JOURNAL OF BACTERIOLOGY, Feb. 1990, p. 1155-1156
Vol. 172, No. 2
0021-9193/90/021155-02$02.00/0 Copyright ©D 1990, American Society for Microbiology
Genetic Mapping of the Structural Gene for Phospholipase C of Pseudomonas aeruginosa PAO VIVEKA LINDGREN,' RACHEL M. OSTROFF,2 MICHAEL L. VASIL,2
AND
BENGT WRETLIND1*
Department of Clinical Bacteriology, Danderyd Hospital, S-18288 Danderyd, Sweden,' and Department of Microbiology and Immunology,
University of Colorado
Medical School, Denver, Colorado 802622
Received 30 May 1989/Accepted 10 November 1989
An insertion mutation constructed by gene replacement methods was used to map the gene corresponding to the hemolytic phospholipase C (plcS gene) in Pseudomonas aeruginosa PAO1 by R68.45-mediated conjugation. plcS mapped approximately at 67 min on the 75-min chromosomal map (B. W. Holloway, K. O'Hoy, and H. Matsumoto, p. 213-221, in S. J. O'Brien, ed., Genetic Maps 1987, vol. 4, 1987), between the markers pur-67 and pru-375 and considerably distal to the regulatory genes plcA and plcB, which are located at approximately 12 min.
The hemolytic phospholipase C (PLC H) (heat-labile hemolysin [3]) is an extracellular enzyme produced by Pseudomonas aeruginosa under phosphate-limiting conditions along with alkaline phosphatase, an extracellular nonhemolytic PLC (PLC N), and other proteins which are part of the phosphate-scavenging system in this organism (1, 3, 4, 9, 10, 15). Serological studies (2) have shown that PLC is produced during clinical infections, and data from animal experiments indicate that it may play an important role in the pathogenesis of P. aeruginosa infections (10, 15; L. M. Graham, and M. L. Vasil, unpublished observations). The gene encoding
wild-type gene (9, 10). The resulting strain was designated PLC S. Colonies of P. aeruginosa PLC S which carried the plcS::tet insertion were unable to produce a zone of hemolysis on sheep blood agar plates after 24 h (Hly- phenotype). Hemolysis was used to score recombinants for PLC H production because the Tcr phenotype of the conjugative plasmid R68.45 did not allow us to use the Tcr phenotype of the insert in picS, nor could PLC activity be used to score the recombinants because the PLC S mutant still carries the gene encoding the recently described PLC N (10). Other strains used are described in Table 1. Conjugation was
TABLE 1. P. aeruginosa strains and plasmids used Strain or plasmid
Bacterial strains PLC S PA0957 PA0982
Reference(s) or construction
Genotypea or phenotype
PA02022 PA06021 PA07527
plcS::tetAR chl-3 aru-321 aru-247 cys-5605 hisI5075 argA171 proB67 nal-25 proB65 pru-375 cvs-54 pur-67 thr-9001 Strr
PA07528
met-9020 catAl mtu-9002 tyu-9030 plcS::tetAR Strr
PA07529
cys-59 pur-67 thr-9001 Strr
PA07530
met-9020 catAl mtu-9002 tyu-9030 nar-9011 Strr Cb Km/Nm Tc Tra Cma IncPl
Plasmid R68.45
9, 10 7 7 Holloway collection 14
Streptomycin-resistant mutant of PA0944 (Holloway collection) Recombinant of cross PLC S(R68.45) x PA07530; selected marker was nar-9011 Streptomycin-resistant mutant of PA0949 (Holloway collection) Streptomycin-resistant mutant of PA02376 (12) 5
a Genotype symbols are as described by Holloway et al. (6), except for arl (arginine utilization, previously designated orul).
performed by using a membrane filter method. In short, donor (PLC S or PA07528) and recipient strains were grown in brain heart infusion broth (Difco Laboratories) at 37°C in a rotary shaker. The donor and recipient cultures were mixed and filtered through a Millipore filter. The filter was placed on an agar plate (blood agar base; Oxoid Ltd.) and incubated for 2 h (8). The recombinants were selected on agar plates containing minimal medium supplemented with glucose and appropriate growth factors. The same medium without citrate or glucose was used for selection of the utilization markers, with carbon sources added at a concentration of 1 g/liter (except arginine [3.2 g/liter]). Streptomy-
PLC H (plcS gene) has been cloned and sequenced (11, 16) and was found to be part of a multigene operon (10, 13) which is regulated by Pi at the level of transcription (11). Here we describe the mapping of the structural gene for the PLC H in strain PAO. A mutation in picS was constructed in vitro by insertion of a tetracycline resistance cartridge in the StuI site of the cloned PLC gene (9, 10). Gene replacement techniques were used to introduce the mutated plcS gene in place of the *
Corresponding author. 1155
1156
NOTES
J. BACTERIOL.
TABLE 2. Three-factor analysis of R68.45-mediated conjugational crosses with strains PA06021 (proB pru-375) and PA0944 (cys-54 pur-67) as recipients and strain PA07529 (p1cS::tetAR met-9020) as donor" Selected marker Unselected marker No. (%) of recombinants
Pro'
Pru+ Pru+ PruPru-
Hly+ HlyHly+ Hly-
17 16 443 1
(4) (3) (93) (
Vol. 172, No. 2
0021-9193/90/021155-02$02.00/0 Copyright ©D 1990, American Society for Microbiology
Genetic Mapping of the Structural Gene for Phospholipase C of Pseudomonas aeruginosa PAO VIVEKA LINDGREN,' RACHEL M. OSTROFF,2 MICHAEL L. VASIL,2
AND
BENGT WRETLIND1*
Department of Clinical Bacteriology, Danderyd Hospital, S-18288 Danderyd, Sweden,' and Department of Microbiology and Immunology,
University of Colorado
Medical School, Denver, Colorado 802622
Received 30 May 1989/Accepted 10 November 1989
An insertion mutation constructed by gene replacement methods was used to map the gene corresponding to the hemolytic phospholipase C (plcS gene) in Pseudomonas aeruginosa PAO1 by R68.45-mediated conjugation. plcS mapped approximately at 67 min on the 75-min chromosomal map (B. W. Holloway, K. O'Hoy, and H. Matsumoto, p. 213-221, in S. J. O'Brien, ed., Genetic Maps 1987, vol. 4, 1987), between the markers pur-67 and pru-375 and considerably distal to the regulatory genes plcA and plcB, which are located at approximately 12 min.
The hemolytic phospholipase C (PLC H) (heat-labile hemolysin [3]) is an extracellular enzyme produced by Pseudomonas aeruginosa under phosphate-limiting conditions along with alkaline phosphatase, an extracellular nonhemolytic PLC (PLC N), and other proteins which are part of the phosphate-scavenging system in this organism (1, 3, 4, 9, 10, 15). Serological studies (2) have shown that PLC is produced during clinical infections, and data from animal experiments indicate that it may play an important role in the pathogenesis of P. aeruginosa infections (10, 15; L. M. Graham, and M. L. Vasil, unpublished observations). The gene encoding
wild-type gene (9, 10). The resulting strain was designated PLC S. Colonies of P. aeruginosa PLC S which carried the plcS::tet insertion were unable to produce a zone of hemolysis on sheep blood agar plates after 24 h (Hly- phenotype). Hemolysis was used to score recombinants for PLC H production because the Tcr phenotype of the conjugative plasmid R68.45 did not allow us to use the Tcr phenotype of the insert in picS, nor could PLC activity be used to score the recombinants because the PLC S mutant still carries the gene encoding the recently described PLC N (10). Other strains used are described in Table 1. Conjugation was
TABLE 1. P. aeruginosa strains and plasmids used Strain or plasmid
Bacterial strains PLC S PA0957 PA0982
Reference(s) or construction
Genotypea or phenotype
PA02022 PA06021 PA07527
plcS::tetAR chl-3 aru-321 aru-247 cys-5605 hisI5075 argA171 proB67 nal-25 proB65 pru-375 cvs-54 pur-67 thr-9001 Strr
PA07528
met-9020 catAl mtu-9002 tyu-9030 plcS::tetAR Strr
PA07529
cys-59 pur-67 thr-9001 Strr
PA07530
met-9020 catAl mtu-9002 tyu-9030 nar-9011 Strr Cb Km/Nm Tc Tra Cma IncPl
Plasmid R68.45
9, 10 7 7 Holloway collection 14
Streptomycin-resistant mutant of PA0944 (Holloway collection) Recombinant of cross PLC S(R68.45) x PA07530; selected marker was nar-9011 Streptomycin-resistant mutant of PA0949 (Holloway collection) Streptomycin-resistant mutant of PA02376 (12) 5
a Genotype symbols are as described by Holloway et al. (6), except for arl (arginine utilization, previously designated orul).
performed by using a membrane filter method. In short, donor (PLC S or PA07528) and recipient strains were grown in brain heart infusion broth (Difco Laboratories) at 37°C in a rotary shaker. The donor and recipient cultures were mixed and filtered through a Millipore filter. The filter was placed on an agar plate (blood agar base; Oxoid Ltd.) and incubated for 2 h (8). The recombinants were selected on agar plates containing minimal medium supplemented with glucose and appropriate growth factors. The same medium without citrate or glucose was used for selection of the utilization markers, with carbon sources added at a concentration of 1 g/liter (except arginine [3.2 g/liter]). Streptomy-
PLC H (plcS gene) has been cloned and sequenced (11, 16) and was found to be part of a multigene operon (10, 13) which is regulated by Pi at the level of transcription (11). Here we describe the mapping of the structural gene for the PLC H in strain PAO. A mutation in picS was constructed in vitro by insertion of a tetracycline resistance cartridge in the StuI site of the cloned PLC gene (9, 10). Gene replacement techniques were used to introduce the mutated plcS gene in place of the *
Corresponding author. 1155
1156
NOTES
J. BACTERIOL.
TABLE 2. Three-factor analysis of R68.45-mediated conjugational crosses with strains PA06021 (proB pru-375) and PA0944 (cys-54 pur-67) as recipients and strain PA07529 (p1cS::tetAR met-9020) as donor" Selected marker Unselected marker No. (%) of recombinants
Pro'
Pru+ Pru+ PruPru-
Hly+ HlyHly+ Hly-
17 16 443 1
(4) (3) (93) (
