with biotin-labeled pSUP5011. A biotin-labeled HindIII digest of phage λ DNA was included as the molecular size marker (not shown). Lanes: 1, KO2-1; 2, KO2-2. (B) Southern hybridization analysis probed with pKTn-1. Lanes: 1, KCT001 genomic DNA restricted with EcoRI; 2, plasmid pKTn-1 as the positive control. (C) Agarose gel electrophoresis analysis of endonuclease-restricted plasmid constructs. Lanes: 1, EcoRI-restricted pKTn-1; 2, BamHI-restricted pKTn-1; 3, BamHI-restricted pPM-1; 4, EcoRI- and BamHI-restricted pPM-2; 5, 1-kb DNA ladder. (D) Physical and genetic map of recombinant constructs. The circular map of pKTn-1 shows the insertion of the 9-kb EcoRI fragment (dashed circular line with arrow at the ends) of Tn5-mob flanked with genomic DNA cloned from the mutant KO2-1. Linear maps show the construction of the subclones pPM-1 and pPM-2. Thick black lines adjacent to the insert ends denote the multiple cloning site (restriction sites which were not used in this study are not shown), and the thin line denotes the part of the vector pBluescript. Grey, dotted, and empty boxes depict mob, Tn5, and genomic DNA of KO2-1, respectively. B, BamHI; E, EcoRI.

Mapping of Tn5-mob insertion mutations. (A) Agarose gel electrophoresis analysis of the PCR amplicons generated with different sets of primer combinations (in parentheses). Lanes: 1, 1-kb DNA ladder; 2, KCT001 (soxAF plus soxAR); 3, KO2-1 (soxAF plus TnG); 4, KO2-2 (TnG plus soxAR); 5, KO2-7 (soxAF plus TnG); 6, KO2-8 (soxAF plus TnG); 7, KO2-10 (soxAF plus TnG); 8, KO2-12 (soxAF plus TnG). (B) Southern hybridization analysis of PCR amplicons (description as given for panel A) probed with the 263-bp PCR product from wild-type strain KCT001. Lanes: 1, KCT001; 2, KO2-1; 3, KO2-2; 4, KO2-7; 5, KO2-8; 6, KO2-10; 7, KO2-12. The arrowhead indicates the hybridized band of the 263-bp PCR product from KCT001, which has been used as the probe. Size markers are not shown; compare relative migration in panel A. (C) Agarose gel electrophoresis analysis of the EcoRI-restricted PCR amplicons (description as given for panel A). Lanes: 1, KCT001; 2, KO2-1; 3, KO2-2; 4, KO2-7; 5, KO2-10; 6, KO2-12 (KO2-8 also generated the expected EcoRI restriction product, but this is not shown). The arrow indicates the shared 422-bp EcoRI product. (D) Southern hybridization analysis of SalI-restricted genomic DNA of Tn5-mob insertion mutants probed with pSUP5011. Lanes: 1, KO2-1; 2, KO2-2; 3, KO2-7; 4, KO2-8; 5, KO2-10; 6, KO2-12. (E) Map of six independent Tn5-mob insertion mutations in a sox locus of KCT001. The inset at the upper left corner is the restriction map of Tn5-mob. The open arrows indicate the position and direction of Tn5-specific primer TnG (not to scale). The insertion position of Tn5-mob in each mutant is shown with vertical arrows coming down from a triangle. The respective nucleotide position of the Tn5-mob insertion is noted along the line of the vertical arrow in landscape layout. The thick horizontal line of each triangle represents the Tn5-mob, and S is SalI. The number inside the triangle denotes the mutant strain as follows: 2, KO2-2; 1, KO2-1; 7, KO2-7; 10, KO2-10; 12, KO2-12; 8, KO2-8. The PCR amplicons (as described for panel A) are shown with thick horizontal bars aligned below the insertion map. The number below each bar indicates the size of the amplicon (in base pairs). The hollow ends indicate the part of the amplicon coming from the Tn5 sequence when the TnG primer was used.

(A) Nucleotide and deduced amino acid sequences of soxA. Arrow length denotes the respective primer sequences, and arrowheads indicate direction of extension from each primer. Two potential Shine-Dalgarno (SD) sequences, −10 and −35, of the putative promoter for soxA are shown. Downstream from soxA, another ORF with a potential SD sequence is shown. Start codons are shown in boldface. The 9-bp shared sequence of pPM-1 and pPM-2 is boxed. (B) Multiple alignment of KCT001 soxA-encoded protein (KCT) with the available partial SoxA sequence (PD) of P. denitrificans GB17 (EMBL X79242), a putative protein sequence (AE) of A. aeolicus (AE000757), and a protein sequence of cytochrome c₅₅₁ of C. limicola (CL [19]). Conserved residues are shaded in grey. The consensus heme-binding domains and the cleavage sites of signal sequences are shown within boxes and with arrowheads, respectively.