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Reversion of an ilvB mutation in Pseudomonas aeruginosa in the presence of a derivative of RP1 plasmid
Gene, 25 (1983) 151-154 Elsevier
151
GENE 868 Reversion of an ilvB mutation in Pseudomonas aeruginosa in the presence of a derivative of RPI plasmid (R-prime formatioa; interspecies conjugation; broad-host-range vector; filamentous morphology)
Stephen Guss and Sunil Palehaudhuri * Department of hnmunology and Microbiology. Wayne State University School of Medicine. Detroit. MI 48201 (U.S.A.) Tel. (313) 577-1313 (Received April 29th, 1983) (Revision received July 6th, 1983) (Accepted July 12th, 1983)
SUMMARY We have isolated a derivative of RP i, a broad-host-range plasmid, in whose presence the ih,B 112 mutation ofPseudomonas aeruginosa strain PU21 reverts at a high frequency. This derivative of RP 1 (RP l-ih,B ÷ complex) may have arisen by a fusion of the P. aeruginosa ilvB gene with RP1 during their co-transfer into strain PU21. The RPI derivative is not very stable in the PU21 background but it can apparently be stabilized by its integration into the host chromosome, resulting in an Hfr-type donor strain, SP500.
INTRODUCTION
EXPERIMENTAL AND DISCUSSION
RPI is a broad-host-range plasmid, belonging to the incompatibility group P-1. We have obtained derivatives of RPI from Pseudomonas aerug;.nosa strain PAO25 which seem to contain some ilv genes It is possible that the RPI-i/v derivatives, although phenotypically l l v - , facilitate the integration of RPl-ilv into the host chromosome through generalized recombination. According to this hypothesis, our newly constructed strain fly ÷ derivative of PU21[RPI ], referred to as SP500, is analogous to Hfr derivative that has RP1 integrated near the ilv genes.
P. aeruginosa strain PAO25 (argG leu- I0) containing the plasmid RP 1 (AprKm~Tcr) was mated with P. aeruginosa strain PU21 (ilvB112/eu-l) and exconjugants were selected for carbenicillin resistance. The purified exconjugants were llv- Leu-, but showed a reversion from llv - to llv ÷ at a frequency of 10 -4. The frequency of reversion of the ilvB112 mutation itself is 10 - 7 to 10 - 8. This suggested that transfer of RPl from PAO25 into PU21 has caused the ilvB mutation to revert at a high frequency. This effect was specific to the transfer of RP 1 from strain PAO25, since when I~PI was transferred from the Escherichio coil K-12 strain JC1553, the resulting exconjugants remaa'ned stable auxotrophs (llv- and Leu - ); in this case the reversion frequency of these mutations was about 10-8.
* To whom reprint requests should be addressed. Abbreviations:Ap, ampicillin;Cb, carhenicillin;r, resistance; s, sensitivity; [ ], indicates plasmid-carrierstate. 0378-1119/83/$03.00 © 1983ElsevierScience Publishers
152 TABLE i "l'ransfe," o f R P I , iiv + genes Cross
~
Selected markers
Transfer frequency b
% Coinheritance o f unselected markers //v +
Cb"
SP500 x AT753
Cbr llv +
5 6
x 10 - 2 x 10- 4
5 -
95
SPS00 x S P I 0 2 P U 2 1 | R P I i x AT753
Cb" ilv + Cb"
1.0 0 4.9 x 10- 2
0 0
-
A T 7 5 3 [ R P I ilv + ] x PU21
ilv + Cb ~
0 4
x 10 - 2
0
-
fly + Cb r llv +
0 3 x 10- ! 4.5 x 10- 3
0 15 -
100
AT753[RPI ilv ÷ ] x JC1553
" P. aerugino~a strain SPS00 is llv * derivative of P U 2 1 [ R P I ] . AT753 and JC1553 are E. c o l i r e c A . SPS00 and all other strains carrying
R P I are C b r. Matings were done by the modified technique. I ml of the log phase culture was washed twice with 0.85~i, sterile saline and ttlen resuspended in selective media ( M M O + thiamine) (Razin, 1973). It was then grown for another 3 h without aeration (starvation). 0.1 ml of these starved cells was mixed with 0.4 mi of 24-h grown recipient cells (1:5 ratio), and the mixture was transferred o n t e a sterile Millipore filter (size 0.45 # m x 47 mm). Bacteria on the sterile fdter disc were placed on L-agar m e d i u m and incubated for 5 min at 32°C, and finally resuspended in 0.85~/o sterile saline (equal volume). T h e suspension was then plated on selective and nonselective media. Controls were maintained by spreading both parents separately on the same selective media and were useful in determining the frequency of reversion. b Per 10a donor cells.
We carried out a series of mating experiments using llv + revertant of P. aeruginosa strain (PU21[RP1 ]), referred to as SP500, as donor and recA E. coil K-12 and P. putida strains as recipients (Table I). In the mating between SP500 and an E. coli recipient, AT753 recA -, with selection for Cb resistance on medium containing isoleucine-valine, about 5% of the exconjugants were found to be isoleucine-valine-independent. The Ilv ÷ exconjugants showed a high rate of segoegation, resulting in Cb r IIv- progeny after growth in nonselective medium. This explains why the majority of Cb r transconjugants became liv- Cb r and, as subsequently shown, why the transfer of Cb r appears to occur at a much higher frequency than the Ilv + character when the Ilv + exconjugants are used as donors in a conjugation experiment. Similarly, selection of isoleucine-valine independence (fly +) brought exconjugants at a frequency o f 10- 4, most of which are also carbenicillin-resistanti: In both cases, the llv + exconjugants retained the other auxotrophic requirements of the recipient, AT753. We
next determined if E. coil AT753[RPI'ilv + ] would transfer RPl'ilv + to a P. putida strain, SPI02 (histrp- ilv- ), having an ilv mutation. Markers on RP 1 showed a normal transfer frequency but the concomitant transfer ofisoleucine-valine was not detectable within 48 h. A similar result was obtained when the donor was SP500 (Table I). It appeared that the ilv mutation in SPl02 is not complemented by the P. aeruginosa genes carried by RPl'ilv+. Furthermore, the liv- PU21[RPI] exconjugant that does not revert to llv + showed a normal transfer of RP 1 markers, but not of the ilv genes (Table I). E. coil strain AT753 containing the presumptive RP l'ilv + plasrnids showed abnormal growth characteristics. As shown in Fig. 1, they formed filamentous cells. Filamentous morphology was not observed in ilv- segregants or in isogenic strains carrying an R P l ' h i s + plasmid. The latter plasmid was constructed by Olsen and Gonzalez (1974). It is already known that some ilv genes (ilvA + and ilvB +) are mobilized by a plasmid at high frequency in matings between P. aeruginosa strains (Loutit
153
" •. I ~ K ~ / . t
iiin~
~.t " , , -:. :,~-~, timbAL-----~
~
s
;/..t
-
¢. ,.-
,,'
"~..r.z~..-~il~ ~
.
r-"
..~
\
-,~ t
-; I
.,-
. •
"
~
,.~
e,
"I"."
,
..
k
~/
¢ ,1
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-
_~l
J
.,.
,
tl
~ ..~ i , ~ ~ : .
LI" •
J
-k
~
~
"'
-
t~, -
~'
,
Fig. I. Morphology ofPseudomonas aeruginosa mutant strains. All strains were grown in I 0.0 ml of L-thy broth (Palchaudhuri et al., 197b } {i:25 dilution) for 8 h at 37°C with aeration. Cultures were then washed and resuspended in sodium phosphate buffer (pH 7.0}. Resuspended cells were subsequently placed on glass slides, stained and photographed• (a} SP500. (b) AT753 recA strain without plasmid DNA. (c) AT753 recA [RPI 'his + ]. (d) AT753 recA [RPI 'ilv ÷ 1. Filamentous morphology follows the transfer of Pseud,,nonas genes {ilv" ) into E. coli AT753 recA.
etal., 1968). Thus the plasmid RP1 could have integrated into the chromosome of PU21 by generalized recombination if RP1 carried the//v genes from the PAO25 donor. In fact, the llv + revertants of PU21[RP1 ], donors did not show the presence of any autonomous plasmids even though they were resistant to carbenicillin, kanamycin and tetracycline (not shown). This suggested that RP 1 had integrated into the PU21 chromosome. The chromosomal segment containing the ilvB genes of PAO25 apparently does not contain the promoter for the//v genes, since the transconjugant PU21 ilvB[RPl-ilvB + ] is llv-, but reverts to llv +. Furthermore, the RP l-ilvB derivative is highly unstable in the PU21 strain (not shown), and therefore the same RPl-ilvB complex either integrates into the chromosome or is cured in nonselective medium, resulting in IIv + Hfr-type
donors (SP500) or RP1 ÷ Ilv- segregants, respectively. Association of the ih,B segment with such high instability is reminiscent of the instability of the E. coli minichromosome carrying the replicon regions, including oriC (Buysse and Palchaudhuri, 1982).
ACKNOWLEDGEMENTS
We are grateful to Dr. R. Olsen for his constructive suggestions. This work is supported by ACS Grant No. NP 333 to Sunil Palchaudhuri.
154 REFERENCES Buysse, J.M. and Palchaudhuri, S.: Chromosomal incompatibility in E. co/i K-12: A new explanation for the observed instability of minichromosome inheritance. Biochem. Biophys. Res. Commun. 106 (1982) 748-755. Loutit, J,S., Pearce, L.E. and Marinus, M,D.: Investigation of the mating system ofPseudomonas aeruginosa strain 1, I. Kinetic studies. Genet. Res. Comb. 12 (1968) 29-36. Olsen, R.H. and Gonzalez, C.: Escherichia coil gene transfer to unrelated bacteria by a histidine operon-RPl drug resistant
plasmid complex. Biochem. Biophys. Res. Commun. 59 (1975) 377-385. Palchaudhuri, S., Maas, W.K. and Ohtsubo, E.: Fusion of two F-prime factors of E. coli studied by electron microscope heteroduplex analysis. Mol. Gen. Genet. 146 (! 976) 215--23I. Razin, S.: Physiology of mycoplasmas, in Rose, A.H. and Tempest, D. (Eds.), Advances in Microbial Physiology, Vol. 10, Academic Press, New York, (1973) pp 1-80. Communicated by A.I. Bukhari.
151
GENE 868 Reversion of an ilvB mutation in Pseudomonas aeruginosa in the presence of a derivative of RPI plasmid (R-prime formatioa; interspecies conjugation; broad-host-range vector; filamentous morphology)
Stephen Guss and Sunil Palehaudhuri * Department of hnmunology and Microbiology. Wayne State University School of Medicine. Detroit. MI 48201 (U.S.A.) Tel. (313) 577-1313 (Received April 29th, 1983) (Revision received July 6th, 1983) (Accepted July 12th, 1983)
SUMMARY We have isolated a derivative of RP i, a broad-host-range plasmid, in whose presence the ih,B 112 mutation ofPseudomonas aeruginosa strain PU21 reverts at a high frequency. This derivative of RP 1 (RP l-ih,B ÷ complex) may have arisen by a fusion of the P. aeruginosa ilvB gene with RP1 during their co-transfer into strain PU21. The RPI derivative is not very stable in the PU21 background but it can apparently be stabilized by its integration into the host chromosome, resulting in an Hfr-type donor strain, SP500.
INTRODUCTION
EXPERIMENTAL AND DISCUSSION
RPI is a broad-host-range plasmid, belonging to the incompatibility group P-1. We have obtained derivatives of RPI from Pseudomonas aerug;.nosa strain PAO25 which seem to contain some ilv genes It is possible that the RPI-i/v derivatives, although phenotypically l l v - , facilitate the integration of RPl-ilv into the host chromosome through generalized recombination. According to this hypothesis, our newly constructed strain fly ÷ derivative of PU21[RPI ], referred to as SP500, is analogous to Hfr derivative that has RP1 integrated near the ilv genes.
P. aeruginosa strain PAO25 (argG leu- I0) containing the plasmid RP 1 (AprKm~Tcr) was mated with P. aeruginosa strain PU21 (ilvB112/eu-l) and exconjugants were selected for carbenicillin resistance. The purified exconjugants were llv- Leu-, but showed a reversion from llv - to llv ÷ at a frequency of 10 -4. The frequency of reversion of the ilvB112 mutation itself is 10 - 7 to 10 - 8. This suggested that transfer of RPl from PAO25 into PU21 has caused the ilvB mutation to revert at a high frequency. This effect was specific to the transfer of RP 1 from strain PAO25, since when I~PI was transferred from the Escherichio coil K-12 strain JC1553, the resulting exconjugants remaa'ned stable auxotrophs (llv- and Leu - ); in this case the reversion frequency of these mutations was about 10-8.
* To whom reprint requests should be addressed. Abbreviations:Ap, ampicillin;Cb, carhenicillin;r, resistance; s, sensitivity; [ ], indicates plasmid-carrierstate. 0378-1119/83/$03.00 © 1983ElsevierScience Publishers
152 TABLE i "l'ransfe," o f R P I , iiv + genes Cross
~
Selected markers
Transfer frequency b
% Coinheritance o f unselected markers //v +
Cb"
SP500 x AT753
Cbr llv +
5 6
x 10 - 2 x 10- 4
5 -
95
SPS00 x S P I 0 2 P U 2 1 | R P I i x AT753
Cb" ilv + Cb"
1.0 0 4.9 x 10- 2
0 0
-
A T 7 5 3 [ R P I ilv + ] x PU21
ilv + Cb ~
0 4
x 10 - 2
0
-
fly + Cb r llv +
0 3 x 10- ! 4.5 x 10- 3
0 15 -
100
AT753[RPI ilv ÷ ] x JC1553
" P. aerugino~a strain SPS00 is llv * derivative of P U 2 1 [ R P I ] . AT753 and JC1553 are E. c o l i r e c A . SPS00 and all other strains carrying
R P I are C b r. Matings were done by the modified technique. I ml of the log phase culture was washed twice with 0.85~i, sterile saline and ttlen resuspended in selective media ( M M O + thiamine) (Razin, 1973). It was then grown for another 3 h without aeration (starvation). 0.1 ml of these starved cells was mixed with 0.4 mi of 24-h grown recipient cells (1:5 ratio), and the mixture was transferred o n t e a sterile Millipore filter (size 0.45 # m x 47 mm). Bacteria on the sterile fdter disc were placed on L-agar m e d i u m and incubated for 5 min at 32°C, and finally resuspended in 0.85~/o sterile saline (equal volume). T h e suspension was then plated on selective and nonselective media. Controls were maintained by spreading both parents separately on the same selective media and were useful in determining the frequency of reversion. b Per 10a donor cells.
We carried out a series of mating experiments using llv + revertant of P. aeruginosa strain (PU21[RP1 ]), referred to as SP500, as donor and recA E. coil K-12 and P. putida strains as recipients (Table I). In the mating between SP500 and an E. coli recipient, AT753 recA -, with selection for Cb resistance on medium containing isoleucine-valine, about 5% of the exconjugants were found to be isoleucine-valine-independent. The Ilv ÷ exconjugants showed a high rate of segoegation, resulting in Cb r IIv- progeny after growth in nonselective medium. This explains why the majority of Cb r transconjugants became liv- Cb r and, as subsequently shown, why the transfer of Cb r appears to occur at a much higher frequency than the Ilv + character when the Ilv + exconjugants are used as donors in a conjugation experiment. Similarly, selection of isoleucine-valine independence (fly +) brought exconjugants at a frequency o f 10- 4, most of which are also carbenicillin-resistanti: In both cases, the llv + exconjugants retained the other auxotrophic requirements of the recipient, AT753. We
next determined if E. coil AT753[RPI'ilv + ] would transfer RPl'ilv + to a P. putida strain, SPI02 (histrp- ilv- ), having an ilv mutation. Markers on RP 1 showed a normal transfer frequency but the concomitant transfer ofisoleucine-valine was not detectable within 48 h. A similar result was obtained when the donor was SP500 (Table I). It appeared that the ilv mutation in SPl02 is not complemented by the P. aeruginosa genes carried by RPl'ilv+. Furthermore, the liv- PU21[RPI] exconjugant that does not revert to llv + showed a normal transfer of RP 1 markers, but not of the ilv genes (Table I). E. coil strain AT753 containing the presumptive RP l'ilv + plasrnids showed abnormal growth characteristics. As shown in Fig. 1, they formed filamentous cells. Filamentous morphology was not observed in ilv- segregants or in isogenic strains carrying an R P l ' h i s + plasmid. The latter plasmid was constructed by Olsen and Gonzalez (1974). It is already known that some ilv genes (ilvA + and ilvB +) are mobilized by a plasmid at high frequency in matings between P. aeruginosa strains (Loutit
153
" •. I ~ K ~ / . t
iiin~
~.t " , , -:. :,~-~, timbAL-----~
~
s
;/..t
-
¢. ,.-
,,'
"~..r.z~..-~il~ ~
.
r-"
..~
\
-,~ t
-; I
.,-
. •
"
~
,.~
e,
"I"."
,
..
k
~/
¢ ,1
/
-
_~l
J
.,.
,
tl
~ ..~ i , ~ ~ : .
LI" •
J
-k
~
~
"'
-
t~, -
~'
,
Fig. I. Morphology ofPseudomonas aeruginosa mutant strains. All strains were grown in I 0.0 ml of L-thy broth (Palchaudhuri et al., 197b } {i:25 dilution) for 8 h at 37°C with aeration. Cultures were then washed and resuspended in sodium phosphate buffer (pH 7.0}. Resuspended cells were subsequently placed on glass slides, stained and photographed• (a} SP500. (b) AT753 recA strain without plasmid DNA. (c) AT753 recA [RPI 'his + ]. (d) AT753 recA [RPI 'ilv ÷ 1. Filamentous morphology follows the transfer of Pseud,,nonas genes {ilv" ) into E. coli AT753 recA.
etal., 1968). Thus the plasmid RP1 could have integrated into the chromosome of PU21 by generalized recombination if RP1 carried the//v genes from the PAO25 donor. In fact, the llv + revertants of PU21[RP1 ], donors did not show the presence of any autonomous plasmids even though they were resistant to carbenicillin, kanamycin and tetracycline (not shown). This suggested that RP 1 had integrated into the PU21 chromosome. The chromosomal segment containing the ilvB genes of PAO25 apparently does not contain the promoter for the//v genes, since the transconjugant PU21 ilvB[RPl-ilvB + ] is llv-, but reverts to llv +. Furthermore, the RP l-ilvB derivative is highly unstable in the PU21 strain (not shown), and therefore the same RPl-ilvB complex either integrates into the chromosome or is cured in nonselective medium, resulting in IIv + Hfr-type
donors (SP500) or RP1 ÷ Ilv- segregants, respectively. Association of the ih,B segment with such high instability is reminiscent of the instability of the E. coli minichromosome carrying the replicon regions, including oriC (Buysse and Palchaudhuri, 1982).
ACKNOWLEDGEMENTS
We are grateful to Dr. R. Olsen for his constructive suggestions. This work is supported by ACS Grant No. NP 333 to Sunil Palchaudhuri.
154 REFERENCES Buysse, J.M. and Palchaudhuri, S.: Chromosomal incompatibility in E. co/i K-12: A new explanation for the observed instability of minichromosome inheritance. Biochem. Biophys. Res. Commun. 106 (1982) 748-755. Loutit, J,S., Pearce, L.E. and Marinus, M,D.: Investigation of the mating system ofPseudomonas aeruginosa strain 1, I. Kinetic studies. Genet. Res. Comb. 12 (1968) 29-36. Olsen, R.H. and Gonzalez, C.: Escherichia coil gene transfer to unrelated bacteria by a histidine operon-RPl drug resistant
plasmid complex. Biochem. Biophys. Res. Commun. 59 (1975) 377-385. Palchaudhuri, S., Maas, W.K. and Ohtsubo, E.: Fusion of two F-prime factors of E. coli studied by electron microscope heteroduplex analysis. Mol. Gen. Genet. 146 (! 976) 215--23I. Razin, S.: Physiology of mycoplasmas, in Rose, A.H. and Tempest, D. (Eds.), Advances in Microbial Physiology, Vol. 10, Academic Press, New York, (1973) pp 1-80. Communicated by A.I. Bukhari.