Mating Medium Research Articles

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

The iron-responsive, GATA-type transcription factor Cir1 influences mating in Cryptococcus neoformans.

Mating and sexual development have been associated with virulence in various fungal pathogens including Cryptococcus neoformans. This fungus is a significant pathogen of humans because it causes life-threatening cryptococcal meningitis in immunocompromised people such as AIDS patients. The virulence of C. neoformans is known to be associated with the mating type of the cells (α or a), with the α mating type being predominant among clinical isolates. However, the mechanisms by which mating and sexual development are controlled by environmental conditions and their relationship with virulence require further investigation. Cir1 is a GATA-type transcription factor that regulates the expression of genes required for utilization of essential metals such as iron and copper, and also genes required for major virulence factors including the polysaccharide capsule and melanin. Here we investigated the role of Cir1 in the mating of C. neoformans. Our results demonstrate that mutants lacking CIR1 are defective in mating, and that Cir1 contributes to copper mediated enhancement of sexual filamentation. Furthermore, we found that Cir1 influences the expression of mating pheromone genes suggesting that this protein plays a role in the early phase of sexual development on V8 mating medium.

Mating Pheromone in Cryptococcus neoformans Is Regulated by a Transcriptional/Degradative “Futile” Cycle

Sexual reproduction in fungi requires induction of signaling pheromones within environments that are conducive to mating. The fungus Cryptococcus neoformans is currently the fourth greatest cause of infectious death in regions of Africa and undergoes mating in phytonutrient-rich environments to create spores with infectious potential. Here we show that under conditions where sexual development is inhibited, a ∼17-fold excess of MFα pheromone transcript is synthesized and then degraded by a DEAD box protein, Vad1, resulting in low steady state transcript levels. Transfer to mating medium or deletion of the VAD1 gene resulted in high level accumulation of MFα transcripts and enhanced mating, acting in concert with the mating-related HOG1 pathway. We then investigated whether the high metabolic cost of this apparently futile transcriptional cycle could be justified by a more rapid induction of mating. Maintenance of Vad1 activity on inductive mating medium by constitutive expression resulted in repressed levels of MFα that did not prevent but rather prolonged the time to successful mating from 5-6 h to 15 h (p < 0.0001). In sum, these data suggest that VAD1 negatively regulates the sexual cell cycle via degradation of constitutive high levels of MFα transcripts in a synthetic/degradative cycle, providing a mechanism of mRNA induction for time-critical cellular events, such as mating induction.

Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics

The conjugation protocols in myxobacterium Sorangium cellulosum are often inapplicable due to the strain-specific sensitivity to the presence of Escherichia coli cells or the resistances to many antibiotics. Here we report that the conjugative transfer of the mobilizable plasmid pCVD442 from E. coli DH5alpha (lambda pir) to Sorangium strains could be greatly increased by the presence of low doses of dual selection antibiotics in the mating medium. The improvement was efficient in either E. coli-tolerant or sensitive Sorangium strains. For those phleomycin and hygromycin tolerant Sorangium strains, chloramphenicol-resistance gene was developed as a new selectable marker by driving the resistance gene with the aphII promoter. Using the improved protocol, the epothilone biosynthetic pathway was inactivated by an insertion mutation in the biosynthetic genes of the producing Sorangium strains.

Mucilage secretion regulated by sex pheromones in Closterium peracerosum-strigosum-littorale complex.

When mating-type plus (mt+) and minus (mt-) cells of the Closterium peracerosum-strigosum-littorale complex were mixed in nitrogen-depleted mating medium, secretion of mucilage containing uronic acid from cells was markedly activated and the mucilage accumulated around the cells. Substances with the ability to stimulate mucilage secretion from mt+ and mt- cells were detected in media in which mt- and mt+ cells had been separately cultured, respectively. We designated the active substances secreted from mt+ and mt- cells mucilage secretion-stimulating pheromone (MS-SP)-plus and MS-SP-minus, respectively. Activity of MS-SP-plus and MS-SP-minus decreased to 20% level by incubation at 80 degrees C for 10 min. Light was indispensable for the secretion of mucilage. The secretion of MS-SP-plus and MS-SP-minus decreased with dark treatment. MS-SP-plus eluted at around 95 k from a gel filtration column, and reacted with antibodies against two subunits of protoplast-release-inducing protein (PR-IP), which induces protoplast release from mt- cells. MS-SP-minus eluted at around 20 k from a gel filtration column, and reacted with an antibody against the PR-IP inducer, which induces the secretion of PR-IP from mt+ cells. In addition, purified PR-IP and PR-IP inducer stimulated mucilage secretion from mt- and mt+ cells, respectively. These results strongly suggested that MS-SP-plus and MS-SP-minus were the same molecules as the PR-IP and the PR-IP inducer, respectively.

Genetic transfer of IncP, IncQ and IncW plasmids to four Thiobacillus ferrooxidans strains by conjugation

The physiological function of some proteins involved in Thiobacillus ferrooxidans energetic metabolism has been difficult to assess. Our long-term goal is to construct and to analyse mutants in which these proteins are no longer synthesized in order to determine in which respiratory chains they are involved. However, up to now, there is no report on the construction of mutants in this microorganism because of the lack of genetic tools. Recently, the transfer by conjugation from Escherichia coli of IncP plasmids and by mobilization of IncQ plasmids into private T. ferrooxidans strains has been reported, but the transfer frequency was very low. We have extended this research by establishing a genetic transfer system by conjugation of IncQ (pJRD215), IncP (pJB3Km1) and IncW (pUFR034) group plasmids from E. coli not only to two private T. ferrooxidans strains (BRGM1 and Tf-49), but more interestingly, to the two collection strains ATCC33020 and ATCC19859, from which most of the T. ferrooxidans genes characterized to date, originate. IncQ plasmid pJRD215 had the higher transfer frequency and was stable in these four T. ferrooxidans strains. The conjugation conditions were optimized for the ATCC33020 strain by analysing the influence on the transfer frequency of: (i) the donor strain; (ii) the plasmid to be transferred; (iii) the ratio of donor/recipient cells; (iv) the adaptation of the donor and the recipient cells to the mating medium; (v) the composition of the mating medium. Under the best conditions, the transfer frequency could be increased from 10 −7 to 5×10 −5.

A novel quantitative mating assay for the fungal pathogen Cryptococcus neoformans provides insight into signalling pathways responding to nutrients and temperature.

Cryptococcus neoformans is a fungal pathogen that causes a lethal meningitis in immunocompromised individuals. Several factors are associated with virulence of this fungus, including its mating type; however, the mechanism by which mating type affects virulence is unknown. C. neoformans is a basidiomycete that exists in two mating types called a and alpha that can fuse to form an a/alpha dikaryon. A mating assay was developed that allowed a quantitative analysis of cryptococcal mating physiology. Interestingly, the efficiency of mating appeared to be dependent on temperature, being highest at 30 degrees C and almost completely absent at 37 degrees C. Thus, while mating type itself may be associated with virulence (which must occur at 37 degrees C), the ability to mate is probably not a virulence factor. Mating efficiency was increased by altering the carbon or nitrogen sources to give so-called starvation media. The addition of various drugs also seemed to alter the frequency of mating, depending on the composition of mating medium. The data suggested that cAMP, 8-bromo-cAMP and caffeine increased mating on starvation medium but only cAMP and 8-bromo-cAMP stimulated mating on rich medium; caffeine was unable to stimulate mating on rich medium. Aluminium fluoride, an activator of heterotrimeric GTP-binding proteins (G-proteins), was also found to stimulate mating, suggesting the involvement of a G-protein that may regulate the level of cAMP.

Host Range, Stability and Compatibility of Broad Host-Range-Plasmids and a Shuttle Vector in Moderately Halophilic Bacteria. Evidence of Intrageneric and Intergeneric Conjugation in Moderate Halophiles

Broad-host-range plasmids, belonging to the incompatibility groups IncP (pVK102), IncQ (pKT230), IncW (pGV1124), and IncN (pCU1), as well as pHS15 (a shuttle vector based on an endogenous plasmid of the moderate halophile Halomonas elongata) were transferred by conjugation from E. coli to moderately halophilic strains of the genera Chromohalobacter, Deleya, Halomonas, Vibrio and Volcaniella, to test their utility as cloning vectors for these extremophiles. Several factors affecting the efficiency of conjugation (cell growth phase, mating time, ratio donor:recipient, and composition and salinity of the mating medium) were evaluated. The highest frequencies (from 10-1 to 10-4 transconjugants/recipient cell) were achieved when exponentially grown donor and recipient cells were mixed and incubated for 16 h at 30 °C on SW-2 saline medium at a ratio 1:1 (for Deleya, Halomonas and Volcaniella strains) or SW-5 saline medium at a ratio 2:1 (for Chromohalobacter and Vibrio strains). Whereas IncP, IncQ, and pHS15 plasmids were able to replicate in all strains tested, the IncW plasmid pGV1124 was only maintained in H. elongata, and pCU1 could not be established in any of the moderate halophiles tested. The IncQ plasmid pKT230 was the most stably maintained in the majority of the moderate halophiles. The shuttle vector pHS15 revealed to be compatible with each of the broad-host-range plasmids assayed. Finally, both intrageneric and intergeneric conjugation between moderate halophiles have been demonstrated by using the self-transmissible IncP plasmid RK2. Transfer of RK2 between Halomonas spp. was observed at 2–7% total salts, yielding the highest transfer frequencies (from 1.2×10-3 to 2.8×10-4) on SW-2 medium. Intergeneric conjugation on SW-2 between Halomonas and other moderate halophiles such as Volcaniella (transfer frequency of 2×010-5) and Deleya (5×10-5) were also found. This is the first report on the existence of conjugation between moderately halophilic bacteria.

Quantification of the effect of substrate concentration on the conjugal transfer rate of the TOL plasmid in short-term batch mating experiments.

Batch mating experiments with Pseudomonas putida PAW 1 (TOL) as a donor and Pseudomonas aeruginosa PAO 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient. The impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain. When the donor strain was harvested in exponential growth phase, no impact was observed; when the donor strain was taken from the stationary phase, however, a strong impact of the substrate concentration was measured: a 10-fold reduction in the substrate concentration decreased the observed plasmid transfer rate by 55%.

Two Novel Endopeptidases Released into the Medium during Mating of Gametes of Chlamydomonas reinhardtii

During the mating reaction between mt+ and mt- gametes of Chlamydomonas reinhardtii, two novel endopeptidases, each of which was able to digest the B chain of insulin, were released into the culture medium, together with a gamete lytic enzyme (GLE) which is responsible for digestion of the gametic cell walls. Both endopeptidases and GLE were copurified from the mating medium by column chromatography on DEAE-cellulose and concanavalin A. Gel filtration separated the peptidases, which were unable to digest gametic cell walls, into two fractions, endopeptidase-1 and endopeptidase-2. These enzymes were also separated from GLE, which was unable to digest the B chain of insulin. Endopeptidase-1, with a molecular mass of about 200 kDa, cleaved the B chain of insulin at the Ala14-Leu15 peptide bond, and this activity was inhibited by EDTA. Endopeptidase-2, with a molecular mass of about 110 kDa, digested the peptide at the Leu15-Tyr16 peptide bond and was sensitive to iodoacetate and chymostatin. When the cell walls of gametes of either mating-type were digested prior to mating with exogenously added GLE, the two endopeptidases were released into the medium, a result that suggests that they are stored, like GLE, outside the plasmalemma.

Double staining of mitochondria and mitochondrial nucleoids in the living yeast during the life cycle.

Mitochondria and mitochondrial nucleoids (mt-nucleoids) of the yeast, Saccharomyces cerevisiae were vitally double-stained with two fluorochromes, 3, 3′-dihexyloxacarbocyanine iodide [DiOC6(3)] and 4′, 6-diamidino-2-phenylindole (DAPI), respectively in different types of cells during the life cycle. Budded cells at logarithmic phase had a single, branched, giant mitochondrion, a part of which migrated into the daughter cell prior to nuclear division. Mt-nucleoids appeared as a string-of-beads within a giant mitochondrion. Single cells at stationary phase had numerous fragmented mitochondria with dot-like mt-nucleoids. When stationary-phase cells were transferred to the sporulation medium, fragmented mitochondria fused with each other and a network of mitochondrion changed its configuration as the meiotic division proceeded. Finally, mitochondria formed four rings which enclosed each daughter nucleus in premature spores. Mitochondria were hardly stained with DiOC6(3) in mature spores. Fused mitochondria were clearly observed again in cells at germination and outgrowth. When cells of opposite mating type were mixed in mating medium, zygote formation and first bud emergence occurred in 4h. Just after mating, string-like and fragmented mitochondria transmitted from each parent cell were separately visible on both sides of a dumbbell-shaped zygote. In contrast, extensive fusion of mitochondria occurred in zygotes with a first bud.

Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.

Enhanced conjugative transfer of plasmid DNA fromEscherichia colitoStaphylococcus aureusandListeria monocytogenes

Transfer of mobilizable shuttle cloning vectors by conjugation from Escherichia coli to Staphylococcus aureus occurred at a very low frequency (10(-9) transconjugants per donor colony-forming unit after the mating period). It was observed that subinhibitory concentrations of penicillins (oxacillin or penicillin G) in the mating medium resulted in increased transfer frequency by conjugation of the shuttle vector pAT18 from E. coli SM10 to S. aureus 80CR5 Str (54-fold) and to Listeria monocytogenes LO17RF (45-fold). These results were interpreted as indicating that the cell wall of Gram-positive bacteria constitutes an important barrier for conjugative transfer of genetic information delivered from E. coli. It was also demonstrated that presence of a restriction system(s) in S. aureus recipients represented a major barrier to introduction of foreign DNA.

Enhanced conjugative transfer of plasmid DNA from Escherichia coli to Staphylococcus aureus and Listeria monocytogenes

Transfer of mobilizable shuttle cloning vectors by conjugation from Escherichia coli to Staphylococcus aureus occurred at a very low frequency (10−9 transconjugants per donor colony-forming unit after the mating period). It was observed that subinhibitory concentrations of penicillins (oxacillin or penicillin G) in the mating medium resulted in increased transfer frequency by conjugation of the shuttle vector pAT18 from E. coli SM10 to S. aureus 80CR5 Str (54-fold) and to Listeria monocytogenes LO17RF (45-fold). These results were interpreted as indicating that the cell wall of Gram-positive bacteria constitutes an important barrier for conjugative transfer of genetic information demonstrated that presence of a restriction system(s) in S. aureus recipients represented a major barrier to introduction of foreign DNA.

The role of calcium in the Chlamydomonas reinhardtii mating reaction.

The mating reaction of Chlamydomonas reinhardtii entails a rapid series of cell-cell interactions leading to cell fusion. We have demonstrated (Pasquale, S. M., and U. Goodenough. 1987. J. Cell Biol. 105:2279-2293) that cAMP plays a key role in this process: gametic flagellar adhesion elicits a sharp increase in intracellular cAMP, and presentation of dibutyryl-cAMP to unmated gametes elicits all known mating responses. The present study evaluates the role of Ca2+ in this system. We document that the mating-induced increase in cAMP, and hence the mating responses themselves, are blocked by a variety of drugs known to interfere with Ca(2+)-sensitive processes. These data suggest that Ca(2+)-mediated events may couple adhesion to the generation of cAMP. Such events, however, appear to be localized to the flagellar membrane; we find no evidence for the mating-related increase in cytosolic free Ca2+ that has been postulated by others. Indeed, by monitoring the length of the Ca(2+)-sensitive centrin-containing nucleus-basal body connector, we show that cytosolic free Ca2+ levels, if anything, decrease in response to cAMP signaling. We confirm a previous report that Ca2+ levels increase in the mating medium, but document that this represents a response to augmented cAMP levels and not a prelude. Finally, we show that IP3 levels remain constant throughout the mating reaction. These results are discussed in terms of the various signal transduction systems that have now been identified in Chlamydomonas.

Tetracycline enhances Tn 916-mediated conjugal transfer

Pregrowth of the donor on medium containing tetracycline increased conjugative transposition of Tn 916 and the transposon-dependent mobilization of pC194 19- to 119-fold in matings between Bacillus subtilis and Bacillus thuringiensis subsp. israelensis. Tn 916 and pC194 transferred independently under these conditions. When Enterococcus faecalis was the donor and B. thuringiensis subsp. israelensis the recipient, pregrowth in tetracycline increased the conjugative transposition frequency by approximately 15-fold. Tetracycline-enhanced conjugation appeared during matings as short as 3 h in length. Pregrowth in tetracycline did not enhance conjugation in Bacillus sphaericus × B. thuringiensis subsp. israelensis or B. thuringiensis subsp. israelensis × B. subtilis matings. Incorporation of tetracycline into the mating medium, at concentrations that did not inhibit growth of the B. thuringiensis subsp. israelensis recipient, resulted in conjugation frequencies similar to those obtained by pregrowth of the B. subtilis donors in antibiotic-containing medium. The data suggest stimulation of donor function by tetracycline.

ANALYSIS OF GAMETIC PROTOPLAST RELEASE IN THE CLOSTERIUM PERACEROSUM‐STRIGOSUM‐LITTORALE COMPLEX (CHLOROPHYTA)1

ABSTRACTA biologically active glycoprotein (protoplast‐release‐inducing protein; PR‐IP), which induces the release of gametic protoplasts from mating type minus (mt‐) cells of the Closterium peracerosum‐strigosum‐littorale complex, was prepared from a medium in which mt‐ and mt+ cells had been previously incubated together. The process of PR‐IP‐inducing protoplast release was analyzed. Induction of protoplast release was dependent upon the duration of both PR‐IP treatment and preincubation in nitrogen‐deficient mating medium before PR‐IP treatment. Low cell density in the preculture stage had a significant stimulative effect upon the induction of protoplast release. Light was necessary for protoplast release, especially just before PR‐IP treatment. Chloramphenicol and 3‐(4‐chlorophenyl)‐1,1‐dimethylurea (CMU) exerted inhibitory effects on protoplast release, especially when they were applied to the preculture stage but not when they were applied to the protoplast‐releasing stage after the PR‐IP treatment. We suggest that preculture at a low cell density under continuous light conditions that may cause metabolic changes in the chloroplast is a very important stage for gametic protoplast release in this Closterium.

Biotic and abiotic factors affecting plasmid transfer in Escherichia coli strains.

The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.

Biochemical and physiological properties of a protein inducing protoplast release during conjugation in theClosterium peracerosum-strigosum-littorale complex

When mating-type minus (mt(-)) and plus (mt(+)) cells of theClosterium peracerosum-strigosum-littorale complex were mixed together in a nitrogen-deficient mating medium, cells of both types released protoplasts, this release being the first step in the process of conjugation. Release of protoplasts by mt(-) cells also proceeded without pairing in a medium in which mt(-) and mt(+) cells had previously been cultured together. A protein with the ability to induce the release of protoplasts was purified from this medium by sequential column-chromatographic steps, and named PR-IP (protoplast-release-inducing protein). The PR-IP had an apparent molecular mass (Mr) of 95000 on gel filtration and could be separated into several isoforms by anion-exchange chromatography. Each isoform consisted of two glycopolypeptides of Mrs 42000 and 19000, while the deglycosylated polypeptides had Mrs of 34000 and 18000, respectively. From an analysis of dose-response curves, the numbers of PR-IP molecules required for the release of a protoplast by a single cell was calculated as 1.5·10(9) and the concentration required for 50% of the maximum response (ED50) as 4.1·10(-9)M. We suggest that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt(-) cells of thisClosterium complex.

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.