Download Cloning and Genetic Analysis of Six Pyrroloquinoline

Journal of General Microbiology ( 1989), 135, 29 17-2929.
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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
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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.
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PQQ biosynthesis genes in M . organophilum
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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
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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
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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
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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.
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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
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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
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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.
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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
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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
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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. This work was supported by grants from the Centre National de la Recherche
Scientifique (URA 1129) and from the EEC (ST2J 0478) to A. Danchin.
REFERENCES
ALLEN,L. N. & HANSON,
R. S. (1985). Construction of
broad host range cloning vectors: identification of
genes necessary for growth of Methylobacterium
organophilum on methanol. Journal of Bacteriology
coenzyme pyrroloquinoline quinone from Acinetobacter calcoaceticus. Journal of Bacteriology 169,
303-307.
GOOSEN,
N., HORSMAN,
M. P. A., HUINEN,R. G. M. &
VAN DE PUITE, P. (1989). Acinetobacter calcoaceticus
161, 955-962.
AMEYAMA,
M., NONOBE, M., SHINAGAWA,E.,
genes involved in biosynthesis of the coenzyme
pyrroloquinoline quinone : nucleotide sequence and
MATSUSHITA,
K., TAKIMOTO,
K. & ADACHI,0.
(1986). Purification and characterization of the
expression in Escherichia coli K 12. Journal of
quinoprotein D-glucose dehydrogenase apoenzyme
Bacteriology 171, 447-455.
in Escherichia coli. Agricultural and Biochemical
GROEN,B. W., VANKLEEF,M. A. G. & DUINE,J. A.
Chemistry 50, 49-57.
(1986). Quinohaemoprotein alcohol dehydrogenase
apoenzyme from Pseudomonas testosteroni. BioANTHONY,
C. (1986). Bacterial oxidation of methane
and methanol. Advances in Microbial Physiology 27,
chemical Journal 234, 61 1-615.
1 13-209.
HOMMES,
R. W. J., POSTMA,P. W., NEIJSSEL,0. M.,
BIVILLE,F., MAZODIER,
P., GASSER,F., VAN KLEEF,
TEMPEST,
D. W., DOKTER,P. & DUINE,J. A. (1984).
Evidence of a quinoprotein glucose dehydrogenase
M. A. G. & DUINE,J. A. (1988). Physiological
properties of a PQQ mutant of Methylobacterium
apoenzyme in several strains of Escherichia coli.
organophilum. FEMS Microbiology Letters 52,53-58.
FEMS Microbiology Letters 24, 329-333.
BOYER,H. W. & ROULLAND-DUSSOIX,
D. (1969). A HOUCK,D. R., HANNERS,J. L. & UNKEFER,C. J.
complementation analysis of the restriction and
(1988). Biosynthesis of pyrroloquinoline quinone.
1. Identification of biosynthetic precursors using 13C
modification of DNA in Escherichia coli. Journal of
labelling and NMR spectroscopy. Journal of the
Molecular Biology 41, 459-472.
American Chemical Society 110, 6920-692 1.
CHANG,A. C. Y. & COHEN,S. N. (1978). Construction
HUMPHREY,
G. O., WILLSHAW,
G. A. & ANDERSON,
and characterisation of amplifiable multicopy DNA
E. S. (1975). A simple method for the preparation of
cloning vehicles derived from the P15A cryptic
large quantities of pure plasmid DNA. Biochimica et
miniplasmid. JournalofBacteriology 134, 1141-1 156.
biophysica acta 383, 457-463.
DIITA, G., STANFIELD,
S., CORBIN,D. & HELINSKI,
D.
(1980). Broad host range DNA cloning system for KLECKNER,N., ROTH, J. & BOTSTEIN,D. (1977).
Genetic engineering in vivo using translocatable
gram-negative bacteria : construction of a gene bank
drug-resistance elements. New methods in bacterial
in Rhizobium meliloti. Proceedings of the National
genetics. Journal of Molecular Biology 116, 125-1 39.
Academy of Sciences of the United States of America
MACHLIN,
S. M., TAM,P. E., BASTIEN,
C. A. & HANSON,
77, 7347-7351.
DUINE,J. A., FRANK,J. & VAN ZEELAND,
J. K. (1979).
R. S. (1988). Genetic and physical analysis of
Methylobacterium organophilum X X genes encoding
Glucose dehydrogenase from Acinetobacter calcomethanol oxidation. Journal of Bacteriology 170,
aceticus : a quinoprotein. FEBS Letters 108,443-446.
DUINE,J. A., FRANK,J. & JONGEJAN,
J. A. (1987).
141-1 48.
D. G., OUSBY,J. C., VASEY,R. B. &
Enzymology of quinoproteins. Advances in MACCLENNAN,
COITON,N. T. (1971). The influence of dissolved
Enzymology 59, 69-2 12.
Fox, C. F. & WILSON,G. (1968). The role of a
oxygen in Pseudomonas AM 1 grown on methanol in
continuous culture. Journal of General Microbiology
phosphoenolpyruvate dependent kinase system in
beta-glucoside catabolism in Escherichia coli.
69, 395-404.
T., FRITSCH,E. F. & SAMBROOK,
J. (1982).
Proceedingsof the National Academy of Sciences of the MANIATIS,
Molecular Cloning: a Laboratory Manual. Cold
United States of America 59, 988-995.
Spring Harbor, NY: Cold Spring Harbor
GOOSEN,
N., VERMAAS,
D. A. M. & VANDE PUT~E,
P.
Laboratory.
(1987). Cloning of the genes involved in synthesis of
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
MARMUR,
J. (1961). A procedure for the isolation of
deoxyribonucleic acid from microorganisms. Journal
of Molecular Biology 3, 208-2 18.
MAZODIER,
P., BIVILLE,
F., TURLIN,E. & GASSER,F.
(1988). Localization of a pyrroloquinoline quinone
biosynthesis gene near the methanol dehydrogenase
structural gene in Methylobacterium organophilum
DSM 760. Journal of General Microbiology 134,
25 13-2524.
MILLER, J. H. (1972). Experiments in Molecular
Genetics, p. 433. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
M. E. (1986~).Isolation
NU", D. N. & LIDSTROM,
and complementation analysis of 10 methanol
oxidation mutant classes and identification of the
methanol dehydrogenase structural gene of Methylobacterium sp. strain AM 1. Journal of Bacteriology
166, 581-590.
NU", D. N. & LIDSTROM,
M. E. (19863). Phenotypic
characterization of 10 methanol oxidation mutant
classes in Methylobacterium sp. strain AM 1 . Journal
of Bacteriology 166, 591-597.
2929
PRIEFER,U. B., SIMON,R. & PUHLER,A. (1985).
Extension of the host range of Escherichia coli vectors
by incorporation of RSFlOlO replication and mobilization functions. Journal of Bacteriology 163,
3 24-3 30.
SIMON,R., PRIEFER,U. & PUHLER,A. (1983). A broad
host range mobilization system for in uiuo genetic
engineering : transposon mutagenesis in Gram negative bacteria. Biotechnology 1, 784-791.
VAN KLEEF, M. A. G. & DUINE, J. A. (1988).
L-Tyrosine is the precursor of PQQ biosynthesis in
Hyphomicrobium X. FEBS Letters 231, 91-91.
VANDER MEER,A. & DUINE,J . A. (1986). 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
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