FIG. 2.

(A) Line diagram depicts the DNA sequence determined from sequencing pIMVS27. The solid box shows the mip gene from L. longbeachae serogroup 1 ATCC 33462, selected restriction sites in the mip gene, and the stem loop structure at the end of the ORF. The inset sequence is the DNA sequence upstream of the ATG start site for translation in L. longbeachae serogroup 1 and L. pneumophila serogroup 1, showing the −10 and −35 promoter regions determined by primer extension analysis. The shaded box indicates the −35 promoter region proposed for L. longbeachae. The nonshaded box is the −35 region proposed in reference 16. The start site for transcription is shown with a solid arrow. (B) Southern hybridization demonstrating mutagenesis by allelic exchange of the L. longbeachae serogroup 1 mip gene. DNA was digested with KpnI and probed with DIG-labeled pIMVS27. Lanes: a, L. longbeachae serogroup 1 A5H5; b, B8. The solid arrow indicates the 7.3-kb fragment generated in B8 due to the loss of an internal KpnI site which generates 1- and 7-kb fragments in the parent strain. A similar pattern was observed for L. longbeachae serogroup 1 ATCC 33462 (data not shown).