Structure of the als operon and location of als-lacZ⁺ and yjcT-lacZ⁺ insertions. Mutagenization of the alsRBACE operon and yjcT was performed as follows. Strain CC118 (Δlac) harboring pTP680 (alsR⁺B⁺A⁺C⁺E⁺ yjcT⁺) or pHO390 (yjcT⁺) was infected with λ::TnphoA′-1 (12). TnphoA′-1 contains a promoterless lacZ⁺ allele, and a β-galactosidase-producing mutant has acquired an operon fusion. Mutants were selected as kanamycin resistant. Plasmid DNA was isolated and transformed back into strain CC118 and screened for production of β-galactosidase activity by the presence of 5-bromo-4-chloro-3-indolyl galactoside (40 mg liter⁻¹). Insertion of a transposon into the als operon was ascertained by restriction endonuclease analysis. Allele replacement of plasmid-harbored als::TnphoA′-1 or yjcT::TnphoA′-1 mutations and the chromosomal als or yjcT genes was performed by homologous recombination. Restriction endonuclease-linearized plasmid DNA was transformed into an recD strain (TP1904) by selection for kanamycin resistance. Genetic mapping ensured the location of the inserted DNA at 92.8 min on the linkage map. Recombinational switching among transposons was performed as previously described (21). The insertion of TnphoA′-1 generated polar mutations. A recombinational switching, using TnphoA-132 (encoding tetracycline resistance) followed by Tn5-112 (encoding kanamycin resistance), resulted in the isolation of a nonpolar version of each als allele or yjcT8, essentially by removing a transcription terminator located within the right-hand IS50 element of the transposon. The plasmids used were pTP680, which contained a wild-type version of the alsRBACE operon and yjcT in a 7.8-kb DNA fragment of chromosomal origin in pUC19 (22), or pHO390, which contained a PCR-amplified wild-type yjcT allele ligated to the BamHI site of pBR322 (4). The inserted yjcT sequence was confirmed by sequencing. (A) Boxes indicate open reading frames of the alsI and alsRBACE operons and yjcT. Staggered boxes indicate open reading frames with possible overlapping translation (alsA and alsC, and alsE and yjcT). Shaded boxes indicate intercistronic regions. The angled arrows indicate the transcription initiation points before the alsI and alsR cistrons (20). Vertical arrows above the boxes indicate the positions of insertions of als-lacZ⁺ or yjcT-lacZ⁺ operon fusions. The alsI-lacZ⁺ fusion was generated by in vitro techniques (Fig. 2). The presumed gene product of each cistron is indicated below the bar. The plasmids constructed were pTP908 (yjcT8::TnphoA′-1), pTP911 (alsE11::TnphoA′-1), pTP919 (alsE19::TnphoA′-1), pTP922 (alsE22::TnphoA′-1), pTP924 (alsE24::TnphoA′-1), and pTP925 (alsR21::TnphoA′-1), which were isolated from pTP680; and pTP926 (yjcT26::TnphoA′-1), pTP927 (yjcT27::TnphoA′-1), and pTP928 (yjcT28::TnphoA′-1), which were isolated from pHO390. (B) Nucleotide sequence of the points of insertion of TnphoA′-1. Sequencing was performed at the Botanical Institute, University of Copenhagen, in an Applied Biosystems model 377 sequencer by cycle sequencing with dye terminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit; Perkin-Elmer) and with the oligodeoxyribonucleotide 5′-GCAGTAATTTCCGAGTCCC-3′ as a primer (Hobolth DNA Syntese, Hillerød, Denmark). A vertical arrow indicates an insertion point. Nucleotides to the left of the arrow originate from the als or yjcT cistrons. Nucleotides to the right of the arrow originate from TnphoA′ sequences. The codon where insertion occurred is indicated together with the nucleotide position of insertion. The latter numbers refer to the nucleotide sequence reported in the database sequence under accession no. AE00482 (3).

Construction of an alsI-lacZ⁺ gene fusion. Open reading frames are indicated by open double lines, vector sequences are indicated by thin lines, and flanking DNA sequences or intercistronic regions are shown as black or shaded double lines. Relevant restriction endonuclease recognition sites are included. (A) The plasmid pKIS212 contains the alsR and alsI genes (20). The plasmid pRS415 contains a promoterless lac operon, which includes a wild-type lacZ gene with translation initiation sequences. Intercistronic regions, which are not drawn to scale, are shown in black. The shaded region contains part of the trp operon as well as the original W205 fusion (17). (B) Construction of a plasmid-borne gene fusion. DNA of pKIS212 was digested with restriction endonuclease BstEII, followed by incubation with the large fragment of E. coli DNA polymerase I in the presence of the four deoxyribonucleoside triphosphates and digestion with BclI. Plasmid pRS415 was digested with endonucleases SmaI and BamHI. The two DNA species were ligated. Transformation followed by selection for ampicillin resistance in the presence of 5-bromo-4-chloro-3-indolyl galactoside to screen for β-galactosidase synthesis resulted in the isolation of pYYC205. The insert of pYYC205 contained 129 nucleotides of the N-terminal encoding end of the alsR reading frame, the 358 nucleotides of the alsR-alsI intercistronic region (cross-hatched), and 28 nucleotides of the N-terminal encoding end of the alsI reading frame. P_alsI indicates the promoter driving transcription of the alsI gene, and an angled arrow indicates the transcription initiation point. alsR′ and alsI′ indicate deletion of the C-terminal encoding ends of the alsR and alsI genes, respectively. The various DNA elements of pYYC205 are not drawn to scale. (C) Isolation of a bacteriophage λ-borne gene fusion by homologous recombination. Bacteriophage λRS45 contains a version of the lacZ cistron that is deleted for the promoter-proximal two-thirds (ΔlacZ_SC), wild-type versions of the lacY and lacZ cistrons, and a truncated bla gene (bla′). Thus, λRS45 forms white plaques, and lysogens of λRS45 form white colonies in the presence of 5-bromo-4-chloro-3-indolyl galactoside. The lac-bla sequence of λRS45 is homologous to sequences of pYYC205. Consequently homologous recombination (indicated by X) which occurred among plasmid and bacteriophage replicons within the bla sequence and within the lac sequence resulted in the formation of a bacteriophage λ genome carrying the gene fusion. Host strain P90C harboring pYYC205 was infected with λRS45 to allow recombination and to generate a lysate. Strain P90C was infected with this lysate and plated on NZY broth containing 5-bromo-4-chloro-3-indolyl galactoside. Blue plaques, which appeared at a frequency of approximately 4 × 10⁻³, were restreaked, and one isolate, λYYC205, was kept for further analysis. Insertion of the prophage at the attλ site at 17 min, rather than at alsI at 92.8 min on the linkage map, was confirmed by genetic mapping.