APPLiED MICROBIOLOGY, Dec. 1975, p. 1046-1047 Copyright C 1975 American Society for Microbiology

Vol. 30, No. 6 Printed in U.S.A.

Improved Method of Selection for Mutants of Pseudomonas putida GEORGIANN CARHART' AND GEORGE HEGEMAN* Department of Microbiology, Indiana University, Bloomington, Indiana 47401 Received for publication 9 July 1975

Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methylN'-nitro-N-nitrosoguanidine mutagenesis with an improved u-cycloserine selection. Pseudomonads are widely distributed in soil and surface waters and are known for their catabolic versatility. The enzymology and regulation of many of their catabolic pathways has been extensively studied (5). Considerable interest has been shown recently in complementing these physiological studies with genetic analysis (1). Such an analysis requires the isolation of stable mutant strains, which are blocked in various steps of biochemical sequences. Ornston et al. (6) reported a procedure for isolation of spontaneous mutants of Pseudomonas putida, which described a selection method requiring at least three "lytic cycles," each consisting of harvesting cells, treating with z-cycloserine and penicillin G for 4 to 6 h, harvesting, washing, and resuspending, allowing an overnight growth period, and then repeating the cycle. After three lytic cycles, mutants were found at a frequency of about 1%. Gaudy and White described methods for the isolation of mutants of Pseudomonas aeruginosa (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G180, p. 60). They reported an increased efficiency of selection by a second addition of the antibiotic D-cycloserine to the selection medium and decreased aeration, followed by plating on solid medium containing a poorly utilized carbon source. This note reports a procedure which is a combination of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved method of selection modified from the Gaudy and White procedure. (A preliminary report of this work was presented at the Annual Meeting of the Genetics Society of America, Bloomington, Ind., August 1974.) The minimal medium used throughout the

isolation procedure was a modification of Hutner's mineral base (2) for the cultivation of pseudomonads (3). Solid medium was prepared by incorporation of 1% (wt/vol) Ionagar no. 2S (Wilson Diagnostics Inc.). Plates for the visual identification of auxotrophs were prepared by including DL-histidine at a final concentration of 0.25 ,ug/ml and fructose at a final concentration of 0.2%, autoclaved and added separately to the minimal medium. Luria broth (L-broth; 4) was the complete medium used in this study. Stock solutions included: i-cycloserine (6.6 pg/ml; Sigma Chemical Co.), 0.1 M citrate buffer (pH 5.5), and N-methyl-N'-nitro-N-nitrosoguanidine in citrate buffer (2.5 ug/ml). The mutagenesis and selection procedure is outlined in Table 1. It was found that the efficiency of enrichment was greatly increased by addition of a poorly utilized carbon source (fructose), decreased aeration, and successive additions of the antibiotic u-cycloserine at 6, 8, and 11 h, followed by dilution and plating at 24 h. Visual identification of auxotrophs as small colonies on minimal medium, including fructose, was achieved by addition of a growth-limiting amount of histidine. The use of a poorly utilized carbon source in the visual identification medium was reported by Gaudy and White to reduce loss of auxotrophs due to residual effects of the D-cycloserine (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G180, p. 60). Colorimetric measurement of the D-cycloserine concentration in the medium during selection was performed by measuring hydroxamic acid formation. These measurements indicated that the successive additions resulted in a gradual increase in concentration of the antibiotic in the medium. This procedure has been used successfully for the isolation of auxotrophs of P. putida ATCC 12633. A reconstruction experiment was per-

' Present address: Department of Microbiology, Indiana University Medical Center, Indianapolis, Ind. 46202. 1046

VOL. 30, 1975

NOTES

TABLE 1. Mutagenesis and selection procedurea Day

Procedure

0

Inoculate 25 ml of L.brothb and allow to grow overnight.

1

Divide the exponential culture in half and wash by centrifugation (10 min, 3,440 x g) and resuspension in minimal medium. Resuspend each in 4.9 ml of citrate buffer. Add to each 0.1 ml of NTG for 30 min and centrifuge (3,440 x g) for 15 min. Wash each, as before, twice with minimal medium and resuspend in 5.0 ml of L-broth. Divide each into 0.5-ml aliquots and add 0.5 ml of Lbroth. Incubate overnight.

2

Wash with minimal medium. Resuspend in 1.8 ml of minimal medium containing 0.2% fructose. Add 0.2 ml of D-cycloserine. Incubate with decreased aeration (shaking slow enough to affect growth rate) and make successive additions of 0.1 ml of D-cycloserine at 6, 8, and 11 h.

3

After 24 h of D-cycloserine treatment, dilute 105-fold in minimal medium and plate on identification medium.

4

Pick small colonies and replica plate to verify identity as Pseudomonas, and confirm histidine auxotrophy (only one mutant from each of the 20 aliquots is used to prevent isolation of sibling mutants, which might confuse subsequent genetic analysis).

* All incubations are performed at 30 C, and all operations are performed aseptically. NTG, N-methyl-N'-nitro-

subjected to the selection procedure. Upon plating, it was determined that his- cells, which gave rise to small colonies when compared with the wild-type, were present in a ratio of 102:1, resulting in an enrichment of at least 106-fold. The mutagenesis and selection procedure described has been very successful in our laboratory and involves much less time and manipulation than procedures currently being employed. This procedure may have utility for other species of Pseudomonas and for isolation of mutants blocked in catabolism and may have potential for the isolation of mutant strains of other genera. This investigation was supported by Public Health Service Research Grant HD-7314 from the National Institute of Child Health and Human Development.

LITERATURE CITED 1. Chakrabarty, A. M., and B. W. Holloway. 1974. Genetic

2.

3.

N-nitrosoguanidine.

4.

L-broth (4) contained, per liter: 10 g of tryptone (Difco); 5 g of yeast extract (Difco); and 10 g of NaCl. Adjust final pH to 7.0 before autoclaving with dilute KOH.

5.

formed in which cells of a stable histidinie auxotroph were mixed with the wild-type cells at a ratio of 1:104. This mixture was then

1047

6.

linkage map of Pseudomonas aeruginosa, p. 643-646. In A. I. Laskin and R. A. Lechevalier (ed.), Handbook of microbiology, vol. 4. Chemical Rubber Co. Press, Cleveland. Cohen-Bazine, G., W. R. Sistrom, and R. Y. Stanier. 1957. Kinetic studies of pigment synthesis by nonsulfur purple bacteria. J. Cell. Comp. Physiol. 49:2568. Hegeman, G. D. 1966. Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91: 1140-1154. Luria, S. E., and J. W. Burrows. 1957. Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74:461476. Ornston, L. N. 1971. Regulation of catabolic pathways in Pseudomonas. Bacteriol. Rev. 35:87-:16. Ornason, L. N., M. K. Ornston, and G. Chou. 1969. Isolation of spontaneous mutant strains of Pseudomonas putida. Biochem. Biophys. Res. Commun. 36: 179-184.

Improved method of selection for mutants of Pseudomonas putida.

Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nit...
254KB Sizes 0 Downloads 0 Views