Butyramide-utilizing Mutants of Pseudomonas aeruginosa 8602 which Produce an Amidase with Altered Substrate Specificity

Abstract

S U M M A R YMutants of Pseudomonas aeruginosa 8602 were isolated which, unlike the wild type, were able to grow with butyramide as a carbon source. Six mutants derived from the constitutive strain c 1 1 were shown to produce an enzyme (B amidase) with altered electrophoretic mobility and altered substrate specificity. The apparent K," for butyramide of the B amidase was about a tenth of that of the A amidase and the V,,,,,, was about ten-fold greater. A further mutation produced mutants able to grow on valeramide. I N T R O D U C T I O NPseudomonas aeruginosa 8602 grows in a minimal medium containing either acetamide or propionamide as a carbon + nitrogen source, and both these amides induce the synthesis of an aliphatic amidase (acylamide amidohydrolase EC 3.4.14). This organism is unable to utilize butyramide for growth and Kelly & Clarke (1962) found that acetamide-grown bacteria (and extracts prepared from them) had very little amidase activity with butyramide as a substrate and that butyramide did not induce amidase synthesis in a succinate + minimal salt medium. The magno-constitutive mutant c 1 1 (Brammar, Clarke & Skinner, 1967) is also unable to grow on butyramide and produces an enzyme which is indistinguishable from the wild-type amidase.We have now obtained mutants of strain c 11 which grow on agar plates and in liquid medium with butyramide as the carbon source. The amidase produced by one of these butyramide-utilizing mutants has been purified and compared with that produced by strain c 11. M E T H O D SOrganisms. The parent strain was Pseudomonas aeruginosa 8602. Strain c 11 was a spontaneous mutant, constitutive for amidase (Brammar et al. 1967) and was used to isolate the butyramide-utilizing mutants designated B I to B 6 (see later for genetic descriptions) .Media. The minimal salt medium described by Brammar & Clarke (1964) was used for all growth experiments; solid media contained 1 . 2 % (w/v) Oxoid No. 3 agar. Lactate and pyruvate media were prepared by adding sterile solutions of the sodium or potassium salts aseptically to autoclaved minimal salt medium. Butyramide plates contained 0 . 1 % (w/v) and valeramide plates 0.2y0 (w/v) of the specific amide, and were prepared by adding sterile solutions of the amides to minimal agar. Other media were prepared as described by Brammar et al. (1967). 18-2

Biochemical and Immunological Comparison of Aliphatic Amidases Produced by Pseudomonas Species

“…A washed suspension of an acetamide-grown culture of 277 was used to assay amidase hydrolase and transferase activities. For the transferase reaction the rates for acetamide:proprionamide were in the ratio 1o0:23 which was similar to that previously obtained for the PACI amidase A (Brown et al 1969). The relative rates of amide hydrolysis, determined by the Conway method, were also similar to those for PAC amidase A ( Table 4).…”

“…Much greater differences had previously been observed between the wild-type PACI amidase and the B and A1 amidases produced by mutants of this strain (Brown et al 1969;Brown & Clarke, 1972).…”

“…PAC128, grew readily on B plates because they were resistant to butyramide repression and produced large amounts of the wild-type A amidase which has low but significant activity towards butyramide (Brown & Clarke, 1970). The constitutive mutant PACI I I was the parent of strain P A C~~I which produced the altered B amidase with a much higher activity for butyramide as substrate than the wild-type A enzyme (Brown et al 1969). The succinate+lactamide (S/L) plates were used to isolate mutants which were resistant to catabolite repression; lactamide is a very good inducer but a poor substrate so that a high amidase concentration per bacterial cell is required for growth in a medium in which succinate is the major carbon source (I %, w/v), and lactamide at a low concentration (0.02 %, w/v) provides the nitrogen source.…”

“…Cell-free extracts were examined by starch gel electrophoresis and tested for immunological cross-reactions with antiserum prepared against a purified preparation of P. aeruginosa amidase from a constitutive mutant of strain PACI used in our previous studies. We have isolated a number of mutants with altered substrate and inducer specificities (Brown, Brown & Clarke, 1969;Brown& Clarke, 1972) which can grow on amides not available to the wild-type strain. The properties of the aliphatic amidases produced by the four different Pseudomonas species were compared with those of the mutant P. aeruginosa strains.…”

“…Antiserum (10 pl) prepared against Pseudomonas aeruginosa A amidase (Brown et al 1969) was placed in the centre well of a gel prepared on a microscope slide (2 ml I %, w/v, Difco agar Noble in barbitone+acetate buffer). The wells were cut with a no.…”