The MacintoshTM computer programs used were DNA. Strider 1.0 and GeneWorksm 2.2.1. The eMail servers of. NCBI running the BLAST program (Altschul et ...
Eur. J. Biochem. 226, 201-210 (1994) 0 FEBS 1994
Expression of the mau genes involved in methylamine metabolism in Paracoccus denitrijkans is under control of a LysR-type transcriptional activator Rob J. M. VAN SPANNING', Carol J. N. M. van der PALEN2,Dirk-Jan SLOTBOOM', Willem N. M. REIJNDERS', Adriaan H. STOUTHAMER' and Johannis A. DUINE' Department of Microbiology, Biological Laboratory, BioCentrum Amsterdam, Vnje Universiteit, Amsterdam, The Netherlands
* Department of Microbiology and Enzymology, Delft University of Technology, Delft, The Netherlands (Received August 9, 1994) - EJB 94 1216/1
Expression of methylamine dehydrogenase in Paracoccus denitrifcans and its concomitant ability to grow on methylamine is regulated by a substrate-induction mechanism as well as by a catabolite-repression-like mechanism. Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy. The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine. Repression is not observed when the growth medium contains methylamine and either choline or methanol. Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene. This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster. The mauR gene encodes a LysR-type transcriptional activator. Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine. The mutation is completely restored when the mauR gene is supplied in trans. The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane-embedded protein. Inactivation of the gene results in the inability of cells to grow on methylamine. Downstream from mauF and in the same transcription direction, mauB is located. This gene encodes the large subunit of methylamine dehydrogenase.
Paracoccus denitrifcans is a Gram-negative soil bacterium that is able to grow under a variety of growth conditions. This flexibility is the result of a potential to adapt its respiratory network to the prevailing growth condition. Constitutive elements of the network are NADH dehydrogenase, ubiquinone-10, a cytochrome bc, complex, and cytochromes c (John and Whatley, 1977; Vignais et al., 1981). During aerobic growth, the electron fluxes are directed to at least three oxidases, one of which is an aa,-type cytochrome c oxidase, the second is a cbb,-type cytochrome c oxidase, and the third is a bb,-type quinol oxidase (De Gier et al., 1994). During anaerobic growth, synthesis of the oxidases is repressed and a number of terminal oxido-reductases are induced to enable electron transport to nitrate, nitrite, nitric oxide and nitrous oxide (Stouthamer, 1991). P. denitrifcans is also able to grow methylotrophically on reduced one-carCorrespondence to R. J . M. Van Spanning, Department of Microbiology, Biological Laboratory, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, The
Netherlands Fax: +31 20 444 7123. Phone: +31 20 444 7179. Abbreviations. ORF, open reading frame; HTH, helix turn helix. Enzyme. Methylamine dehydrogenase (EC 1.4.99.3). Note. The novel nucleotide sequence data mentioned here have been submitted to the GenBank sequence data bank and are available under accession number U12464.
bon compounds (Harms and Van Spanning, 1991). For growth on methanol, methanol dehydrogenase and its obligate acceptor cytochrome c551iare induced. For growth on methylamine, methylamine dehydrogenase and its obligate acceptor amicyanin are induced. Both branches operate in the periplasm and are connected to the respiratory network at the level of cytochromes c (Bamforth and Quayle, 1978; Bosma et al., 1987; Husain and Davidson, 1985,1986,1987; Van Spanning et al., 1990b, 1991a). Although it is obvious that the organization of the network is strongly influenced by environmental factors, little is known about the molecular background of the regulation. Only recently has a detailed study on the regulation of methanol metabolism been presented (Harms et al., 1993). From these studies, it was demonstrated that expression of the mox operon encoding methanol dehydrogenase and cytochrome q5',, is regulated by a set of regulatory genes that belongs to the family of two-component regulatory systems. The study described here focuses on understanding of the regulation of methylamine metabolism. The genes involved in methylamine metabolism are clustered in the mau locus. A thorough description of the organization and function of the mau locus of Methylobacterium extorquens AM1 has been presented by the group of Lidstrom (Chistoserdov et al., 1990 ; Chistoserdov and Lidstrom, 1991; Chistoserdov et al., 1992; Lidstrom and Chistoserdov, 1993). The rnau genes of this locus are organized in the tran-
202
Table 1. Bacterial strains and plasmids. Sm, streptomycin ; Km, kanamycin ; Rif, rifampicin ; Spec, spectinomycin. Strain or plasmid
Relevant characteristics
Source or reference
Bacteria E. coli S17-1 TG1
Sm‘, pro, r-, m+, RP4-2 integrated (Tc::Mu)(Km::Tn7) supE, hsdA5, thi, A(1ac-proAB), F’, (traD36 proAB laclq, lacZ AM15)
Simon et al., 1983 Sambrook et al., 1989
R denitrificans Pdl222 Pd41.21 Pd41.51 Pd47.21 Pd54.21 Pdup.21
DSM4 13, Rip, Spec‘, enhanced conjugation frequencies Pd1222, m a d : : Km’ Pd1222, AmauC Pdl222, mauR : : Km’ Pd1222, rnauF:: Km’ Pd1222, OW1 : : Km’
De Vries et al., 1989 Van Spanning et al., 1990b Van Spanning et al., 1990h this work this work this work
Plasmids pGRPdl PEG400 pEG.mauR pUC18/19 pUC4K pRTd47.2 1 pRTd54.21
oriV (colEl), oriT (RK2), Amp‘, Smr (Tn1831) IncP, Sm‘, Spec‘, pUC12/13 mcs, l a d ’ pEG400 derivative, mauR Amp‘, lacZ’ Km’ (Tn903) pGRPdl, mauR: : Km’ pGRPdl, mauF::Km’
van Spanning et al., 1990;i Gerhus et al., 1990 this work Yanisch-Perron et al., 1985 Pharmacia this work this work
scriptional order mauFBEDACJGLMN. The large and small subunits of methylamine dehydrogenase are encoded by mauB and m a d , amicyanin is encoded by mauC. MauG is a peroxidase-like enzyme suggested to be involved in maturation of the tryptophan tryptophylquinone cofactor. MauF, E, D, J, L, M and N are proteins with thusfar unknown functions. Comparable mau loci or parts of it have been found in a number of methylamine-utilizing organisms, including Thiobacillus versutus and P. denitrifcans (Chistoserdov et al., 1992; Huitema et al., 1993; Lidstrom and Chistoserdov, 1993; Ubbink et al., 1991; Van Spanning et al., 1990b). However, up to now, no genes involved in regulation of these mau clusters have been isolated. From earlier studies it has been shown that methylamine dehydrogenase in €? denitrifcans is specifically induced during growth on methylamine (Husain and Davidson, 198.5, 1987). During the course of this study, it has been reported by others that the synthesis of methylamine dehydrogenase is repressed when succinate is present in the growth medium in addition to methylamine. Repression was not observed in the simultaneous presence of choline (Page and Ferguson, 1993). In this paper, it will be shown that the results of our studies on the regulation of methylamine metabolism are consistent with these data. In line with these studies, it will be demonstrated that methylamine-induced expression of the mau genes is under control of mauR, a gene encoding a LysR-type activator of transcription.
MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions The strains and plasmids used are listed in Table 1. Escherichia coli strains were grown aerobically at 37°C in batch with YT. k? denitrificans was grown aerobically at 30°C in batch or on plates either with brain heart infusion broth (BHI) or in mineral salts medium with either 100 mM methylamine, 100 mM methanol, 20 mM choline, or 25 mM succinate as the carbon and energy source, unless stated otherwise. The mineral salts medium was as described by Chang and Morris (Chang and Morris, 1962) but supplemented with 1.0 ml
trace element solutiodliter. The carbon sources were added from filter-sterilized stock solutions. When necessary, antibiotics were added to final concentrations of 40 pg rifampid ml, 25 pg kanamycidml, 25 pg streptomycidml, and 50 pg ampicillinlml.
Preparation of cell extracts For the isolation of cell extracts, cells were suspended in 10 mM potassium phosphate, pH 7.0, to give an A,,,, of 1.0 upon a SO-fold dilution, then broken in a French pressure cell (American Instrument Company). Membranes were removed by centrifugation for 60 min at lOOOOOXg at 4°C. Cell extracts were routinely stored at -80°C at a concentration of 25 mg proteidml. The concentration of protein was determined by the method of Lowry et al. (Lowry et al., 1951), with bovine serum albumin as a standard.
Gel electrophoresis and immunoblotting Protein suspensions were incubated for 15 min at 100°C in 0.0625 mM Tris/HCl, pH 6.8, 2% (mass/vol.) SDS, 10% (by vol.) glycerol, 0.001 % (masdvol.) bromophenol blue and 700 mM 2-mercaptoethanol, according to the method of Laemmli (Laemmli, 1970). SDS/polyacrylamide gel electrophoresis was carried out on 15% slab gels at 4°C in the dark. For immunoblotting experiments, proteins were transferred to 0.45-pm pore-size nitrocellulose filters (BA 85, Schleicher & Schuell). The possible presence of amicyanin or methylamine dehydrogenase was checked for by immunochemical staining of the nitrocellulose filters with polyclonal antisera raised against the purified counterparts from Thiobacillus versutus as primary antibodies (Ubbink et al., 1991) and goat anti-rabbit alkaline phosphatase (Promega Corp.) as a secondary antibody, exactly as described by Bollag and Edelstein (Bollag and Edelstein, 1991).
Methylamine dehydrogenase activities Methylamine-grown cells were harvested, washed with 1OmM TrisMC1, pH7.0, and resuspended in the same
203 buffer. This suspension gave an A660 of 1.0 upon a 100-fold dilution. The assay mixture (3 ml) contained 0.03 ml of this cell suspension, 100 mM Tris/HCI, pH 7.0, 1 mM KCN, 0.1 mM 2,6-dichlorophenolindophenoland 0.1 mM phenazine methosulfate. The reaction was started by the addition of 10 pl 2.5 M methylamine. The changes at A,,, were recorded using an Aminco DW-2 UVNis spectrophotometer (American Instrument Company) with the reference wavelength set at 750nm. Enzyme activities were expressed in nmol 2,6-dichlorophenolindophenolreduced min-' mg protein-'.
grown in mineral medium supplemented with different carbon sources or combinations of those. At the end of the exponential phase of growth, cells were harvested and analyzed for the presence and activity of methylamine dehydrogenase. The results are presented in Table 2. With respect to growth on single substrates, active methylamine dehydrogenase is found in the wild-type strain only after growth on methylamine or ethylamine. The enzyme is not found in cells grown on succinate, methanol or choline. These results show that methylamine or ethylamine are essential for induction of the methylamine-oxidizing respiratory branch. As a consequence, for analyses of methylamine dehydrogenase subunits in the mauC mutants which are unable to grow on methylDetermination of succinate amine or ethylamine, a second substrate has to be supplied Succinate was determined according to the method of to the growth media as a source of carbon and energy in addition to the inducing substrate itself. In the presence of Holdeman et al. (Holdeman et al., 1977). both succinate and methylamine, growth of the wild-type strain is biphasic (Fig. 1A). During the first phase of growth, DNA manipulations succinate is fully consumed. Neither methylamine dehydroGeneral cloning techniques were carried out essentially genase subunits nor dye-linked activity were detectable imas described by Maniatis et al. (Maniatis et al., 1982). Plas- mediately after this phase. After a lag phase, a second growth mid DNA was isolated from E. coli by the cleared-lysate phase was observed. Cells harvested at the end of this phase method (Van Embden and Cohen, 1973) and purified by contained active methylamine dehydrogenase. These results using Qiagen. For rapid screening, plasmid DNA was iso- demonstrate that expression of the methylamine-oxidizing related by the alkaline lysis method (Maniatis et al., 1982). spiratory chain is repressed during oxidation of succinate. Chromosomal DNA of P. denitrificans was isolated as de- MauC mutant strains grown under a similar condition were scribed earlier (Van Spanning et al., 1990a). DNA restriction only able to metabolize succinate. A second phase of growth fragments were purified from agarose gels using Geneclean was not observed in these cultures (results not shown). In (Bio 101). Digested chromosomal DNA ( 5 pg/lane) was addition, cells from these cultures did not contain methylloaded onto 1% agarose gels, denatured, and transferred to amine dehydrogenase at the end of the exponential phase of positively charged nylon membranes (Boehringer Mann- growth on succinate. In contrast to the situation described heim) according to the method of Southern (Southern, 1975). above, growth of the wild-type strain on a mixture of choline Southern analysis of chromosomal restriction fragments was and methylamine is monophasic (Fig. 1B). Both at the midcarried out by random-primed DNA labeling of cloned se- dle as well as at the end of the exponential phase of growth, quences with digoxigenin and subsequent detection of hy- methylamine dehydrogenase subunits are synthesized. In adbrids by an enzyme immunoassay according to the protocol dition, the cells displayed dye-linked methylamine dehydroof the manufacturer (Boehringer GmbH). Conjugations were genase activity at both stages of growth. MauC mutant strains carried out by streaking cells of donor and recipient strains were grown under the same condition. As compared to the on BHI plates. After 1 day of incubation at 3 0 ° C cells were wild-type strain, cell densities of these cultures at the stationcollected and plated on selective plates. In vitro and in vivo ary phase of growth were lower (A660 2.3 compared to A660 gene-exchange experiments were carried out as described 1.4), obviously as a consequence of the inability to metaboearlier (Van Spanning et al., 1990a). Sequence reactions were lize methylamine. However, albeit these strains were unable performed on single-stranded M13mp18 and M13mp19 to use methylamine for growth, they did synthesize methylclones (Sanger et al., 1977; Sanger et al., 1980) using the amine dehydrogenase subunits both at the middle as well as dye-primer and dye-terminator cycle luts from ABI. Samples at the end of the exponential phase of growth. Surprisingly, containing the labeled fragments were loaded and analyzed dye-linked methylamine dehydrogenase activity was obby an ABI 373A fluorescent sequencer (Applied Biosystems served in the mauC deletion mutant strain Pd41.51 but not in Pd41.21. A comparable set of experimental data was found Perkin and Elmer). when the strains were grown on a mixture of methanol and methylamine. Apparently, growth on either methanol or choDNA sequence analyses line does not affect the methylamine-induced expression of The MacintoshTMcomputer programs used were DNA methylamine dehydrogenase. For analysis of the structural Strider 1.0 and GeneWorksm 2.2.1. The eMail servers of proteins encoded by the mau genes, mau mutant strains deNCBI running the BLAST program (Altschul et al., 1990), scribed in this study were grown in mineral medium with the FASTA and BLITZ server at Heidelberg, Germany and methylamine and choline, the first substrate as inducer of the the BLOCKS server were used for comparison of sequences mau gene cluster, the latter substrate as source of carbon and energy. with the international databases.
RESULTS Growth-condition-dependentsynthesis of methylamine dehydrogenase P. denitrificans wild-type strain Pd1222 and mauC insertion and deletion mutant strains Pd41.21 and Pd41.51 were
Analysis of the region encompassing the start of the mau gene cluster In an attempt to understand the mechanism of induction in more detail, our initial studies were focused on a thorough description and mutational analysis of the locus encompassing the genes involved in methylamine metabolism. In a
204 Table 2. Growth-condition-dependentsynthesis of methylamine dehydrogenase of I? denitnficans wild-type and mau mutant strains. MA, methylamine; EA, ethylamine; MeOH, methanol; Succ, succinate; Chol, choline. MADH, Western analysis of large and small methylamine dehydrogenase (MADH) subunits; +/-, subunits present or absent; n.g., no growth. Activities are dye-linked methylamine dehydrogenase activities in whole cells. Analysis of methylamine dehydrogenase was performed on cells at the stationary phase of growth. In the case of Succ+MA, analysis was at the end of the first exponential phase of growth (see Fig. 1)
Strain
Growth with Succ, or MeOH, or Chol
EA
MA
MADH activity
MADH activity
MeOH + MA
Succ +MA
________
~
~
Chol+ MA ~~
MADH activity
MADH activity
MADH activity
nmol DCPIP min-' mg-'
nmol DCPIP min-'
nmol DCPIP min-' mg-' 180
MADH activity nmol DCPIP min-' mg-' 210 n.g. n.g.
+
Pdl222 Pd41.21 Pd41.51
n.g. n.g.
nmol DCPIP min-' mg-'
nmol DCPIP min-' mg-' 160 n.g. n.g.
+
n.g. n.g.
-
-
-
-
-
-
-
-
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-
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190
-
170
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0 ;
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Fig. 1. Growth-condition-dependentsynthesis of methylamine dehydrogenase in P. denitrificans. Cells were grown in mineral salts medium with succinate and methylamine (A), or choline and methylamine (B). Absorbances at 660 nm are indicated by filled circles, succinate concentrations by open circles. Western analysis and dye-linked activities of methylamine dehydrogenase were determined in cells harvested at the time points marked by arrows. Absence or presence of active methylamine dehydrogenase is indicated by - or +, respectively.
previous paper, we described the isolation and mutational analysis of the mauC gene encoding amicyanin (Van Spanning et al., 1990b). Chromosomal DNA of the mauC insertion mutant strain Pd41.21, in which mauC had been interrupted by the gene encoding kanamycin resistance, was isolated and restricted with BamHZ. Restriction fragments were subsequently ligated in pUC19, after which the constructs were used to transform competent E. coli TG1. Transformants were isolated on plates supplemented with kanamycin. Plasmid DNA from these transformants, designated pMAUl, was isolated and further analyzed. From restriction enzyme and Southern analyses it was shown that the approximately 16-kb BamHI chromosomal fragment contained the interrupted mauC gene with upstream and downstream located flanking regions of 13.0-kb and 1.7-kb, respectively. DNA located upstream of mauC was subcloned into M13 for sequence analysis. The sequence data we obtained were
consistent with the partial sequence published by the group of Lidstrom (Chistoserdov et al., 1992). They presented the sequence data for m a d and mauB, encoding the small and large subunits of methylamine dehydrogenase, respectively. This work focuses on the region located upstream of mauB. A map of part of the P denitrificans rnau locus is presented in Fig. 2 along with the sequencing strategy for analysis of the 3-kb SalI-XhoI fragment covering the 5' part of mauB and its upstream region. The sequence of this region, along with the deduced amino acid sequences, are presented in Fig. 3. The data revealed that, upstream of mauB, two additional open reading frames (ORFs) are located and the 5' part of a third OW, the codon usage of which are in agreement with the common gene codon preference in I? denitri3cans (Steinriicke and Ludwig, 1993). In order to establish whether or not the putative genes are involved in methylamine metabolism, mutant strains with mutations in
205
Fig. 2. Physical map (A) and sequencing strategy (B) of a P denitrijkans BamHI fragment containing part of the mau gene cluster. Genes are presented as open rectangles, in which sequences encoding signal sequences are indicated by filled bars. The direction of transcription is indicated by the direction of arrows under the genes. Restriction fragments were subcloned in M13mp18/19 after which the sequences were determined in a polymerase reaction either by using commercially available dye primers or by using the dye-terminator cycle kit in a reaction primed by synthesized primers. The direction and extent of the nucleotide sequences are indicated by the direction and length of the arrows in the bottom panel. Positions of chromosomal insertions are indicated by vertical arrows.
either of the ORFs located downstream of mauB were constructed. The correctnesses of gene replacements were checked by Southern analyses (results not shown). The positions of the insertional mutations are shown in Fig. 2.
mauF The first ORF upstream of mauB, is tentatively designated mauF. The transcription of mauF is in the same direction as mauB. Both genes are separated by 79 nucleotides. This intergenic region contains an inverted repeat and the Shine Dalgarno sequence 5'-AGGAGG-3' located in front of mauB. mauF is preceded by the Shine Dalgarno sequence 5'-GAGAGGAGG-3' and encodes a putative protein of 277 amino acids with a molecular mass of 28328 Da. The MauF protein is predicted to have three relatively large hydrophobic segments at residues 28-77, 100-152 and 183-235. A search in the international data bases revealed no apparent similarity with other proteins sequenced thus far. Only recently, the sequence of the MauF counterpart from M. extorquens AM1 has been published (Chistoserdov et al., 1994). Comparison studies revealed an overall identity of 52% at the protein level. Apart from the first 70 amino acids at the N-terminus, the identity even reached a value of 62%. The mauF gene was inactivated by insertion of a kanamycin resistance cartridge in the unique Hind111 site. The resulting mutant strain Pd54.21 was unable to grow on methylamine or ethylamine. These data demonstrate that mauF is essential for methylamine metabolism in P. denitrificans.
mauR Upstream of mauF and separated by 182 nucleotides, an ORF with the transcription direction opposite to that of mauF
and mauB was found. The gene is tentatively designated mauR. mauR is preceded by the Shine Dalgarno sequence 5'GGAGA-3'. The putative MauR protein is 283 amino acids in size with a predicted molecular mass of 32070Da. The protein is overall hydrophilic and does not contain a signal sequence. These data indicate that the protein is located in the cytoplasm. A search in the GenBank and EMBL data banks revealed that MauR has high similarity to a number of LysR-type transcriptional activators. Highest similarities were found with a hypothetical 33.8-kDa protein from E. coli (Nishimura, Komine, Miyamoto, Kitabatake, Mathunaga, Hisano, Miki and Inokuchi, GenBank accession number P30864), CbbR from Xanthobacter flavus, activator of the autotrophic CO, fixation genes (Van den Bergh et al., 1993) and LysR from E. coli, activator of the gene encoding diaminopimelate decarboxylase (Stragier et al., 1983). An alignment of MauR with these transcriptional activators is presented in Fig. 4. The consensus sequence derived from these four proteins shows a relatively high similarity at the aminoterminal regions. This finding is in agreement with the result from alignment studies on a number of LysR-type transcriptional activators (Schell, 1993). These studies revealed that proteins belonging to this class of transcriptional activators contain three more or less conserved regions. An alignment of these regions with the corresponding regions in MauR is presented in Fig. 5. The first region is located at the amino terminus ranging over residues 6 to 66. This amino-terminal region covers the 20-residue DNA-binding helix-turn-helix (HTH) motif located at positions 23-42 (Henikoff et al., 1988). Secondary-structure analyses also revealed a HTH motif in MauR at a similar position. The most highly conserved residues in MauR are found at HTH positions 5 (Ala) and 15 (VaVLeuDle). Similarity with the second conserved region, which is located at residues 98 - 150, is less promi-
206 GTCGACCTCGGGGAGGCCCTTGATCGGCGGCGGGGACAGCCGGCCATCGCGATATAGCCGCAACTGGCCGGGACGTTCGGAGATCAGCAGCCCGCCATCGGGC D V E P L G K I P P S L R G D R Y L R L Q G P R E S I L L G G D P AUlAGCGCCATGCCCCAGGGATGGCGCAGATTCCTCGGTCAGTACGGTGCGGATCAGACGCATCGCCTGCGGCATGACCGGCGCGCGGGTCTGGCCGGCAA
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2000
TGCGGCCCTTGCCGGGGCGGCGGGCGGGGTGGCGCTGGCATCGGCCGCCGGTCCGCAGCCGCTCTGGGCGGTGCTCGGCGCGGCGGCGGTGGCGGGCGGG A A L A G A A G G V A L A S A A G P Q P L W A V L G A A A V A G G
2100
CTGTTGTCCACCTGGTCGCCCTGCGGCTATTCGTCGATCTCGCTTCTGCGGCCGGATGGGCGGGGGCTGCGCGCGGTGGCGGGATTGGCTGCCGACATTCG L L S T W S P C G Y S S I S L L R P D G R G L R A V A G W L P T F A
2200
CCATGCACGGTGCGGGCTACGGCCTGGGCGCGCTGATGCTGGGCGGCCTGCTGGGCGGCATCGGCCTGATCGCCGGCTTTTCCGGTTTCGGCAGCACGGC M H G A G Y G L G A L M L G G L L G G I G L I A G F S G F G S T A
2300
CCTGCTGGTGCTGGGGCTGGTCGGCCTGGCTTACGGTGCGCATCAGCTTGACTTCCTGCGCGTGCCCTATCCGCAGCGCCGGGCGCAGGTGCCGCATGAC
2400
L
L
V
L
G
L
V
G
L
A
Y
G
A
H
Q
L
D
F
L
R
V
P
Y
P
Q
R
R
A
Q
V
P
H
D
GCACGCCAGCGTTTCCCGAAATGGGTCATCGGCGGGCTTTACGGGCTGTCGCTGGGGCTCGATTACCTGACCTATGTGCAGACGCCGCTGCTTTACATGA A R Q R F P K W V I G G L Y G L S L G L D Y L T Y V Q T P L L Y M M
2500
TGACGCTGGCGGCGGTCTTTACCGGCAATATCGCCCATGCCATCGCCATTGTCGCGCTGTTCAACCTGGGCCGGTTCCTGCCGGTCGCGGTCAACGCGCT T L A A V F T G N I A H A I A I V A L F N L G R F L P V A V N A L
2600
GCCGATCCCCGATTACCGGGTGCAGGCATGGCTGGCCCGGCACCAGG~CGCCGCGCTGGCCGATCGGCGCCATCCTGACCGCGCTTGGCGCGGGCTTC P I P D Y R V Q A W L A R H Q E N A A L A D G A I L T A L G A G F
2700
ACGGTGCTGGCGCTGATCTGAACCACCACCCCGACTCTCGATCCCATTCATACGGCGATGCCCGCGTGTCGCGGGTGTCCTGCTGCCATCAC~CG T V L A L I . mpua
2800
ATGGCCCTTCCACCCTTTCATGCCGCTGTTCCGGGCTTCGCTGATCGGGCTTGGGCTGGGCTGCTCGGCGCTTGCGCTGGCGGCCAGCGCC~GGATCG M A L P P N F M P L F R A S L I G L G L G C S A L d L A A S A Q D A
2900
CGCCCGAGGCCGAGACCCAAGCCCAGGAAACCCCAACCAGGCCAGGCCGCTGCCCGCGCCGCCGCGGCCGAC~TGCCGCCGGCCAGGACGACGAGCCGCGCAT
3000
P
E
CCTCGAG L E
A
E
T
Q
A
Q
E
T
Q
G
Q
A
A
A
R
A
A
A
A
D
L
A
A
G
Q
D
D
E
P
R
I 3007
Fig.3. Nucleotide sequence of the 5' part of ORFl, moxR and moxF, and the 5' part of mauB. Amino acid sequences for the aminoterminal part of ORFl and its signal sequence, and MauR were deduced from the strand opposite that presented. Amino acid sequences for MauF, and the amino-terminal part of MauB and its signal sequence were deduced from the strand as shown. The putative signal sequences are in italics, Shine-Dalgarno sequences are underlined, and dyadic sequences are indicated by arrows.
207 --helix-turn-helix--
EKVIGLK EGSLGCV EMKLGVS EEDIGVP
LysR. Ec MauR .P d ORF EC CbbR Xf
MAPHWTLRQLRLVALAAASG
Consensus
-.......* L.*..**..*GS***AA..L..****V*R.*.*LE..*G*.
. .
LFERVRGRLHPTVQGLRLFEEVQRSWYGLDRIVSAAESLREFRQGELSIA
49 46 48 50 50
99 95 98 100
LysR. Ec MauR.Pd ORF. EC CbbR .Xf
LFQAVDGQRRPTEQCRALLQPLV-LMEQAAEAITLQLERQERPLRNFRLT LLNRTTRQLSLTEEGERYFRRVQSILQENAAAESEIMETRNTPRGLLRID MFERVDGRLRPTAAGQELLSAQERIARALSEAEFC+IAALKSPERGSVVVG
Consensus
LF*RV.G.L*PT.*G
LysR.Ec MauR.Pd ORF .EC CbbR .Xf
CLPVFSQSFLPQLLQPFLARYPDVSLN1VPQESPLLEEWLSAQFM)LGLT
TIDAIAQHYLAPTLADLLIAEPELSLQL---ETSDDNVDMARWHADIAIR AATPVVLHFLMPLIKPFRERYPEVTLSL---VSSETIINLIERKVDVAIR WSTAKYFAPMALA-AFREIELRLIIGNREDIIRGIVSLDFDVAIM
149 142 145 149
Consensus
.***....*
150
..L...**....
.**. A.*.. ...**... G.*.**
L.*L*.*F..R.PE**L.L...****....*...*.D*AI.
100
LysR .Ec MauR.Pd ORF .EC CbbR Xf
LGRPRRGNFTMRRVGEMRFNLVLPRGAAPEDLVLAAYPDPLMEVPEMQDF AGTLTDSSLRARPLFNSYRKIIASPDYISRYGKPETIDDLKQHICLGFTE GRPPPALEAETRLIGDHPHIWAPVDHPLFKRRKRITPADLTRESLLVRE
199 192 195 199
Consensus
.*..*.*....R.*.*. ...*V.P. ..*....*..*.. D......*....
200
LysR.Ec MauR .P d ORF .Ec CbbR .Xf
TDSYRQLLDQLFTEH--QVKRRMIVETHSAASVCAMVPAGVGISVVNPLT
247 226 243 249
Consensus
*. S...,........
LysR. EC MauR. P d ORF .EC CbbR .Xf
ALDYAASGLWR---RFSIAVPFTVSLIRPLHRPSSALVQAFSGHLQAGL IDKEIARGELVELMADKVLPVEMPFSAVYYSDRAVSTRIRAFIDFLSEHV VAAEVADGRLRVL-EVEGLPVVRQWLAVRAFOKRLLPAGQALMDFLEREG
294 275 293 298
Consensus
....*A.G .*...-....**
300
LysR. EC MauR.Pd ORF.Ec CbbR .Xf
PKLVTSLDAILSSATTA ETLAGDAV KTAPGGAVREA ASFLPQMPGGEGGRCYLPDHVSGSTPAKAVARDPV
311 283 304 333
Consensus
.*. .*. . .
335
.
ETLHTPAGTERTELLSLDEVCVLPPGHPLAVKmTPDDFQGENYISLSR
QQYFPGRQA--------------RLRSANLRVIRVLVDSGRPDFL
PASLNTWPIARSDGQ--LHEVKYGLSSNSGETLKQLCLSGNGIACLSDYM PGSGTRILMERVFEEAGAPNPPIAMEIGSNETIKQSVMAGLGLAFISAHT
-- .......*.*.
S..***.*V.*G.G*A.**...
SVDLVGDERFQV---HRLPAIWLLAQPHLRDDPLARN”JNWCADLFA
V...*..*.*..R..**...A....L*...
250
Fig. 4. Alignment of MauR with the LTTR’s LysR from E. coli (Stragieret al., 1983), an LTTR-like ORF from E. coli (Nishimura, Komine, Miyamoto, Kitabatake, Mathunaga, Hisano, Miki and Inokuchi, GenBank accession number P30864), and CbbR from X . fzavus (Van den Berghet al., 1993). The HTH motifs in these proteins are boxed. The consensus sequence shows the residues that are similar in at least three of the four proteins. Conservative substitutions are indicated by asterices. Amino acid residues were grouped as follows: STAPG, DENQ, WYF, HRK, VLIM, C.
nent. The third conserved region is located at the carboxy terminus residues 236-246, which is thought to be involved in the response to coinducer or DNA binding. A region homologous to the conserved region is also found at the carboxy terminus of MauR. mauR was inactivated by exchange of the internal SalI fragment for the kanamycin-resistance cartridge. The resulting mutant strain Pd47.21 was unable to grow on methylamine or ethylamine as sole sources of carbon and energy, nor could it use these C, substrates as nitrogen source. In addition, the mutant strain did not synthesize either of the methylamine dehydrogenase subunits or amicyanin (Fig. 6,
lanes 2 and 5 ) , and did not display any in-vitro dye-linked methylamine dehydrogenase activity. Growth of the mutant strain on mineral medium with succinate, choline or methanol was unaffected. The mutation could be complemented by providing the mutant strain with the mauR gene in trans. For this, a 3.2-kb SmaI-Hind111 fragment from plasmid pMAU8, containing the concerning O W and its upstream located promoter region, was isolated and cloned in the broad-hostrange vector pEG400. This construct was transferred to Z? denitrijicans strain Pd47.21. Plasmid containing ex-conjugant strains were shown to synthesize methylamine dehydrogenase and amicyanin again (Fig. 6, lanes 3 and 6), displayed
208 LTTR cons (6-66)
T ST TR N hpLRpLRxFxxhxpppphSxAApxLphSQPAhSxQhppLEpxLGxxLFxRxp~hxxxT~
MauR (1-61)
EGSLGCVLFQAVDGQRRPTEQC MNWDDLRWAAINRCGS FNRAAKMLNVEETTIARRLA~
LTTR cons (98-150)
LxIGxxxxxxxxhLPxxxxxxxxxxPxxxxxLxxxxxxxxxxxLpxxphDhh
MauR (92-140)
FRLTTIDAIAQHYLAPTLADLLIAEPELSLQL---ETSDDNVDMARWHADIA
..
.. .
.**
**. *..
*
*. *
*
.
.** ** **
.
*
.
. *.
L I V LTTR cons (236-246) VxxGxGVxVLP
***
* *
MauR (213-223)
VDSGRAAAVLP
Fig. 5. Alignment of MauR protein regions with the conserved parts of the LTTR consensus sequence (Schell, 1993). HTH motifs are boxed. Identical residues are indicated by an asterisk. Dots represent a match with either the group of hydrophobic (h) amino acids (VLIM) or hydrophilic (p) residues (TSNQDEKRH). The symbol x stands for any residue.
1 116.5 80.0 49.5 32.5 27.5 18.5
2
3
4
5
6
-
-
-
- a-MADH
- BMADH
- amicyanin
Fig. 6. Western analysis of methylamine dehydrogenase subunits (A) and amicyanin (B) in l? denitrificans wild-type strain (lanes 1 and 4), the mauR mutant strain Pd47.21 (lanes 2 and 3,and Pd47.21 complemented with the mauR gene on the broad host range vector pEG400 (lanes 3 and 6). The positions of the marker proteins are indicated by their relative molecular masses. The mobilities of the large and small methylamine dehydrogenase subunits and amicyanin are as shown.
dye-linked methylamine dehydrogenase activity, and grew in media with methylamine as sole source of carbon and energy, just like the wild-type strain. These data demonstrate that the mauR gene is essential for induction of the genes involved in methylamine metabolism. In many cases, DNA-binding sites of LysR-type transcriptional activators are characterized by partially dyadic sequences that contain a conserved T-N11-A motif (Goethals et al., 1992; Schell, 1993). A search in the mauR-mauF intergenic region did not reveal sequences that exactly fulfilled these criteria. However, two partially dyadic sequences that resemble the consensus sequence were found. The first one, 5’-CTGCA-N4-TGCAG-3’, is located directly in front of the mauR gene. The second sequence, 5’-CTGG-N6-CCAG-3’, resembles the first and is located 16 nucleotides downstream of it. In these sequences, the T and A residues are separated by 10 nucleotides instead of 11.
ORFl Downstream of rnauR, relative to its transcription direction, and separated by 535 bp, the 5’ part of an additional ORF was found. The ORF is preceded by the Shine-Dalgarno sequence 5’-GAGGA-3’, and ProSite analysis revealed the presence of a signal sequence in the deduced amino acid sequence. For mutational analysis of this ORF, plasmid pMAU8 was partially restricted at the Nrul site located in
ORFl after which the 1.3-kb Hincll fragment of pUC4 K encoding kanamycin resistance was inserted. The interrupted gene was subsequently isolated as a 2.6-kb EcoRV-Sphl fragment and cloned into pGRPdl. The resulting construct was used in a gene-exchange experiment. Proper ex-conjugant strains were isolated and analyzed. With respect to the ability to synthesize active methylamine dehydrogenase and to grow on methylamine, this mutant strain behaved just like the wild-type strain. This finding demonstrates that ORFl is not essential for methylamine metabolism. Apparently. mauR is the most distant gene at this side of the mau locus.
DISCUSSION The results described in this paper demonstrate that induction of the rnau genes involved in methylamine metabolism in P. denitrificans is under control of MauR. MauR is encoded by the mauR gene, which is located 182 bp in front of, but with the transcription direction opposite, to that of the structural rnau genes. Up to now, no genes comparable to mauR have been found in the vicinity of the mau gene cluster in M . extorquens AM1. Instead, an ORF has been identified in front of this cluster, the function of which, however, is not known (Lidstrom and Chistoserdov, 1993). Insertional inactivation of mauR results in the inability to synthesize methylamine dehydrogenase and amicyanin with the concomitant loss of the ability to use methylamine as a source
209 of carbon, energy or nitrogen. Since the transcription directions are opposite to each other, a possible cis effect of the insertion of mauR on transcription or translation of these structural mau genes can be excluded. A possible effect of the mutation on genes located downstream of mauR with respect to its transcription direction can be excluded too, since insertion mutations in this region did not affect the ability to grow on methylamine. Furthermore, the mutation in mauR was fully restored by supplying an intact copy of the gene in trans. This result demonstrates that mauR is indispensable for induction of the rnau genes. Since growth on other carbon and energy sources was unaffected, it appears that only the cluster of rnau genes is the target of MauR. Protein sequence comparisons revealed that MauR belongs to the family of LysR-type transcriptional activators (Henikoff et al., 1988). Especially at the amino-terminal region of MauR, a high similarity is found with the proteins belonging to this class of regulatory proteins. This region contains the HTH motif that is involved in DNA binding. The available physiological and biochemical data suggest that methylamine and ethylamine are effectors of MauR, since methylamine dehydrogenase and amicyanin are found only in methylamine-grown or ethylamine-grown cells. This induction principle is different from that of the mox genes encoding the methanol oxidative enzymes. Induction of these genes is under control of a two-component regulatory system that is activated during growth on methanol, methylamine or choline. Since formaldehyde is produced under all these growth conditions, it has been suggested that not the growth substrate methanol but its oxidation product formaldehyde is the effector of the sensor protein in this specific regulation mechanism (Harms et al., 1993). In contrast to this difference in the induction of the mox and the rnau genes, a common feature is that transcription of both gene clusters is repressed when succinate is added to the growth medium in addition to the C, substrate (Harms and Van Spanning, 1991; this study). The results from this study revealed that repression of transcription of at least the rnau genes is not observed when either methanol or choline are added in addition to methylamine. These data are in agreement with the findings of the group of Ferguson (Page and Ferguson, 1993). Apart from mauR, the organization of mau genes in l? denitrificans appears to be similar to that in M. extorquens AM1 where the mau genes are organized in the order FBEDACJGLMN. According to the studies presented here, and those described by Chistoserdov (Chistoserdov et al., 1992) and Van Spanning (Van Spanning et al., 1990b), at least the mauFBEDAC genes are found in this order in l? denitrijicans. Analyses of mauC insertion and deletion mutant strains further suggest the presence of additional rnau genes downstream of mauC. The mauC deletion mutant strain Pd41.51 synthesizes active methylamine dehydrogenase, whereas the insertion mutant strain Pd41.21 synthesizes inactive forms of the enzyme. These results demonstrate that introduction of the kanamycin resistance cartridge has a cis effect on the transcription of genes located downstream of the mutation in mauC, whereas an unmarked mutation has no effect. In M . extorquens AM1, mauG, which encodes a heme-c-containing cytochrome c peroxidase, is located downstream of mauC. Mutational analysis suggested the enzyme to be involved in the processing of the tryptophan tryptophylquinone cofactor of the small subunit (Lidstrom and Chistoserdov, 1993). This is in agreement with the results of Ferguson et al., who showed that a I? denitrificans mutant strain impaired in the synthesis of heme c, was still able to synthesize methylamine
dehydrogenase subunits, albeit with an unprocessed cofactor (Page and Ferguson, 1993). Since none of the mau mutant strains analyzed in this study was able to use methylamine and ethylamine either as the source for carbon and energy or as source of nitrogen, it must be concluded that oxidation of these alkyl amines is predominantly and perhaps solely performed by methylamine dehydrogenase. The results further emphasize that amicyanin is the obligatory and sole electron acceptor of methylamine dehydrogenase in I? denitrificans. The mauC unmarked mutant strain Pd41.51 is unable to synthesize amicyanin but does synthesize active methylamine dehydrogenase. Nevertheless, the mutant strain is unable to grow on methylamine or to use it as a nitrogen source. This finding is in agreement with the results of in vitro studies which also support this claim. Firstly, the report that amicyanin is absolutely required to mediate the transfer of electrons from the dehydrogenases to cytochromes (Husain and Davidson, 1986), and secondly the characterization of crystal structures of complexes of amicyanin with the dehydrogenase (Chen et al., 1992, 1994). This is different from the situation in M . extorquens AM1 where cytochromes may take over the role of amicyanin (Lidstrom and Chistoserdov, 1993). The authors wish to thank Prof. G. Canters for supplying the antibodies raised against 7: versutus methylamine dehydrogenase and amicyanin. Part of this research was funded by the Dutch Organization for Scientific Research (NWO).
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