Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob.

RK2::Mu plasmids and transposon Tn5-Mob were used to mobilize the Legionella pneumophila chromosome. Plate matings between L. pneumophila donors that contained RK2::Mu plasmids and auxotrophic recipients yielded recombinants at frequencies ranging from 10(-6) to 10(-7) per recipient for the markers tested. The presence of a Mu insertion in the chromosome of donors that harbored RK2::Mu plasmids increased the frequency of chromosome transfer of certain selected markers as compared with strains that contained RK2::Mu alone. Cotransfer experiments with Mu-containing donors and a thymidine and tryptophan auxotroph failed to reveal any linkage between the thy and trp loci in L. pneumophila. A strain that contained a chromosomal Tn5-Mob insertion and helper plasmid pRK24.4 transferred chromosomal markers at frequencies of 10(-7) per recipient. These findings suggest that RK2::Mu plasmids and Tn5-Mob may be useful for genetic mapping experiments with L. pneumophila.     

Isolation and characterization of a conjugative plasmid from Legionella pneumophila.

The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied. To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe. The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L. pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli. Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L. pneumophila plasmids. There was no detectable DNA homology between pCH1 and L. pneumophila chromosomal DNA. Additional mating experiments revealed that pCH1 was unable to mobilize the L. pneumophila chromosome. The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis. These results suggest that pCH1 does not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.     

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: Inverse transposition

Summary Upon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.     

Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.     

Serospecific antigens of Legionella pneumophila.

Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria.     

Ecological distribution of Legionella pneumophila.

Bacteria were concentrated 500-fold from 20-liter water samples collected from 67 different lakes and rivers in the United States. The data suggest that Legionella pneumophila is part of the natural aquatic environment and that the bacterium is capable of surviving extreme ranges of environmental conditions. The data further demonstrate the effectiveness of the direct fluorescent-antibody technique for detecting L. pneumophila in natural aquatic systems. Smears of the concentrated samples were screened microscopically for serogroups of L. pneumophila by the direct fluorescent-antibody technique. Virtually all of the 793 samples were found to be positive by this method. The 318 samples containing the largest numbers of positive bacteria which were morphologically consistent with L. pneumophila were injected into guinea pigs for attempted isolations. Isolates were obtained from habitats with a wide range of physical, chemical, and biological parameters. Samples collected monthly from a thermally altered lake and injected into guinea pigs demonstrated a seasonality of infection, with the highest frequency of infection occurring during the summer months.     

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.     

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species.

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.     

Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

The rpsL gene of Escherichia coli was inserted into the BamHI site of transposon Tn5. This transposon was called Tn5-rpsL. Tn5-rpsL may be useful in microbiological studies when one wants to cure various bacterial genera of certain plasmid(s). A streptomycin-resistant (SmR) derivative of the host bacterial strain is first isolated. The plasmid(s) later to be cured are then labelled with Tn5-rpsL, which makes the cells Sm-sensitive. These cells can regain their resistance to Sm if they lose the Tn5-rpsL-tagged plasmid. Thus, plasmid-free bacteria are easily selected among SmR survivors. The frequency of occurrence of the plasmid-less variants of plasmid-containing wild-type Salmonella typhimurium measured by this method is given as an example.     

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.     

Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila

BACKGROUND: Legionella pneumophila is a gram-negative bacterial pathogen that is the cause of Legionnaires' Disease. Legionella produces disease because it can replicate inside a specialized compartment of host macrophages. Macrophages isolated from various inbred mice exhibit large differences in permissiveness for intracellular replication of Legionella. A locus affecting this host-resistance phenotype, Lgn1, has been mapped to chromosome 13, but the responsible gene has not been identified. RESULTS: Here, we report that Naip5 (also known as Birc1e) influences susceptibility to Legionella. Naip5 encodes a protein that is homologous to plant innate immunity (so-called "resistance") proteins and has been implicated in signaling pathways related to apoptosis regulation. Detailed recombination mapping and analysis of expression implicates Naip5 in the Legionella permissiveness differences among mouse strains. A bacterial artificial chromosome (BAC) transgenic line expressing a nonpermissive allele of Naip5 exhibits a reduction in macrophage Legionella permissiveness. In addition, morpholino-based antisense inhibition of Naip5 causes an increase in the Legionella permissiveness of macrophages. CONCLUSIONS: We conclude that polymorphisms in Naip5 are involved in the permissiveness differences of mouse macrophages for intracellular Legionella replication. We speculate that Naip5 is a functional mammalian homolog of plant "resistance" proteins that monitor for, and initiate host response to, the presence of secreted bacterial virulence proteins.     

Conjugation-mediated genetic exchange in Legionella pneumophila.

Genetic exchange mechanisms, to our knowledge, have not been reported for Legionella pneumophila, and consequently, studies on the genetic organization of L. pneumophila have not appeared in the literature. Here, we describe gene transfer mediated by broad host range conjugative plasmids in Legionella spp. Escherichia coli strains carrying plasmids RP1 and R68.45 (IncP1), S-a (IncW), and R40a (IncC), but not plasmids of incompatibility groups FI, FII, and FV, served as donors in matings with L. pneumophila Knoxville 1 (LPK-1). Transconjugants selected by resistance to kanamycin (RP1, R68.45, and S-a) and carbenicillin (R40a) were observed at frequencies of 6.6 X 10(-3), 4.7 X 10(-3), 2.2 X 10(-4), and 5.4 X 10(-5), respectively. Plasmid transfer was not affected by DNase added to the mating medium. After plasmid transfer, LPK-1 stably maintained RP1, R68.45, and S-a, but not R40a. Plasmid-containing LPK-1 isolates also served as donors in agar plate matings with E. coli W1485-1 and naladixic acid-resistant mutants of LPK-1, Legionella micdadei, and Legionella longbeachii. Recombinational exchange of a chromosomal trait was demonstrated when a thymidine auxotroph of L. pneumophila was repaired by R68.45-mediated chromosomal mobilization of a prototrophic donor strain.     

Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5.

We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5. Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti. The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in the same cell. The broad host range features of RK2 make this system of oriented chromosome mobilization applicable to most gram-negative bacteria.     

Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.     

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.     

Laboratory studies of disinfectants against Legionella pneumophila.

Legionella pneumophila suspended in tap water was exposed to biocides recommended for inhibiting biological growth in cooling towers and evaporative condensers of air-conditioning systems. Chlorine, 2,2-dibromo-3-nitrilopropionamide, and a compound containing didecyldimethylammonium chloride and isopropanol were effective in destroying concentratiois of 10(5) to 10(6) viable cells per ml. Formulations consisting of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, disodium ethylene bis(thiocarbamate) and sodium dimethyl dithiocarbamate, and a phenolic with pentachlorophenate and sodium salts of other chlorophenols were less effective.     

Genetic structure of populations of Legionella pneumophila.

The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources. Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. Principal coordinates and clustering analyses demonstrated that isolates received as L. pneumophila were a heterogeneous array of genotypes that included two previously undescribed species. For 50 ETs of L. pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources. Cluster analysis revealed four major groups or lineages of ETs in L. pneumophila. Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs. Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total. Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET. The genetic structure of L. pneumophila is clonal, and many clones apparently are worldwide in distribution. The fact that L. pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole.     

Growth of Legionella pneumophila in continuous culture.

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.