Molecular Microbiology (1992) 6(21), 3109-3119

Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes C. Clepet,^ F. Borne,' V. Krishnapillai,^ C. Baird,^ J. C. Patte^ and B. Camr* 'Laboratoire de Chimie Bacterienne, Centre National de la Recherche Scientifique, 31 chemin J. Aiguier, BP 71 13277. Marseille. France. ^Department of Genetics and Developmental Bioiogy, Monash University, Clayton, Victoria 3168. Australia. Summary Three genes from Pseudomonas aeruginosa involved in threonjne biosynthesis, hom, ihrBand thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The ihrBgene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.

Introduction Pseudomonas aeruginosa has been studied intensively for its well-known catabolic properties. With respect to biosynthetic pathways, genetic experiments have focused mainly on tryptophan (Crav^ord, 1986; 1989) and arginine (Haas et ai., 1990) biosynthesis. Further nnolecular studies of anabolic genes in this bacterial species are of interest for several reasons. For example, many biosynthetic genes are concentrated in one half of the P. aeruginosa chromosome (Holloway and Morgan, 1986). Biosynthetic Received 13 December, 1991: revised 22 May. 1992; accepted 28 May, 1992, 'For correspondence. Tel. (16)91 71 16 96; Fax (16) 91 71 89 14.

genes belonging to the same pathway are often scattered rather than being organized in operons on the chromosome (Holloway and Morgan, 1986; O'Hoy and Krishnapillai, 1987), which is in contrast to the situation in Escherichia coli. Moreover, repression of biosynthetic enzymes in P. aeruginosa is less common than in £ coii (Crawford, 1986; 1989; Haas etal., 1990). We have undertaken to study the genes involved in threonine biosynthesis in P. aeruginosa. Of particular interest in terms of evolution is the organization of genes for enzymes in a particular pathway, the fusion of specific genes to form multifunctional enzymes, and the different strategies for the regulation of gene expression. In all cells studied to date, five enzymatic steps are involved in the conversion of aspartic acid to threonine as schematized in Fig. 1. The two first steps catalysed by aspartokinase and aspariate semi-aldehyde dehydrogenase are common to diaminopimelate, lysine and methionine biosynthesis, whereas the third enzyme, homoserine dehydrogenase, is also used for methionine biosynthesis. Threonine biosynthesis is achieved through successive catalysis by homoserine kinase (HK) and threonine synthase (TS). In E. coli, four enzymes of thr biosynthesis are encoded by the //jr>ASC operon (Saint-Girons and Margarita, 1978), The product of the thrA gene is a single polypeptide chain containing both the aspartokinase 1 (AK1, N-terminal region of the polypeptide) and homoserine dehydrogenase 1 (HDH1, C-terminal region) enzyme activities (Cohen, 1983). Homoserine kinase and TS are encoded by thrB and thrC. respectively. Regulation of the £ coli threonine operon is accomplished by an attenuation mechanism sensitive to threonine and isoleucine {Gardner, 1979; reviewed by Lynn and Gardner, 1983). In Serratia marcescens, the genes are organized in a manner similar to that in £ coii (Komatsubara et ai., 1979). In Baciilus subtiiis there is a cluster of three genes, with the order hom-thrC-thrB, encoding homoserine dehydrogenase (devoid of aspartokinase activity), TS and HK, respectively; these genes may form an operon (Parsot, 1986; Parsot and Cohen, 1988). The HK is repressed by threonine (Skarstedt and Greer, 1973). In Corynebacterium giutamicum two unlinked genetic loci exist: (i) the hom-thrB operon, and (ii) the thrC gene. They encode homoserine dehydrogenase, HK, and TS, respectively. Expression of the hom-thrB operon is

3110 C. C/epefetal. aspartyl- phospate

aspartic semiadehyde

dihydropicolinate

homoserine

homoserine phosphate

L threonine

0-succinyl homoserine a-ketobutyrate

I L-mediionine

LL diaminopimelate

I I L isoleucine

L-lysine

1-aspartokinase 2-aspartic semialdehyde dehydrogenase 3-homoserme dehydrogenase

4-homoserine kinase 5-threonme synthase

Fig. 1. The threonine biosynthetic pathway and its derivative. The biosynthetic pathways leading to lysine, methionine and isoieucine are schematically represented Irom the branch-point metabolites of the threonine pathway presented on the top line. Numbers above the arrows refer to the enzymes, the names of which are indicated at the bottom of the figure.

repressed by methionine (Follettie et al, 1988), A similar genetic organization is observed in Brevibacterium lactofermentum (Han ef a/., 1990).

of presumptive thrB mutants could be made. This is not because of the poor sensitivity of the method, sinoe aspartokinase activity, determined by the same coupled assay with aspartate as a substrate, could be measured easily.

Results Mutants of P. aeruginosa exhibiting a Thr phenotype We have examined a large number of mutants described as threonine auxotrophs (listed in Table 1). Their phenotype was checked and the HDH- and HK-speoific activities were determined in crude extracts (we were unable to assay TS activity accurately in crude extracts of P. aeruginosa using the assay proposed by Daniel (1976), by separation of radioactive phosphohomoserine as a substrate, and produced threonine after barium precipitation). Mutants were grouped into three classes. Representative strains of each class are shown in Tables 2 and 3. Mutants requiring threonine alone for grovi/th. Thr" mutants could be affected either in thrB or in thrC. Although HK activity was easily measurable when highly expressed (see below), activities obtained from crude extracts of wild-type PA01 were not significantly different from the blank value obtained without substrate with the coupled assay used. Therefore, no clear-out identification

Mutants requiring homoserine aione. or threonine + mefA7/on/ne, TheHom"(orThr~ -i- Met") phenotype corresponds to mutants affected in the hom gene as proved by the absence of HDH activity in crude extracts. Mutants requiring threonine + homoserine (or threonine •+• methionine). In these mutants homoserine alone did not allow growth and HDH activity could not be detected. The (Hom" -I- Thr") or (Thr" Met") phenotype could be due to a polar mutation that would simultaneously affect expression of both the hom gene and the thrB and/or the thrC gene, organized in a multicistronic operon. These results and the faot that only one thr locus has been mapped on the P. aeruginosa chromosome (at 31 min; Holloway et al., 1990), lead us to suppose the possible existence of a multicistronic gene organization. Molecular cloning of hom, thrB and thrC Isolation of the Pseudomonas genes was performed by both homologous complementation of P. aeruginosa

Pseudomonas aeruginosa threonine genes 3111 auxotrophs and heterologous complementation of £ coli auxotrophs (see the Experimentai procedures). The results of the heterologous complementation experiments are summarized in Table 3 (upper part). They demonstrate that pUC19-derived plasmids p14 and pST7 (see the legend of Table 3, and Fig, 5) complement all of the thrB mutants whereas cosmid pM0010316 (see the legend of Table 3) complements hom and thrC mutants, but not thrB mutants. For homologous complementation, plasmid pM7 was derived from pST7 in the shuttle vector pMMB66EH (see the legend of Table 3, and Fig. 5). Plasmid pM7 and cosmid pM0010316 were transferred by conjugation to different mutant strains of Pseudomonas. All classes of mutants (Thr", Hom", Hom" + Thr") were complemented by the cosmid whereas none was complemented by plasmid pM7 (Table 3, lower part). We postulate that all Thr" Pseudomonas mutants are thrC mutants, that no thrB mutant has been isolated so far, and that cosmid pMOOl 0316 carries hom and thrC. To test this hypothesis, the following experiments were performed. The £ coli thrB or thrC genes were cloned on

PAO2770

thr-70, srgFlO, teu-)0::Tn7

PAO2829

thr-62, mtU'2

PAO2830

thr-63. mtu-2

PAO2831

thr-64. mtu-2

PAO2832

thr-65. mtu-2

PAO2833

thr-66. mtu-3

PAO6093

thr-6093..Jn5-751. met-9011. amiE200. StrA

E. coli C600

GT28

hsdR. supE44. thi-1. leuBB. lacYI. tonA21. thrB 1023 thrBIOOO. metU005. lysC1004. prO'1001.serB22 thrA1015, metLMW05. lysC1004. pro-1001.serB22 HirH. thrClOW

GT869

thrB1004. pro. thi. strA. hsdS.

K38

HfrC(x)

POM 734

F

GT12 GT15

RRIiMIS

RRI-5 Genotype/Characteristics

P. aeruginosa PAOl Prototrophic PAO213 thr-56. met-28. trp-6. lysA12. proA882, iiv-226. his-4 PAO277 thr-46. met-28. trp-6 PAO278

thr-47, met-29. trp-6

PAO760

thr-61. pyrF

PAO874

thr-2. pur-66. his-151. thi-1. ura-21

PAO887 PAO1016

thr-22,pro-71.his-1S1. pyrB21. thi-1 thr'348

PAO1278

thr-9001. rif-125

PAO1480

(/ir-5ft:Tn5, trpCD64, rif-5, fon-1

PAO1490

f/7/--60::pfyiO514 (Cb, Km, Tc, Sm. Sp, Su, R68trfA (Js)::Tn2521) trpCD54. rif-5. fon-1 f/7r-7J;:pMO514, trpCD54. rif-5, fon-1

PAO1501

PA02114 PA02115 PAO2162 PAO2169

pur-66,

thr-9002. met-9020. hex-9001. leu9006. arg-9012. nar-9001 thr-9003, met-9020. hex-9001. leu9006. arg-9012. nar-9001 thr'9005. met-9024, hutC107. ami151 thr-9006, met-9025. hutC107. ami151

Reference/Source S17-1 Holloway (1955) Royleefa/. (1981) Holloway collection Molloway collection Holloway collection Holloway collection Holloway collection Leisinger collection Holloway collection O'Hoy and Krishnapillai (1987) O'Hoy and Krishnapillai (1987) O'Hoy and Krishnapillai (1987) Holloway collection Holloway collection Holloway collection Holloway collection

Cossart ef al. (1979) Theze ef ai. (1974b) Theze et al. (1974b) Theze ef al. (1974b) Parsot (1986)

traD36, proA ' , proB')

Table 1. Strains and plasmids used. Strain/ Plasmid

Holloway collection Holloway collection Hollo way collection Holloway collection Holloway collection Holloway collection RellaefaA(1985)

,araD139.ara::iMucts)MacX74. galU. galK. rps'Mudll-1734 leu. pro. thi, rpsL, hsdR. hsdM. lacZMVI15. F'llacI''. lacZAM15. pro'] Leu' Thr transductant of RRI-AM15 obtained by PI transduction with donor GT 28 recA, proA. hsdR. (RP4-2, Tcifuiu. Km:tn1)

Plasmid pBR327

Ap, Tc, pMBI

pGP1-2

Ap, pMBI

pHPS45Hg plP3 plP79

Ap, pMBI, Hg

PKT240 pf^MB66EH, HE pNM480,481,482 PRK2013

Cb, Km, RSF1010 Cb, RSF1010

Ap, thrABC. pf^BI

Ap, thrS.pMBI

Russel and Model (1984) de Castilho ef al. (1984) Ruther(1982)

Riither (1982)

Simon ef a/, (1983)

Covarrubias ef al. (1981) Tabor and Richardson (1985) Fellay efa/. (1987) Parsot ef a/. (1983) P. Cossart ef al. (unpublished) Fursteefa/, (1986) Bagdasarian ef a/. (1983)

Ap, lacZa, pMBI

Minton (1984)

Km" Tra ColEI replicon (c, 50kb)

Figurski and Helinski(1979) Tabor and Richardson (1985) Yanisch-Perron ef aA(1985) Stratagene

pT7-6

Tc, Ap, pMBI

pUC19

Ap, lacZa, pMBI

pBluescript M13mp18,19

Ap,/acZa, pMB1,f1 lacZa

Yanisch-Perron ef al. (1985)

Cb, carbenicillin; Km. kanamycin; Tc, tetracyciine; Sm. streptomycin; Sp, spectinomycin; Su, sulphonamide; Ap, ampicillin.

3112

C. Clepet et 3i.

TaUe 2. Enzymatic activity of mutant strains and the effect of piasmids. Specific Enzyme Activity Strain

Phenotype

PAOl wild type PAO2830 thr-63' PAOU80 thr-59°

ThfThr" Hom" or Thr + Met" Hom + Thr Thr + Met

PA01016 Wr-34fl= PAO2169 thr-9006 m0t-9O25 PA01/pM7 PAO1/PM0010316

HDH

HK

AK

150 165