Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or thenpt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when thegfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed toN′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.

To understand the basis of pathogenesis by Legionella longbeachae serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned L. longbeachae serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an Escherichia coli background. DNA sequence analysis of plasmid pIMVS27, containing the entire L. longbeachaeserogroup 1 mip gene, revealed a high degree of homology to the mip gene of Legionella pneumophilaserogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both species, with some differences observed for the −10 and −35 promoter regions. Primers designed from the mip gene sequence obtained forL. longbeachae serogroup 1 ATCC 33462 were used to amplify the mip genes from L. longbeachaeserogroup 2 ATCC 33484 and an Australian clinical isolate of L. longbeachae serogroup 1 A5H5. The mip gene from A5H5 was 100% identical to the type strain sequence. The serogroup 2 strain of L. longbeachae differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic mip mutant in ATCC 33462 and strain A5H5. The ATCCmip mutant was unable to infect a strain ofAcanthamoebae sp. both in liquid and in a potting mix coculture system, while the A5H5 mip mutant behaved in a manner siilar to that of L. pneumophilaserogroup 1, i.e., it displayed a reduced capacity to infect and multiply within Acanthamoebae. To determine if this mutation resulted in reduced virulence in the guinea pig animal model, the A5H5 mip mutant and its parent strain were assessed for their abilities to establish an infection after aerosol exposure. Unlike the virulent parent strain, the mutant strain did not kill any animals under two different dose regimes. The data indicate that the Mip protein plays an important role in the intracellular life cycle ofL. longbeachae serogroup 1 species and is required for full virulence.

All Legionella longbeachae strains, both serogroups of L. bozemanii, and three strains of L. anisa reproducibly infected washed Tetrahymena pyriformis at 30؇C. L. pneumophila serogroup 1 strains infected T. pyriformis less reproducibly than did L. longbeachae. Low-level concentrations of nutrients in cocultures inhibited infection. Four L. micdadei strains and L. anisa ATCC 35292 failed to infect T. pyriformis.

A flagellum-negative mutant, M8.2, of the marine bacterium Vibrio sp. S141 was produced by transposon mutagenesis. Time-lapse video imaging of surface colonisation behaviour and microcolony formation of S141 compared to M8.2 cells was carried out to investigate the role of the flagellum of Vibrio sp. S141 in microcolony formation on agar and glass substrata. On an agar surface, S141 cells formed a tetrad pattern after the first two cell divisions, during initial surface colonisation. Developed microcolonies consisted of tight circular arrangements of cells with infrequent branching of cells from the main body. In contrast, M8.2 cells did not form tetrad patterns and micro-colonies generally showed enhanced branching and did not develop circular arrangements of cells. On a glass surface under flow conditions, S141 cells displayed several types of movement behaviours at the surface which may have assisted microcolony formation. M8.2 cells appeared unable to develop micro-colonies, but rather displayed a behaviour which enabled them to spread out across the substratum. Laser scanning confocal microscopy revealed S141 mature biofilms consisted of characteristic towers of bacterial growth with scattered troughs. The flagellum-negative M8.2 biofilm did not form such architecture, displaying a homogeneous distribution of cells throughout the biofilm and across the entire substratum. Although not required for attachment to the glass substratum, the flagellum was required for alignment as well as specific movement behaviours by S141 cells.