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Journal of General Microbiology ( 1989), 135, 29 17-2929. Printed in Great Britain 2917 Cloning and Genetic Analysis of Six Pyrroloquinoline Quinone Biosynthesis Genes in Methylobacterium organophilum DSM 760 By F R A N C I S BIVILLE, E V E L Y N E T U R L I N A N D F R A N C I S G A S S E R * UnitP de RPgulation de I’Expression GPnPtique, Dipartement de Biochimie et GinPtique MolPculaire, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France (Received 2 May 1989; revised 26 June 1989; accepted 9 August 1989) ~~~ After EMS mutagenesis, mutants of Methylobacterium organophilum DSM 760 unable to synthesize pyrroloquinoline quinone (PQQ) were selected among mutants which did not utilize methanol but were still able to use methylamine as growth substrate. Six different pqq genes (pqqA topqqF) were identified by complementation analysis. The genespqqA topqqD, cloned in a single R’ plasmid, were grouped in a 3.9 kb DNA fragment. The genespqqA andpqqB belonged to a single transcription unit independent from the adjacent gene pqqC. The gene pqqD was contained in a short DNA segment of approximately 0.1 kb, separated from pqqC by a region with no apparent role in PQQ biosynthesis. Two other genes were identified : pqqE, which was closely linked to pqqD; and pqqF, located approximately 19 kb from the other genes. Directed mutagenesis by marker exchange provided chromosomal insertion mutations of these genes in M . organophilum. Attempts to express the pqq genes in two heterologous hosts, Escherichia coli and Pseudomonas testosteroni, were unsuccessful, and no plasmid containing all of the pqq genes was isolated. INTRODUCTION Pyrroloquinoline quinone (PQQ) is the prosthetic group of a recently discovered category of NAD(P)-independent oxidoreductases known as quinoproteins (reviewed by Duine et al., 1987). Numerous examples of enzymes now recognized as quinoproteins have been characterized in both eukaryotes and prokaryotes. Among the latter organisms, the most extensively studied quinoproteins are alcohol dehydrogenases (e.g. methanol dehydrogenase, MDH, from methylotrophic bacteria; Anthony, 1986) and aldose dehydrogenases (e.g. glucose dehydrogenase, GDH, of Acinetobacter calcoaceticus, an enzyme also found in other aerobic or facultatively anaerobic Gram-negative bacteria; Duine et al., 1979; Van Schie et al., 1987). Other well-studied quinoproteins are methylamine dehydrogenases, associated with methylamine catabolism in several bacteria (Duine et al., 1987) and amine oxidases, linked to the metabolism of lysine in human placenta (Van der Meer & Duine, 1986). Some bacteria produce the apoenzyme of PQQ-dependent dehydrogenases but not the prosthetic group. Examples are the GDH of Escherichia coli (Hommes et al., 1984; Ameyama et al., 1986) and the ethanol dehydrogenase of Pseudomonas testosteroni (Groen et al., 1986). In these organisms, PQQ has the physiological role of a growth factor for the use of substrates dissimilated through quinoprotein activity. The biosynthetic pathway leading to PQQ is not yet known. Recently, however, two laboratories independently identified the structure of PQQ precursors and further suggested that tyrosine and glutamic acid are the initial compounds of its biosynthetic pathway in Methylobacterium extorquens AM1 (Houck et al., 1988) and in Hyphomicrobium X (Van Kleef & Abbreviations: GDH, glucose dehydrogenase; MDH, methanol dehydrogenase; PQQ, pyrroloquinoline quinone. 0001-5567 0 1989 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 2918 F. BIVILLE, E. T U R L I N A N D F. GASSER Duine, 1988). PQQ biosynthesis mutants were first reported in A. calcoaceticus as being unable to oxidize glucose to gluconate (Goosen et al., 1987). These mutants were used for cloning four genes involved in PQQ biosynthesis. DNA sequence analysis failed to give clues to the nature of putative biosynthesis enzymes (Goosen et al., 1989). In some facultative methylotrophic bacteria such as M. extorquens strain AM1, the ability to grow with methanol or methylamine is associated with PQQ-dependent dehydrogenases. Consequently, potential PQQ- mutants cannot grow with either of these substrates. However, this phenotype is also found in all the mutants impaired in the numerous steps of formaldehyde assimilation by the serine pathway, thus complicating the isolation of PQQ- mutants. In a previous paper, we showed that methylamine utilization in Methylobacterium organophilum is not PQQ dependent since methylamine is dissimilated through the methylglutamate cycle and oxidized by methylglutamate dehydrogenase, a flavin-dependent enzyme (Biville et al., 1988). A mutant impaired in PQQ biosynthesis, MTM1, was isolated among mutants unable to grow on methanol medium but able to grow normally with methylamine as substrate and consequently was unaltered in the serine pathway. The gene mutated in MTM1, pqqA, was isolated and localized in the vicinity of moxF, the structural gene of MDH apoenzyme (Mazodier et al., 1988). In this work, the isolation of other PQQ- mutants allowed the identification and the localization of six genetic loci, pqqA-pqqF, involved in PQQ biosynthesis. METHODS Bacterial strains and plasmids. These are listed in Table 1. Media and growth conditions. M . organophilurn was grown on minimal medium (medium A) of MacLennan et al. (1971) containing 0.5% methanol or 0.2% sodium succinate or 0.2% methylamine hydrochloride. For growth of PQQ- mutants, methanol medium was supplemented to 1 VM with filter-sterilized PQQ (Methoxatin; Fluka). E. coli strains were grown on LB medium (Miller, 1972). Antibiotics, when added, were used at the following final concentrations (CLgml- l ) : kanamycin, 25 ; ampicillin, 50 ; rifampicin, 20; tetracycline, 10; chloramphenicol, 25. Biochemical techniques.The preparation of crude extracts, MDH assay, determination of PQQ concentration in crude extracts or in culture supernatants, and protein staining after PAGE have been reported previously (Mazodier et al., 1988). Molecular biology techniques. Plasmid DNA was isolated according to Humphrey et al. (1975). Endonuclease digestion, alkaline phosphatase treatment, ligation and DNA transformation in E. coli were done essentially as described by Maniatis et al. (1982). M . organophilum DNA was purified essentially by the method of Marmur (1961). Mutagenesisand selection of PQQ- mutants. A 2 ml sample of a culture of wild-type M . organophilumDSM 760 in succinate medium was treated during the early exponential phase (ODGo00.6) with 0-2% ethylmethane sulphonate (EMS).After 2 h incubation at 30 "C with shaking, the culture was centrifuged and washed twice with 4 ml minimal medium without carbon source. The final pellet was resuspended in 4 ml succinate minimal medium and the culture incubated at 30 "C for 20 h. This culture was diluted and spread onto succinate minimal medium containing 0.05% allyl alcohol. After incubation for 6-8 d at 30 "C, colonies were re-isolated on the same medium. Resistance to allyl alcohol can result from a mutation which prevents expression of active MDH (MDH oxidizes allyl alcohol to the toxic compound acrolein) or can be acquired independently of the loss of MDH activity (Nunn & Lidstrom, 1986a); the latter mutants are still able to grow on methanol. The colonies growing in the presence of allyl alcohol were patched successively onto minimal medium supplemented with methanol or with methylamine. Only the methanol-negative, methylamine-positive mutants were tested for PQQ-dependent growth on methanol medium. Genetic techniques.The details of most of the techniques have already been described (Mazodier et al., 1988). In this work, genomic banks were constructed in the cosmid pLA2917 (Allen & Hanson, 1985), which contains a unique BglII cloning site in the kanamycin resistance gene. Two sources of DNA were used : chromosomal DNA from M . organophilum,or plasmid R51, which contains DNA from M . organophilum. In both cases the DNA was partially digested by Sau3A. Hybrid DNA from the pLA2917 derivatives was packaged in oitro with Packagen according to the instructions of the supplier (Promega Biotec). After infection of the host strain E. coli S17-1, tetracycline-resistant, kanamycin-sensitive colonies were selected. The M . organophilum gene banks constructed in E. coli were screened by drop mating on minimal medium plates supplemented with 0.5% methanol and 0.01% sodium succinate. The medium composition prevented growth of the donors and allowed growth of the recipient M . organophilum mutants only after complementation of the mutations. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 PQQ biosynthesis genes in M . organophilum 2919 Quantitative conjugation experiments were done after mating to distinguish trans complementation from recombinational events between homologous chromosomal and plasmid DNA. The donor was an overnight culture of E. coli S17-l(pLA2917 : :X),X being a fragment of cloned DNA from M. orgunophilum. The recipient was a PQQ- mutant grown in minimal medium with succinate. Both cultures were centrifuged, washed three times in minimal medium without carbon source and resuspended at OD,,, 1. Donor (0.1 ml) and recipient (0.2 ml) cultures were spread onto a succinate minimal medium plate which was then incubated for 18-20 h at 30 “C.This mating mixture was then harvested in minimal medium without carbon source, diluted and spread onto minimal medium plates supplemented with succinate, succinate plus tetracycline, or methanol. After incubation, the number of colonies growing on each medium was compared. Plasmid entry into the recipient occurred at a frequency of about 1O-* per recipient cell. A ratio of methanol-positive to tetracycline-resistant exconjugants of approximately 1 was taken as an indication of trans complementation. When a recombinational event occurred, this ratio was in the range 0.01-0.05. Before each cross, the frequency of revertant cells was estimated by plating onto minimal methanol medium; it was always lower than lo-’. Consequently reversion and recombinational events could not be confused. Directed mutagenesis by insertion of antibiotic-resistance genes in defined locations of the chromosome of M. organophilumusing derivatives of pSUP106 and the obtaining of R plasmids with the R6845 derivative pJB3J1 have been described (Mazodier et al., 1988). Transposon Tn5 was inserted into plasmid DNA by the method of Kleckner (1977) as previously reported in detail (Mazodier et al., 1988). RESULTS Isolation and characteristics of PQQ- mutants Under the conditions described in Methods, 80% of the cells of M . organophilum DSM 760 were killed by EMS. In preliminary experiments the mutagenized cells were used to inoculate separate small cultures, with the aim of isolating a single mutant strain from each culture, so as to obtain independent mutations. However, the PQQ- mutants obtained by this method were all affected in pqqA, as determined by complementation experiments with plasmids pM0500 and pM0512 (Fig. 1) (Mazodier et al., 1988). This suggested a hot-spot of sensitivity to EMS mutagenesis inpqqA. As a consequence, the isolation of mutants affected in loci other thanpqqA required the screening of a large number of mutant strains. To avoid the burden of numerous small independent cultures, it was decided to risk the isolation of several strains bearing the same mutation by using a single, large culture, as described in Methods. The numerous colonies isolated from this culture on succinate medium containing ally1 alcohol were re-isolated on the same medium. Five per cent retained their ability to grow on methanol medium. Among 1400 colonies unable to grow on methanol medium, 90% could grow with methylamine as sole carbon source and, consequently, were not affected in the serine pathway of carbon assimilation. Seven hundred (55%) of the latter colonies had their growth on methanol medium restored by PQQ addition; 484 phenotype-stable mutants of this class were kept for further study. All these PQQmutants were subjected to complementation tests by drop mating with pM0500 and pM0512 to determine whether they were pqqA mutants or mutants affected in other pqq genes. Only 64 of the mutant strains (13%) were not complemented by pM0500 and pM0512 and therefore were considered as affected in genetic regions other than that of pqqA. The characteristics of representatives of each category of mutant (see below) are similar to those already described forpqqA mutants (Mazodier et al., 1988), i.e. no PQQ could be detected in crude extracts or in culture supernatants and the apo-MDH was practically undetectable by Coomassie blue staining after PAGE (results not shown). Genetic analysis of mutants complemented by R’51 (group I ) Plasmid R’51, a previously isolated derivative of pJB3J1 (Mazodier et al., 1988), complemented all the pqqA mutants and, in addition, 14 of the 64 mutants characterized as non-pqqA mutants. R’5 1 was not a suitable tool for genetic analysis but, since it contains a large amount of M . organophilum DNA (about 100 kb), it was used as a source of DNA to build a genomic library in the cosmid pLA2917. Cosmid pM0550 (not shown), isolated from this library, retained the complementing abilities of R51. Subcloning of various restriction Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 2920 F. BIVILLE, E. T U R L I N A N D F. GASSER Table 1. Bacterial strains, phage and plasmids Bacteria Relevant characteristics Escherichia coli S17-1 recA thi pro, contains an RP4 derivative integrated in the chromosome providing tra functions used in this work for transfer of plasmids pLA2917 and pSUP106 Spontaneous rifampicin-resistant derivative of S17-1 hsd (rB1mi) recAB proA2 S17-1 Rifr HBlOl 1101 Methylobacterium organophilum ptsH1, his62, thi-I, relAl DSM 760 (wild-type strain) Pseudomonas testosteroni M . organophilum PQQmutants Representative mutants obtained with EMS : MTMl 81,452 474 71 430 530 Kanr cartridge insertion mutants : . MDQl MDQ2 MDQ3 MDQ4 MDQ6 MDQ7 MDQ8 MDQ9 Phage A467 ATCC 15667 (wild-tye) Plasmids pLA29 17 pSUP 106 pUC4K pRK290 pJB3J1 R'5 1 pM05 12 pMO5121 pM05 122 Source or reference* Simon et al. (1983) Boyer & RoullandDussoix (1969) Fox & Wilson (1968) Deutsche Sammlung von Mikroorganismen Groen et al. (1986) Mazodier et al. (1988) PQQ+; BamHI insertion near ppqA PQQ-; SalI insertion in pqqA PQQ-; XhoI insertion in pqqB-pqqC PQQ-; XhoI substitution in pqqE PQQ+; BamHI insertion between pqqC and pqqD PQQ+; XhoI insertion between pqqD and pqqE PQQ+; M I insertion between pqqD and pqqE PQQ-; PstI insertion in pqqF Mazodier et al. (1988) Mazodier et al. (1988) b221, cZ857, rex : :Tn5, 029, P80 a replicationdeficient, integrationless phage used to deliver TnS Kleckner et al. (1977) IncP, Tetr, Kan'; a pRK290 derivative with a single BglII site affecting Kanr expression Conjugative cosmid using RSFlOlO origin of replication; Cmpr Tetr Contains a 1400 bp DNA fragment encoding kanamycin resistance with BamHI, SalI and PstI sites at each end IncP1, Tetr; cloning vector with BglII and EcoRI sites A kanamycin-sensitive derivative of R68-45 Allen & Hanson (1985) Priefer et al. (1985) pJB3J1 derivative containing DNA from M . organophilum including moxF, pqqA and the region in-between with a Tn5 insertion close to moxF A 2.5 kb of BglII-XhoI fragment containing pqqA cloned in pSUP106 Same as pM0512 with a Kanr cartridge inserted at the BamHI site near pqqA Same as pM0512 with a Kanr cartridge inserted at the SalI site in pqqA Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 Pharmacia (cat. no. 27-4987-01) Ditta et al. (1980) J. E. Beringer, University of Bristol, UK Mazodier et al. (1988) PQQ biosynthesis genes in M . organophilum 292 1 Table 1 (continued) Plasmids Relevant characteristics Derivatives of pLA2917 containing M. organophilum DNA as indicated in Figs 1 and 2 6 kb of M. organophilum DNA pqqA region pM0500 3.8 kb of BamHI fragment including pqqA, pqqB, pMO552 P49C As pM0552, with a Kan' cartridge inserted at the pM05521 BglII site of pqqB As pM0552, with a Kan' cartridge inserted at the pM05522 XhoI site of pqqB-pqqC As pM0552, with a Tn5 insertion in pqqA pM05525 As pM0552, with a Tn5 insertion in pqqB pM05526 A 2.5 kb BglII fragment containing pqqC, pqqD pM055 1 and part of pqqB As pMO551, with a 0.7 kb SalI deletion pM0553 As pM0551, with a 0.3 kb SulI deletion pM0554 As pM0551, with a Kan' cartridge inserted at the pM05511 Xho site of pqqB As pM0551, with a Kan' cartridge inserted at the pM055 12 BamHI site between pqqC and pqqD About 22 kb of the pqqE region of M . organophipM0200 lum DNA A 7 kb BglII fragment containing pqqE pM0230 As pM0230, with a 1.8 kb BamHI deletion pM023 1 As pM023 1 , with a Kanr cartridge inserted at the pM023 1 1 XhoI site near pqqE As pM0231, with a Kan' cartridge at the SalI pM023 12 site near pqqE A 2.2 kb BglII-Psi1 fragment containing pqqE pM0240 As pM0240, with a Kan' cartridge inserted at a pM02401 XhoI site of pqqE As pM0240, with a Kan' cartridge inserted at the pM02402 BamHI site of pqqE A large BglII-PstI fragment resulting from ligpM0245 ation of the cloned M . organophilum DNA from pM05511 and pM0240 About 22 kb of the pqqE and pqqF regions of M . pM0600 organophilum DNA A 4 kb XhoI fragment containing pqqF pM0611 As pM0611, with a 2 kb BamHI deletion pM0612 A BamHI fragment from pM0611 p M 0 6 13 Source or reference* Mazodier et al. (1 988) * Bacterial strains and plasmids for which no source or reference is given were derived during this work. fragments of pM0550 in pLA2917 provided pM0551, containing a 2.5 kb BglII fragment, and pM0552, containing a 3.8 kb BamHI fragment. Fig. 1 shows restriction maps of the M . organophilum D N A fragments cloned in pM0551 and pM0552 and in the previously isolated two plasmids pM0500 and pM0512 (Mazodier et al., 1988). Mutants with the same pattern of complementation obtained with these plasmids were arranged in different classes as shown in Fig. 1. From these experiments it can be deduced that at least four genes can be recognized: pqqA, pqqB, pqqC and pqqD, which are contained in a 3.9 kb BamHI-BglII fragment of M . organophilum D N A . A more precise physical delimitation of these four pqq genes in this fragment was obtained with various deletion and insertion mutations. It was previously reported that pqqA was located in a 1.5 kb BamHI-BglII fragment at the left-hand end of this region (Mazodier et al., 1988). This information was obtained in complementation experiments with plasmids pM05121 and pM05122, each containing a pUC4K Kanr cartridge insertion in pM0512. We showed in the present work that pqqD was localized in a small BamHI-BglII fragment (about 0.1 kb) at the right-hand end of the 3.9 kb BamHI-BglII fragment, as Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 2922 F . BIVILLE, E . T U R L I N A N D F . GASSER 4/1 [ 1 I 5121 5122 I pM0500 pM05 12 l 1 1 pM0551 1 1 kb I 551 1 5512 pM0553 pM0554 - 5525 ss 5A2 EMS mutants in: P99B Prototype strains : Plasmids pM0500,512 pM055 1 pM0552 81 (2) R - C 452 (3) pqqC pqqD 474 (8) 71 (1) - - - R C C C C - Insertions and deletions in the above plasmids pM0512: 5121 5122 pM0551: 553 554 551 1 5512 pM0552: 5521 5522 5525 5526 pM0552 Insertion mutants MDQ2 MDQ3 C ND - C C C C - ND ND ND ND ND ND ND ND - ND ND C - C Fig. 1. Map of plasmids used in complementation analysis of group I PQQ- mutants, and corresponding chromosomal map of M. orgunophilum. The upper horizontal line represents the bacterial chromosome map, all the other lines the plasmid maps. All the DNA fragments shown were cloned in pLA2917 except pM05121 and pM05122, which were cloned in pSUP106 (Mazodier et al., 1988),and the plasmids carrying a Tn5 insertion, which were cloned in pRK290. Filled triangles indicate the location of the Kanr cartridge of pUC4K, with the identification number of the corresponding plasmid shown below. Open triangles indicate the location of Tn5 insertions, with the arrows above indicating the direction of transcription of the Tn5 resistance operon. The sign associated with each MDQ mutant on the chromosome map indicates whether it can (+) or cannot (-) synthesize PQQ. Each column of the table represents a class of mutants determined after the results of complementation experiments and designated after the number of the prototype strain of each class. The number of mutant strains in each class is given in parentheses. Abbreviations in the table: C, complementation; R, recombination; -, no complementation or recombination ; ND, not determined. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 PQQ biosynthesis genes in M. organophilum 2923 evidenced by the different complementing abilities of pM0551 and pM0552 and by the fact that the pqqD mutant 71 could be complemented by pM05512, a pM0551 derivative with a Kanr insertion at the BamHI site (Fig. 1). The right-hand end ofpqqC was located with the help of deletions of two contiguous SalI fragments in pM0551: a large one (0.7 kb) inside pqqC (pM0553) and a small one (0-3 kb) outside pqqC (pM0554). These two deletions, although located outside pqqD as previously defined, abolished complementation of the pqqD mutant 71. Obviously integrity of the DNA of the two SalI fragments was needed in cis topqqD+. Since the effect of the two SalI deletions on pqqD was equivalent to a polar effect, we tried to obtain Tn5 insertions in this region, but without success despite several attempts. On the basis of the results described above, and since none of the PQQ- mutants isolated mapped in the SalI-BamHI fragment between pqqC and pqqD, it appeared probable that this region is not involved in PQQ biosynthesis, at least as a structure encoding biosynthesis genes. The delimitation of pqqC and pqqB was not clear-cut. Complementation of pqqC mutants by pM0551 was abolished by a Kanr cartridge insertion at the XhoI site of pM0551 (plasmid pM055 1 1). A similar insertion at the same XhoI site of pM0552 (plasmid pM05522) abolished complementation of all the mutants affected in pqqB and pqqC. This experimental observation cannot result from a polar effect of the Kanr cartridge on a single transcription unit containing pqqB-pqqC, since no such polarity effect was observed in M. organophilum (see below). This absence of polarity was confirmed by the lack of a negative effect on pqqC of an insertion at a BglII site in pqqB (pM05521). This suggests that the 0.5 kb BglII-XhoI segment contained an overlapping DNA fragment common to both pqqB and pqqC genes. The table in Fig. 1 shows that there were two classes of mutants corresponding to the genomic designation pqqB. Although this conclusion might have to be reconsidered, it was suggested by the results of complementation experiments with plasmid pM05521, which did not differentiate the two classes. Transposon Tn5, which has polar transcriptional effects, was used to explore the functional organization of the four pqq genes. Tn5 was transposed into the 3.9 kb BamHI fragment of pM0552 cloned in pRK290 and the location of the insertions determined by restriction analysis. In pMO5525, a Tq.5 insertion in pqqA abolished the complementation of both pqqA and pqqB mutants but not pqqC mutants, suggesting the presence of a single transcription unit, pqqA-pqqB. Another Tn5 insertion in pqqB (pM05526) abolished only the complementation of the pqqB mutation of strain 452 and had no effect on pqqC mutants. To avoid the possibility of errors due to multiple mutations caused by EMS, directed mutagenesis was carried out by transferring the Kanr cartridge of pUC4K into the chromosome of M. organophilum, thus creating single sites of mutation. The suicide plasmid pSUP106 was used for this purpose (Mazodier et al., 1988). Various restriction fragments from plasmids pMO5 121, pM05 122, pM055 1 1 and pM055 12, all containing the Kanr cartridge, were cloned in pSUP106 and transformed into E. coli S17-1. After conjugation of the transformants with M. organophilum, kanamycin-resistant colonies were selected, purified, and their growth on methanol medium with or without PQQ tested. Two chromosomal insertion mutants, MDQ2 located in pqqA and MDQ3 located in pqqC, were unable to grow on methanol medium without PQQ. MDQl was already known as a PQQ+ strain (Mazodier et al., 1988). The methanol-positive phenotype of MDQ6 confirmed that a DNA segment located betweenpqqC andpqqD was not involved in PQQ biosynthesis. The table in Fig. 1 shows the results of complementation experiments involving these chromosomal insertion mutants. They confirm the locations of thepqq genes obtained with the EMS mutants. The only discrepancy concerns the mutation in MDQ3 which, although mapping in the overlapping region ofpqqB and pqqC, was complemented by pM055 1, which contains only part of pqqB and does not complement pqqB mutants. Genetic analysis of PQQ- mutants not complemented by R'51 (groups 11and III) In order to isolate cloned DNA able to complement the 49 mutants not complemented by R'51, a gene library was constructed in the cosmid pLA2917 with chromosomal DNA isolated directly from M . organophilum. Two cosmids were isolated from a library of 1200 colonies: pM0200, which complemented 39 PQQ- mutants (group 11, pqqE mutants) and could Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 2924 F. BIVILLE, E . T U R L I N A N D F . G A S S E R recombine with the pqqD mutant 7 1 ; and pM0600, which complemented group I 1 mutants and also the 10 remaining mutants (group 111, pqqF). Further subcloning of pM0200 gave plasmids pM0230, containing a 7 kb BglII fragment, and pM0240, containing a 2.2 kb BglII-PstI fragment (Fig. 2), both retaining the abilities for complementation and recombination of pM0200. In pM0240, two Kan' cartridge insertions (pM02401 and pM02402) abolished the complementation of the group I 1 mutants. For unknown reasons, the insertion of the cartridge in pM02401 prevented recombination with the DNA region of the pqqD mutant 71, perhaps because of the unavoidable deletion of the neighbouring XhoI fragment when the cartridge was cloned at the XhoI site. The Kan' cartridge of pM02401 was transferred into the chromosome of M .organophilum as previously described. The resulting mutant, MDQ4, was unable to synthesize PQQ and could be complemented by pM0240. The conclusion is that the 2.2 kb of cloned DNA in pM0240 contained a new pqq gene, pqqE. There was no definitive evidence for the relative locations ofpqqE andpqqD except the ability of pM0240 to recombine with the pqqD mutant 71. The BglII fragment cloned in pM05511 (Fig. 1) ligated to the BglII end of the BglII-PstI fragment of pM0240 provided a plasmid pM0245 which complemented both pqqD and pqqE mutants, but this does not provide strong evidence for a close physical link between pqqD and pqqE in the chromosome. The sixth pqq gene, pqqF, was localized in a 4 kb XhoI fragment of pM0600 by complementation experiments with plasmids pM0611, pM0612 and pM0613 (Fig. 2). The transfer of a Kanr cartridge into the chromosome of M . organophilum gave a mutant, MDQ9, which was unable to synthesize PQQ. As mentioned above, the cosmid pM0600 complemented both pqqE and pqqF mutants. The size of the DNA fragment separating the latter two genes was estimated to be about 19 kb. pM0200, another cosmid, complemented pqqE but not pqqF mutants, suggesting that pqqE and pqqF were located in pM0600 at each end of the cloned M . organophilum DNA. This assumes that no DNA rearrangements occurred during the formation of this cosmid. The region separating pqqE from pqqF was explored with two chromosomal insertions transferred from pM02311 and pM02312, two plasmids constructed from pM0231, a derivative of pM0230 with a large BamHI deletion removing the XhoI and SalI sites of the pqqE gene. The two insertion mutants MDQ7 and MDQ8 obtained from pM023 11 and pM023 12, respectively, could grow on methanol medium without PQQ, showing that the region betweenpqqE andpqqF was not involved in PQQ biosynthesis. Absence of polar eflect of the insertion of the Kanr gene of pUC4K in M . organophilum Plasmid pUC4K contains the Kanr gene of Tn903 with various restriction sites at each end in order to make an adaptable gene cartridge. This cartridge was used to build insertion mutations at defined restriction sites. To test whether these insertions had a polar effect on transcription in M . organophilum, two plasmids, pFB 132 and pFBl33 (Fig. 3) were used. The Kanr cartridge was inserted in both orientations between the streptomycin-resistance gene of Tn5 and its promoter. The expression of the streptomycin-resistance gene, located downstream of the Kanr cartridge, was observed after conjugation of M . organophilum with E . coli S17-1 containing pFB132 or pFB 133. The M . organophilum exconjugants were selected on methanol minimal medium supplemented with kanamycin. All the Kanr colonies were also resistant to streptomycin, irrespective of the orientation of the insertion. These results demonstrated that the pUC4K Kanr gene had no polar effect in M . organophilum. R' formation in the chromosomal region of the pqq genes Plasmid R'5 1, which contains the genes pqqA, pqqB, pqqC and pqqD, was obtained by using pJB3J1 as a mobilizing vector of chromosomal DNA. The source DNA was M . organophilum MD5, which contains a Tn5 insertion proximal to rnoxF, a genetic region located about 30 kb frompqqA (Mazodier et al., 1988). In order to increase the probability of obtaining R' plasmids containing all the pqq genes, the Kanr cartridge close to pqqA in MDQl (Fig. 1) was used as a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 PQQ biosynthesis genes in M . organophilum - - - - - -B Bg H- 1 P 1X p MS 231 I BBg \I A pM0200 2402 2401 B X I 1 2312 0 2 3 pM06I 1 7 2925 1 BS -------- pM0612 1SX I 4'pM0245 1 Ikb P B S 7 * pM0613 1-1 I ----------- I EMS mutants in 1 I P49D Prototype strains : Plasmids pM0200 pM0600 Derivatives of the above plasmids pM0200: 230 23 1 240 2401 2402 245 pM0600: 611 612 613 P44E MDQ4 MDQ9 C C C C - C 430 (39) R - R R R R C ND ND ND C C Insertion mutants P44F - ND ND ND ND ND C R - - C ND ND C ND ND ND - ND ND ND ND ND ND C - R Fig. 2. Map of plasmids used in complementation analysis of mutants from group I1 (430) and from group 111(530) and corresponding chromosomal map of M.orgunophiium. The DNA fragments shown on this map were cloned in pLA2917. On the chromosome map (upper horizontal line), the sign associated with each MDQ mutant indicates whether it can (+) or cannot (-) synthesize PQQ. Filled triangles indicate the Kanr cartridge insertion sites, as for Fig. 1. Abbreviations in the table: C, complementation; R, recombination; -, no complementation or recombination; ND, not determined. selectable marker. Sixty-four Kanr R' plasmids were tested: all had the same complementing abilities as R'5 1 with respect topqqA andpqqD, and none were able to complement mutations in pqqE and pqqF. The use of strains MDQ7 and MDQ8 (Fig. 2) containing the same Kanr marker located between pqqE and pqqF did not yield any R' plasmid able to comlement mutations in pqqE or pqqF, despite several attempts. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 2926 F . BIVILLE, E . T U R L I N A N D F . GASSER I ................................ ..... ................. ............... P pFB133- slr I I pRK290 pFB132 Fig. 3. Construction of plasmids pFB132 and pFB133. Plasmid pPM131 (Mazodier et ul., 1986) used for this construction was a derivative of pACYC184 (Chang & Cohen, 1978), containing a Tn5 fragment (represented by a double line) including the promoter (P) and the streptomycin-resistance gene (str) of the Tn5 resistance operon. To construct pPM131, a 980 bp PstI fragment of Tn5 was deleted, removing large parts of the kanamycin-resistanceand of the bleomycin-resistancegenes. To construct pFB132 and pFB133, the Kanr cartridge of pUC4K was ligated in both orientations at the PstI site, then the unique EcoRI site of pPM131 was opened and the whole plasmid was ligated to the single EcoRI site of pRK290. As a result, the expressionof str from Tn5 is blocked by the Kanrcartridge if the latter has a polar effect. The arrows indicate the direction of the transcription of the kun gene in pFB132 and pFB133. Attempts at expression of pqq genes in heterologous hosts The expression ofpqq genes in heterologous hosts was obtained by Goosen et al. (1988) with a plasmid containing pqq genes from A . calcoaceticus : the expression of an active GDH in E. coli cells was confirmed by growth on minimal medium supplemented with glucose. The pqq genes cloned from A. calcoaceticus might correspond to the genes pqqA, pqqB, pqqC, pqqD from M . organophilum (see Discussion). Therefore plasmid R 5 1 containing these four genes was introduced into a mutant of E. coli affected in the phosphotransferase transport system (pts) and consequently unable to utilize glucose. No acidification was observed on McConkey medium supplemented with glucose. Negative results were also obtained with plasmids pM0550, pM0200 and pM0600. The apo-GDH was correctly synthesized since a control on McConkey glucose medium supplemented with PQQ yielded red colonies, indicating acidification of the medium. One possible cause of this failure to achieve expression of the pqq genes is the large difference in GC content of the E. coli host (50 mol%) and of the cloned M. organophilum DNA (66 molx). This was overcome by the use of a different host, namely a strain of P . testosteroni which has a GC content of 64 mol%. P . testosteroni synthesizes an apo-ethanol dehydrogenase but not the PQQ needed for enzyme activity. Although this organism can grow poorly in liquid ethanol minimal medium, no isolated colonies could be obtained on solid media unless PQQ was added to the culture medium. P . testosteroni is naturally resistant to ampicillin (50 pg ml-l) and sensitive to chloramphenicol(l0 pg ml-l), tetracycline (10 pg ml-l) and kanamycin (25 pg ml-l). After transfer of pJB3J1 and pLA2917 into P . testosteroni, the stability of antibiotic resistance encoded by these plasmids was confirmed after several subcultures in medium supplemented with antibiotics. E. coli S17-1 containing R'51 or other plasmids was crossed with P . testosteroni and exconjugants were selected on LB medium supplemented with ampicillin to eliminate the donor E. coli and with kanamycin and tetracycline for plasmid selection. Several re-isolated colonies of P . testosteroni containing the plasmids were tested on ethanol minimal medium; none of them could grow on this medium. On the same medium supplemented with PQQ, colonies 2 mm in diameter were obtained in 2-3 d. DISCUSSION The efficient use of EMS to obtain PQQ- mutants was hampered by the presence of a hot-spot of sensitivity to this mutagen in the pqqA gene. UV light was used for mutagenesis of M. organophilum by Machlin et al. (1988), but it did not give satisfactory results in our hands. In spite of this difficulty, the large number of PQQ- mutants isolated, and the ability to discriminate pqqA mutants easily, allowed the recognition of six genetic regions involved in PQQ biosynthesis in M. organophilum. Irrespective of the genes affected, these mutants shared Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 PQQ biosynthesis genes in M . organophilum - -f-. -R- -5 1- - I 2927 - - - -a- -+pM0600 Fig. 4. Map of the six pqq genes and of moxF, the structural gene of MDH in M . organophilum DSM 760. Complementation of both moxF and pqq genes A, B, C and D was obtained with a single plasmid R’51 according to the results of this work and of Mazodier et al. (1988). common characteristics : inability to grow on methanol minimal medium unless supplemented with PQQ, but normal growth on methylamine minimal medium; no detectable PQQ biosynthesis; and barely detectable apo-MDH. The main characteristics mentioned above show that the mutants isolated were affected in PQQ biosynthesis. They are clearly distinct from the mutants affected in genes moxAl, moxA2 and moxA3 characterized in M . extorquens AM1 and ascribed to the PQQ-apo-MDH association (Nunn & Lidstrom, 1986a, b). In these mutants the apo-MDH was synthesized but the characteristic absorption peak at 345 nm of the MDH was displaced. PQQ assay was not carried out, but PQQ biosynthesis was unaffected since methylamine oxidation, which is a PQQ-dependent process in M . extorquens AM1, allowed normal growth on this substrate. Goosen et al. (1989) have characterized four genes of Acinetobacter calcoaceticus, numbered I to IV, involved in PQQ biosynthesis. Several similarities of the latter genes and the genes isolated from M . organophilum in the present work suggest some correspondence. Gene IV of A . calcoaceticus contains 75 bp, and this characteristic small size might correspond to our pqqD gene. pqqA synthesized a 43 kDa protein in maxi-cells (unpublished results), a size consistent with the 42-6 kDa protein deduced from the sequence of the gene I11 of A . calcoaceticus. The overall genetic organization seems similar in both organisms : the orderpqqA-pqqB-pqqC-pqqD in M . organophilum (Fig. l), might correspond to the order 111-11-I-(V)-IV in A . calcoaceticus. Region V in A . calcoaceticus is not involved in PQQ biosynthesis, and a correspondence might exist with a similar region detected between pqqC and pqqD in M . organophilum (Fig. 1). No common restriction sites were detected in these presumptively isofunctional genetic regions, probably because the GC content of the two organisms is so different ( A . calcoaceticus 38 mol%, M . organophilum 66 molx). The overall similarity of the map of pqq genes in both organisms is underlined by another common particularity : in our work the delimitation betweenpqqB andpqqC could not be clearly determined, suggesting an overlapping region of the two genes; and correspondingly the sequence in A . calcoaceticus showed that the open reading frame of gene I overlaps a 16 bp region upstream from the 3’ end of gene 11. The effects of Tn5 insertions in A . calcoaceticus and M . organophilum are nevertheless quite different. In M . organophilum a Tn5 insertion in pqqA abolishes the expression of pqqB but not the expression of pqqC, suggesting a common transcription unit pqqA-pqqB. In A . calcoaceticus an operon 11-1 was detected (corresponding to pqqB-pqqC) and the transcription of gene I11 (corresponding to pqqA) was achieved independently. Beside the region pqqA-pqqD we found two additional genetic regions involved in PQQ biosynthesis in M . organophilum: pqqE, located in the immediate vicinity of pqqD, and pqqFlocated about 19 kb frompqqE. The genetic map in Fig. 4 shows the respective location of pqq genes and of the MDH structural gene moxF, according to the results of this work and those obtained previously (Mazodier et al., 1988). At present no precise function can be assigned to the pqq genes. The three genes pqqA, pqqB and pqqC are large enough to contain genetic information encoding enzyme proteins. Probably no such role can be assigned to pqqD. The sequence of pqq genes of A . calcoaceticus did not provide information on their role (Goosen et al., 1989). Although a tyrosinase activity was suggested by Houck et al. (1988) and by Van Kleef & Duine (1988) to be involved in PQQ Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45 2928 F . BIVILLE, E . T U R L I N A N D F. GASSER biosynthesis, no tyrosinase activity was detected in crude extracts of M. organophilurn.A product of tyrosinase activity and a putative intermediate of PQQ biosynthesis, dihydroxyphenylalanine, did not permit growth of any mutant when added to methanol minimal medium (unpublished observation). The problem remains as to why only fourpqq genes could fulfil the need for PQQ biosynthesis in A. calcoaceticus, whereas two more appear to be necessary in M. organophilurn. A different mode of genetic regulation of the biosynthesis in the two organisms might explain this discrepancy. The PQQ structure used for GDH of A. calcoaceticus and for MDH of M. organophilurn is likely to be the same since biochemical complementation of the mutants of both organisms was obtained with the same commercially available PQQ. We are indebted to Philippe Mazodier for his constant interest in this work and we thank Karl Reich for critical reading of the manuscript. 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Covalently bound pyrroloquinoline quinone is the organic prosthetic group in human placental lysyl oxidase. Biochemical Journal 239, 789-79 1 . VANSCHIE,B. J., DE MOOY,0. H., LINTON,D. J., VAN DIJKEN,J. P. & KUENEN,J. G. (1987). PQQ-dependent production of gluconic acid by Acinetobacter, Agrobacterium and Rhizobium species. Journal of General Microbiology 133, 861-875. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 02:59:45