World Journal

of Microbiology

and Biotechnology

9, 562-565

Isolation and characterization of Pseudomonas aeruginosa mutants deficient in the utilization of the terpenoid citronellic acid M.L. Hector,* M.F. Murphy-Waldorf,

T.B. Giertych, M.J. Hickey and A.A. Haggard

Eighteen non-sibling mutants of Pseudomonas aeruginosa PA0 were isolated that were deficient in the utilization of the j?-methyl branched acid citronellic acid but not in the utilization of the unbranched n-octanoic acid (Cau mutants). These mutants are also deficient in the utilization of citronellol and citronellal. R68.45 plasmid-mediated transfer of chromosomal material has been used to map one of the mutations at about 52 min on the PA0 chromosome and to show linkage of some, but not all, of the other mutations to this region. This system is of interest for bioremediation in oil spill areas since /%methyl branches block normal /&oxidation and cause recalcitrance of organic molecules present in petroleum products. Key words: Bioremediation,

Cau, citronellic

acid, cintronellol,

Although n-alkanes are readily degraded by many microbes, branched-chain alkanes are more recalcitrant and hence tend to accumulate in the biosphere (McKenna 1972; Pimik 1977). Branched molecules have been detected in oil spill areas up to 10 years after the spill (Zafiriou 1973). Molecules with branches at the w-2 position (i.e. anteiso-terminus) are especially recalcitrant since this inhibits normal P-oxidation (Alexander 1973; Bartha & Atlas 1977; Schaeffer et al. 1979; Trudgill 1986). Thus these types of molecules require other metabolic pathways for degradation such as a-oxidation (Lough 1973; Beam & Perry 1974, o-oxidation (Pimik 1977) or /I-alkyl group removal (Seubert & Fass 196410; Cantwell et al. 1978). Much of the pathway for b-alkyl group removal was determined by Seubert and colleagues (Seubert & Remberger 1963; Seubert et al. 1963; Seubert & Fass 1964a,b) in a soil pseudomonad, Pseudomonas cifronellolis, isolated for its ability to grow on citronellol, a /?-methyl branched alcohol. This pathway involves the removal of the methyl group through carboxylation followed by oxidative cleavage of the carboxymethyl group to generate a substrate suitable for P-oxidation (Figure 1). An enzyme unique to this pathway, geranyl-CoA carboxylase (EC 6.4.1.5), is

M.L. Hector, M.F. Murphy-Waldorf. T.S. Giertych, M.J. Hickey and A.A. Haggard are with the Department of Chemistry, Californian State University, Chico, California 95929-0210. USA: fax: 916 696 6624. *Corresponding author. @ 1993 Rapid

Communications

of Oxford

Ltd

mapping,

Pseudomonas aerxginosu, terpene.

induced in other pseudomonads capable of growth on citronellol such as P. aeruginosa and P. mendocina (Cantwell et al. 1978) indicating the presence of this pathway in these pseudomonad species. To study this P-decarboxymethylation we have isolated mutants of Pseudomonas aeruginosa PA0 which have lost the ability to utilize citronellic acid and have designated them Cau (citronellic acid utilization) mutants. Using these mutants as donors in conjugal crosses, one cau gene has been mapped at about 52 min on the PA0 chromosome and shown to be unlinked to at least four other cau lesions.

Materials

and Methods

All bacterial strains used were derivatives of Psetldomonasaeruginosa PA0 and are described in Table 1. Except as noted, minimal medium was Medium R (Cantwell et al. 1978) containing either (% w/v): citronellol (C23190, Pfa]tz & Bauer), 0.1; citronellal (C23150, Pfaltz & Bauer), 0.2; citronellic acid [synthesized from citronella1 (Katsura et al. 1963)], 0.2; octanoic acid (O-390-7, Aldrich), 0.3; succinate disodium salt (V103; Baker), 0.4; xanthine (Sigma), 0.1; hypoxanthine (Sigma), 0.2; or benzoic acid (Mallinckrodt), 0.3. Complex medium was modified Luria broth (Carlson et al. 1983) containing 0.1% citronellol (LBCO medium). For plates, liquid medium was solidified with 2% (w/v) Difco agar. Amino acids were supplied at 50 pg/ml, nucleotides at 20 pg/ml, and the antibiotics used were (pg/ml): kanamycin sulphate (Sigma), 500; tetracycline (Sigma), 200; nalidixic acid (Sigma), 1500; and/or

Pseudomonas

/&&C&OH CllRONELLOL

-

Table

1. Pseudomonas

Designation

pall B

“s

&OSCoA

COSCoA

mltfanfs

aerughosa

nut using

cj~o~el~~f~

strains.

Relevant geno~~lp~no~~*

Reterence, source or derivation

MH503

IeulU

argFf0

Nal

MH518

led0

argFl0,

R68.45t

MH601-618

ieulg argF70 R66.45

cau

/eulO argFlO R68-45

cau-7

Nal,

This study; Nal-resistant derivative of PA025 Haas & Holloway (1976); R68.45 derivative of PA025 This study; Cau derivatives of MH503 containing R68.45

Donor

GERANYLCaA

MH601

hydrame

Nal,

This

study

(see

above)

Recipients*

I

MHf19

his-3 &p-S lys-56 nab501

PA0851

thi-l his-151 trp-150 pyrB21 pur-66 thr-9001 cys-54 pur-67 fbr-~1 pur-67 cys-59 meWOff met-9077 catAl nar-9077 cnu-9001 puuE8

COSCaA

Figure 1. Pathway at. 1978).

carbenicillin (Sigma), chemicals were of sources. All cultures

for the degradation

of citronellol

(Cantwell

1000; (or as indicated in the text). reagent grade and obtained from were grown at 37’C.

et

All other standard

Mufani Isolation Mutants unable to utilize citronellic acid were obtained by adding EMS (60 ~1) to 5 ml of a mid-growth-phase culture of MHS03 (see Table I) in LBCO, which was vortexed vigorously and then incubated for 2 h without shaking. Cells were harvested by centrifugation, suspended in 5 ml fresh LBCO, diluted I to 50 (v/v) in LBCO and incubated overnight with shaking to allow for of mutations. Cells were again harvested by expression centri~gation, washed with 5 ml of 0.9% (w/v) saline, centrifuged and suspended in 2.5 ml Medium R containing citronellic acid (mRCA). This suspension was diluted 1 to 20 (v/v) in mRCA and incubated with shaking for 6 to 8 h. One ml of this culture was added to 9 ml mRCA containing carbenicillin (2 mg/ml) in a 125 ml flask. After overnight incubation with shaking, cultures were appropriately diluted in 0.9% (w/v) saline such that 0.1 ml spread on LBCO plates produced about 200 colonies per plate. These colonies were screened for utilization of citronellic acid and octanoic acid by replica plating. Mutants able to utilize octanoic acid but not citronellic acid were designated Cau (citronellic acid utilization) and numbered sequentially. Eighteen non-sibling Cau mutants were obtained, corresponding to a frequency of approximately 1% of the colonies screened after carbenicillin counterselection. These mutants also could not use citronellol and citronella1 as expected from the pathway outlined in Figure 1. All mutants isolated exhibited a very faint growth on citronellic acid plates, even with highly purified citronellic acid. Although the Cau mutant phenotype was easily distinguishable from the wild type, it was not possible to select wild type colonies on a citronellic acid plate containing a background lawn of Cau mutants. Thus mapping by interrupted mating techniques, using sex factors such as FP2 or HFr donors such as PA0 1000 (Haas et al. 1981), was not possible. Therefore the mutations were mapped by R68.45 plasmid-mediated genetic exchange (Haas &

PA0944 PA0949 PA01834 PA02368

met-28

Pemberton & Clark (1973); Nal-resistant derivatives of PA0283 Pemberton & Holloway (1972) Royle et al. (1981) Royle et a/. (1981) Royle et a/. (1981) Hanne et a/. (1983)

‘arg--Arginine: caf-catechol; cau-citronellic acid utilization; cnu-carnosine utilization; cys-cysteine; his-histidine; leti ieucine; /ys--fysine; mef~ethionine [met-9077 = met-9020; Matsumoto et al. (1978)]; naL--nalidixic acid resistance; naf-nitrate reductase; pur-purine; puu-purine utilization; pyr-pyrimidine; thgthiamine; fhr-threonine; frp-tryptophan. t The R66.45 plasmid confers resistance to carbenicillin, kanamycin, and tetracycline and has chromosome mobilizing ability (cma) (Haas & Holloway 1976). $ All recipients are cau positive.

Holloway markers

1978) using linkage analysis of known map locations.

of the ctlti with

auxotrophic

The plasmid R68.45 was transferred, according to Haas & Holloway (1976), from MH516 (Table 1) to Cau mutants in order to generate donors for the bacterial conjugations described below. Cau recipients containing R68.45 were selected on LBCO plates containing kanamycin (500 pg/ml) and nalidixic acid (1.5 mg/ml) then purified and maintained on LBCO containing kanamycin (500 pg/ml). Purified recipients were tested for antibiotic resistance on appropriately supplemented Medium R plates and for chromosome mobilizing ability (Cma) (Haas & Holloway 1976). Strains which were resistant to tetracycline, carbenicillin and kanamycin and exhibited Cma were selected as donors and maintained on LBCO plates containing kanamycin (500 pg/ml). R68.45 plasmid-mediated conjugations were performed by plate matings (Stanisich & Holloway 1972). Cau (R68.45) strains grown overnight in LBCO liquid containing 500 pglml each of kanamycin and carbenicillin were used as donors and PA0 strains with appropriate auxotrophic or utilization markers were used as

M.L. Hector et al. recipients. Prototrophic ex-conjugants were selected on Medium R containing succinate as carbon source and supplemented with citronellol (mRSucC0) and appropriate amino acids and nucleotides for unselected markers. CatA + colonies were selected on minimal

plates

containing

benzoic

acid

as sole

carbon

source;

PuuE + colonies were selected on minimal plates containing xanthine or hypoxanthine as sole carbon source; Nar+ colonies were selected anaerobically on Vogel/Banner minimal plates (Vogel & Bonner 1956) containing 0.4% (w/v) glucose and supplemented with 0.5% (w/v) KNO,. Ex-conjugates were purified on the selective media, induced by growth on mRSucC0 plates when

necessary,

then

scored

for

citronellic

acid

utilization

by

replica plating.

Results

and Discussion

The results from the matings of MH601 (cuu-I, R68.45) with the recipient strains listed in Table 1 are presented in Table 2. Ex-conjugates were selected for one or two prototrophic markers then screened for cotransfer of cau-1. The highest percentage of coinheritance (80%) of cau-l with a single selected marker was observed with nar-9011 located at 53 min, indicating that cuu-I is very close to nar-9017. Co-inheritance analysis of cuu-I with recipients containing

Table

2. Linkage

Selected

of cau

marker(s)

Linkage

with

mutations

markers.

PA0 chromosome location* (mln)

Linkage

0

1 (l/95) 0 (0195)

2 5

lys-56

9 20

mef-28 trp-6 met-901 cafA 1 nar-9011 cys-64 puuE8 pur-67 cys-59