Eur. J. Biochem.251, 45253 (1998) FEBS1998

Characterisation of a binding-protein-dependent,active transport system for short-chain amides and ureain the methylotrophic bacterium Methylophilus methylotrophus

James MILLS, Neil R. WYBORN, Jacqueline A. GREENWOOD, Steven G. WILLIAMS and Colin W. JONES

Department of Biochemistry, University of Leicester, England

(Received 5 August1997) 2 EJB 971121/1

Three genes (fmdCAB) encoding an outer-membrane porin for short-chain amides and urea, formami-dase, and a putative regulatory protein inMethylophilus methylotrophushave previously been cloned andcharacterised. Three genes have now been identified downstream offmdB, viz fmdD encoding a hydro-philic protein containing an N-terminal signal sequence, andfmdEF encoding hydrophobic transmem-brane proteins. The derived amino acid sequence of mature FmdD (predicted molecular mass 41870 Da)was similar to the cytoplasmic, amide-binding protein (AmiC) fromPseudomonas aeruginosaand toseveral periplasmic, solute-binding proteins from other bacteria. Mature FmdD was purified and shown tobe a monomer (40245 kDa) with the predicted N-terminal amino acid sequence (ADYPTA-). Equilibriumdialysis showed that the purified protein bound short-chain amides and urea with high affinity (Kd 7.2µMfor [14C]urea). SDS/PAGE and western blotting using antiserum to mature FmdD showed it was inducedby short-chain amides and urea, and repressed by excess ammonia. The derived amino acid sequences ofFmdE (32822 Da) and FmdF (incomplete;.25435 Da) were similar to the transmembrane proteinsBraD/LivH and BraE/LivM, respectively, in various leucine/isoleucine/valine transport systems. Uptakeof [14C]urea by washed cells was inhibited by the uncoupling agent carbonyl cyanidep-trifluoromethoxy-phenylhydrazone and unlabelled formamide. It is concluded that FmdDEF comprise part of a high-affinity,binding-protein-dependent active-transport system for short-chain amides and urea inM. methylotrophus.

Keywords:amide-urea transport system;fmdDEF; Methylophilus methylotrophus; urea uptake.

The methylotrophic bacteriumMethylophilus methylo- ing an outer-membrane porin for short-chain amides and urea(mature subunit molecular mass 39 204 Da) (Wyborn et al.,trophus readily uses formamide and acetamide as sources of

nitrogen for growth by virtue of its ability to hydrolyse these1996; Mills et al.,1997).Many species of bacteria, includingM. methylotrophus, areamides to ammonia and the corresponding organic acid using a

cytoplasmic formamidase and acetamidase, respectively. Puri- also able to use urea as a source of nitrogen for growth by virtueof their ability to hydrolyse it to ammonia and carbon dioxidefied formamidase is virtually specific to formamide (high activ-

ity with formamide, very low activity with short-chain amides, using cytoplasmic ureases of various types (see Mobley andHausinger,1989 ; Mobley et al.,1995). These enzymes generallyno activity with urea), whereas acetamidase is less specific (var-

ied activities with short-chain amides, no activity with formam- exhibit relatively poor affinities for urea and usually haveKm

values in the millimolar range. Little is known about urea trans-ide or urea); both enzymes exhibit relatively low affinities fortheir primary substrate (Km 2.1 mM for formamide and1.1 mM port in bacteria, although there is some physiological evidence

for high-affinity, energy-dependent urea uptake byAlcaligenesfor acetamide) (Silman et al.,1989, 1991 ; Wyborn et al.,1994,1996). eutrophus, Klebsiella pneumoniae, and Bacillus megaterium

(Jahns et al.,1988; Jahns and Kaltwasser,1989), and a geneNucleotide sequencing of cloned DNA fromM. methylo-trophushas shown the presence of three genes in close proxim- (ureI) encoding a putative transmembrane protein has been iden-

tified in the Helicobacter pyloriurease operon (Cussac et al.,ity, viz fmdA encoding formamidase (subunit molecular mass44 438 Da),fmdBencoding a putative positive regulator offmdA 1992; Mobley et al.,1995).

In this paper, we report the identification, molecular charac-expression (subunit molecular mass12306 Da) andfmdCencod-terisation, and physiological properties of a high-affinity, bind-ing-protein-dependent, active-transport system for short-chainCorrespondence toC. W. Jones, Department of Biochemistry, Uni-

versity of Leicester, Leicester LE1 7RH, UK amides and urea inM. methylotrophus.Fax: 1 44 116 2523369.E-mail : cwj1@le.ac.uk

MATERIALS AND METHODSAbbreviations.ABC, ATP-binding casette;D, dilution rate; FCCP,carbonyl cyanidep-trifluoromethoxyphenylhydrazone. Bacterial strains and plasmids.The bacterial strain used inEnzymes.Acetamidase (acylamide amidohydrolase) (EC 3.5.1.4);

this work wasMethylophilus methylotrophus(NCIB 10515), andformamidase (EC 3.5.1.49) ; urease (EC 3.5.1.5).the plasmid was pSWl (pUC18 carrying an 8.4-kbpSphI restric-Note.The novel sequence data described here have been submittedtion fragment of DNA fromM. methylotrophus; Mills et al.,to the DDBJ/EMBL/GenBank databases and are available under acces-

sion number Y14964. 1997).

46 Mills et al. (Eur. J. Biochem. 251)

Growth of M. methylotrophus. M. methylotrophuswasgrown at 37°C in methanol-mineral salts medium with ammo-nia, formamide, acetamide or urea as the source of nitrogen,either in batch culture (Silman et al.,1989) or in continuousculture (D 0.1 h21) under various nutrient limitations (Wybornet al.,1994; Mills et al.,1997). Culture supernatants were pre-pared and analysed for ammonia as described previously (Sil-man et al.,1989; Wyborn et al.,1994) ; residual urea was deter-mined by analysing rapidly-filtered culture supernatants forammonia6preincubation with urease for1 h at 37°C.

Preparation of washed cells and measurement of for-mamidase and urease activities.Cells were harvested, washed,resuspended and assayed for formamidase activity using 50 mMformamide as substrate as described previously (Silman et al.,1989). Urease activity was assayed in an identical manner exceptthat 50 mM urea was the substrate and the assay buffer was

Fig.1. Restriction mapping and genetic organisation of part of the100 mM Mes/NaOH, pH 7.0.8.4-kbp Sph1 restriction fragment of M. methylotrophusDNA inMeasurement of [14C]urea transport. Rates of urea trans-pSW1. (A) restriction map; (B) genetic organisation of thefmd genes.port were determined by measuring the disappearance of 50µM

[14C]urea from a reaction mixture containing washed cells. Thereaction was carried out at 37°C in M. methylotrophusgrowth and the concentration of [14C]urea varied over a 20-fold range.medium pH 7.0 (minus carbon and nitrogen sources) containingThe data were analysed by the method of Scatchard (1949). Thewashed cells (0.5 mg dry cells) in a final volume of 0.5 ml. Theeffect of unlabelled amides and urea on the binding of [14C]ureareaction was started by the addition of [14C]urea. Samples was measured using the same procedure but with a 25-fold mo-(50µl) were taken at10 s, 1, 2, 3, and 4 min, and cells were lar excess of unlabelled compounds (32.5µM versus1.3µM).removed by filtration (filter pore size 0.20µm) using a rapid- Preparation of antiserum to mature FmdD. Antiserum tofiltration and sampling manifold which had been modified tomature FmdD was prepared essentially as described earlier forcollect filtered supernatants (Williams et al.,1994). The filters antisera to various sugar-binding proteins and formamidasewere washed in growth medium pH 7.0 (minus carbon and nitro-(Cornish et al.,1989; Wyborn et al.,1996). The antiserum wasgen sources) (3 ml), then samples of the filtrate (100 µl) were stored at220°C until required.added to OptiPhase HiSafe scintillation fluid (4 ml) prior to Polyacrylamide gel electrophoresis.Discontinuous SDS/analysis by scintillation counting. Inhibition by 20µM carbonyl PAGE was carried out as described previously (Silman et al.,cyanidep-trifluoromethoxyphenylhydrazone (FCCP) and unla-1989) using the procedures of Hames (1981). SDS/polyacrylam-belled formamide (5µM) was determined using the same pro-ide gels were stained for protein with Kenacid blue R and, wherecedure but with 2 min and 30 s preincubation, respectively, priorappropriate, subjected to image analysis using a GDS2000 gelto the addition of [14C]urea. documentation system (Ultraviolet Products) linked to an IBM-

Purification of mature FmdD. A formamide-limited con- compatible PC. Western blotting was carried out as describedtinuous culture ofM. methylotrophuswas harvested by centrifu- previously (Greenwood et al.,1990).gation at 12 2003g for 15 min, washed in 20 mM Bistris, N-terminal amino acid sequencing.N-terminal amino acidpH 6.8, re-centrifuged and finally resuspended in the samesequencing of proteins excised from SDS/polyacrylamide gelsbuffer to a cell density of approximately 40250 mg dry cells was carried out as described previously (Wyborn et al.,1996).ml21. The cell suspension was disrupted by passage three timesDNA sequencing and analysis.Double-stranded plasmidthrough an Aminco French pressure cell at approximatelyDNA was sequenced as described previously (Wyborn et al.,9 MPa, then unbroken cells and cell debris were removed by1996). Nucleotide and derived amino acid sequence analysescentrifugation at 400003g for 20 min, and the resultant cell- were carried out using DNA Strider version1.2 and the Univer-free extract was centrifuged at1750003g for 75 min to produce sity of Wisconsin Genetics Computer Group sequence analysisa high-speed supernatant. The latter was carefully removed, ad-package version 8.0 (CODONPREFERENCE, BLAST, GAPjusted to 65% saturation with ammonium sulphate and centri-and ISOELECTRIC programmes). Secondary structure predic-fuged at 400003g for 20 min. The supernatant was dialysedtions were obtained using the PHD and TMpred programmesovernight against 20 mM bistris, pH 6.8, then filtered through(Hofmann and Stoffel,1993; Rost and Sander,1993,1994; Rostan acrodisc filter (0.2µm pore size; Gelman) loaded on to anet al., 1995) and optimal-fold threading was carried out usingFPLC Mono-Q anion-exchange column (Pharmacia) pre-equili-the THREADER programme (Jones et al.,1992).brated with 20 mM bistris, pH 6.8, and eluted using a linear Presentation of results.Results are expressed, where appro-gradient of KCl (021M over 20 min) at a flow rate of 4 ml priate, as the mean6SEM with the number of independent de-min21. The protein eluted from the column as a sharp peak atterminations shown in brackets.approximately115 mM KCl, and was shown by SDS/PAGE andgel scanning to be>95% pure. The identity of the protein as

RESULTSmature FmdD was confirmed by N-terminal amino acid se-quencing. Protein was assayed by the method of Bradford (1976) Nucleotide sequencing and analysis of derived amino acid

sequences.The 8.4-kbpSphI restriction fragment ofM. methylo-using the Bio-Rad protein assay reagent.Equilibrium dialysis. The binding constant and binding trophus DNA in pSWl (Mills et al., 1997) was sequenced in

both directions for approximately 3.2 kbp downstream offmdB.stoichiometry of purified binding protein for [14C]urea was de-termined by equilibrium dialysis at 4°C in 20 mM bistris, Codon preference analysis showed the presence of two complete

open-reading frames (orfD and orfE, later identified asfmdDpH 6.8, essentially as described previously for the binding ofradiolabelled sugars to sugar-binding proteins (Cornish et al., andfmdE) plus an incomplete open-reading frame (orfF, later

identified asfmdF (Fig.1A and B). Each of these was preceded1989). The concentration of the binding protein was 0.83µM

47Mills et al. (Eur. J. Biochem. 251)

Fig. 2. Nucleotide sequence of the 8.4-kbpSph1 restriction fragment of M. methylotrophusDNA in pSW1 downstream of fmdB. The sequenceis shown in the 5′ to 3′ direction. The putative224/212 promotor site and the ribosome-binding sites are singly underlined, thePstI, EcoRI, andSphI restriction sites are doubly underlined, the translational-start codons are overdotted, and the translational-termination codons are overasterisked.Derived amino acid sequences of FmdD, FmdE, and FmdF (incomplete) are shown under the nucleotide sequence, the N-terminal signal sequenceof FmdD is underlined, and the directly determined N-terminal amino acid sequence of mature FmdD is doubly underlined.

by a putative ribosome-binding site (AGGAGA; Fig. 2) which12’ promotor sequences (GG-N10-GC) found upstream of vari-ous genes inK. pneumoniaeand S. typhimurium(Merrick,was very similar or identical to the consensus sequences inE.

coli (AGGAGG) and various methylotrophic bacteria including1988), of amiC in P. aeruginosa(Wilson and Drew,1991) andprobably also offmdCin M. methylotrophus(Mills et al.,1997).Methylophilusspp. (AGGAGA) (see Wyborn et al.,1996). Sev-

eral potential open-reading frames were identified on the oppo- No putative ‘235’/‘210’ promotor sequences corresponding tothe consensus sequences found inE. coli and various methyl-site strand, but none of these encoded proteins of molecular

mass greater than 5000 Da that were also preceded by candidate otrophic bacteria includingMethylophilusspp. were identifiedupstream offmdD. FmdDwas terminated by a TAA stop codon.ribosome-binding sites.The derived amino acid sequence indicated thatfmdD encodeda protein (FmdD) of 412 amino acids with a predicted molecularfmdD. fmdDcontained1236 bp and was preceded by a potential

ribosome-binding site (AGGAGA) that was centred11 nucleo- mass of 44 892 Da.To identify FmdD, washed cells prepared from a formamidetides upstream of the ATG start codon and 51 nucleotides down-

stream from the TAA stop codon offmdB. The potential ribo- limited continuous culture ofM. methylotrophuswere subjectedto SDS/PAGE, and a strongly expressed protein(s) of molecularsome-binding site was preceded by putative ‘224’ (TCGGGC)/

‘212’ (CAGCGA) promotor sequences centred 69 and 58 nu- mass approximately 40 000 was carefully excised and subjectedto N-terminal amino acid sequencing. This revealed two N-ter-cleotides, respectively, upstream of the ATG start codon. These

sequences were very similar to the nitrogen-regulated ‘224’/‘2 minal sequences (GATISF- and ADYPTA-) indicating the pres-

48 Mills et al. (Eur. J. Biochem. 251)

Fig. 3. Hydropathy profiles for FmdD, FmdE, and FmdF (incom-plete) based on Kyte-Doolittle assignments.The ordinate and abscissaof each plot represent the hydropathic index and the amino acid residue,respectively. Mature FmdD starts at residue 31 of FmdD. (A) FmdD;(B) FmdE; (C) FmdF (incomplete).

ence of two major proteins in approximately equal concentra-Fig.4. Amino acid sequence alignment of mature FmdD fromM.tions. One of these sequences (GATISF-) has recently been iden-methylotrophusand AmiC from Pseudomonas aeruginosa.Identicaltified as the N-terminal sequence of an outer-membrane porinamino acids in the two sequences are linked by vertical lines. Upper

sequence,M. methylotrophusmature FmdD; lower sequence,P. aerugi-for short-chain amides and urea encoded byfmdC (Mills et al.,nosa AmiC (Wilson and Drew,1991 ; Pearl et al.,1994). Predicted1997). The other sequence (ADYPTA-) was identical to aminoA-helical (*) andβ-sheet (s) regions are shown for each protein.acid residues 31236 of FmdD, suggesting that nascent FmdD

was processed by removal of a 30-amino-acid signal peptidefrom the N-terminal end of the nascent protein. This peptideexhibited the expected characteristics of a prokaryotic signal se- (LivJ; 21% identical, 44% similar), the leucine-binding proteins

from E. coli (LivK; 21% identical, 43% similar) andS. typhimu-quence (see Goodwin and Anthony,1995) namely a basic N-terminal region (two adjacent arginine residues, no acidic resi-rium (LivK, originally LivC; 22% identical, 43% similar), and

a putative binding protein of unknown function in the methylo-dues), a central region of high hydrophobicity (as evidenced bythe hydropathy profile of FmdD based on Kyte-Doolittle assign- trophic bacteriumMethylobacterium extorquens(AbcA; 18%

identical, 45% similar) (Landick and Oxender,1985; Adams etments; Kyte and Doolittle,1982) (Fig. 3A), a C-terminal do-main containing a helix-disrupting glycine residue, and an AxA al.,1990; Hoshino and Kose,1990; Onishi et al.,1990; Chisto-

serdova and Lidstrom,1997).sequence immediately upstream of the cleavage site.The derived amino acid sequence of mature FmdD indicated Secondary structure predictions (Rost and Sander,1993,

1994) showed that mature FmdD was particularly rich inA-heli-an overall hydropathic index of20.28, no major hydrophobicregions, an isoelectric point of 8.35 and a net charge of14.0. It cal regions (37%A-helix, 22%β-sheet), thus confirming its re-

semblance to AmiC (41% A-helix, 18% β-sheet). Furthermore,was concluded that mature FmdD is a hydrophilic, slightly basicprotein composed of 382 amino acids with a predicted molecular the elevenA-helical regions predicted in mature FmdD were

closely aligned to11 of the 12 A-helical regions predicted inmass of 41870 Da.Comparison of the derived amino acid sequence of mature AmiC, and many of the predictedβ-sheet regions also over-

lapped substantially. Comparison of mature FmdD and AmiCFmdD with other proteins in the databases revealed that it wasclosely related to the cytoplasmic acetamide-binding protein using optimum fold threading (Jones et al.,1992) yielded a

z-score of 24.06, confirming that the two proteins were veryfrom Ps. aeruginosa(AmiC; 31% identical, 53% similar includ-ing conservative replacements; Wilson and Drew,1991) (Fig. 4). closely related.

Analysis of subcellular fractions prepared fromM. methylo-It was also similar to various periplasmic binding proteins, prin-cipally the leucine/isoleucine/valine-binding proteins fromP. trophusshowed that mature FmdD (identified by SDS/PAGE

followed by N-terminal amino acid sequencing and western blot-aeruginosa(BraC; 26% identical, 48% similar) andE. coli

49Mills et al. (Eur. J. Biochem. 251)

Fig.6. Amino acid sequence of FmdF fromM. methylotrophusandBraE from Pseudomonas aeruginosa. Identical amino acids in the twosequences are linked by vertical lines. Upper sequence,M. methylo-trophusFmdF; lower sequence,P. aeruginosaBraE (Hoshino and Kose,1990). Predicted transmembrane regions (d) are shown for each protein.

Fig. 5. Amino acid sequence alignment of FmdE fromM. methylo-trophus and BraD from Pseudomonas aeruginosa. Identical amino

of FmdF showed that the protein contained.232 amino acidsacids in the two sequences are linked by vertical lines. Upper sequence,M. methylotrophusFmdE; lower sequence,P. aeruginosa BraD with a predicted molecular mass of.25435 Da. Hydropathic(Hoshino and Kose,1990). Predicted transmembrane regions (d) are and topological predictions indicated that FmdF was a hy-shown for each protein. drophobic protein containing at least 6 (I2VI) transmembrane

A-helices (Fig. 3C), and was organised in the cytoplasmic mem-brane such that the N-terminus was exposed on the cytoplasmic

ting; see below) was located in the high-speed supernatant frac-side (Kyte and Dolittle,1982; Hofmann and Stoffel,1993; Rosttion. This observation, together with the presence of a putativeet al.,1995).signal sequence in FmdD, indicated a periplasmic location. Comparison of the derived amino acid sequences of FmdE

and FmdF (incomplete) with other proteins in the databases re-vealed that they were very similar to the transmembrane proteinsfmdE and fmdF. fmdE contained 915 bp, was preceded by a

potential ribosome-binding site (AGGAGA) centred eight nucle- in various binding-protein-dependent, leucine/isoleucine/valinetransport systems. FmdE was most closely related to BraD/LivHotides upstream of the ATG start codon and130 nucleotides

downstream from the TAA stop codon offmdD, and was termi- fromP. aeruginosa(BraD; 25% identical, 53% similar includ-ing conservative replacements) (Fig. 5),E. coli (LivH, originallynated by a TAA stop codon. The derived amino acid sequence

of FmdE showed that the protein contained 305 amino acids called livA; 24% identical, 53% similar) andS. typhimurium(LivH; 24% identical, 53% similar) (Nazos et al.,1986; Adamswith a predicted molecular mass of 32 822 Da, an overall hy-

dropathic index of10.88, an isoelectric point of10.26 and a net et al.,1990; Hoshino and Kose,1990; Matsubara et al.,1992).FmdF was most closely related to BraE/LivM fromP. aerugi-charge of17. Hydropathic and topological predictions indicated

that FmdE contained at least 6 (I2VI), and possibly 8 (I2VI, nosa(BraE: 30% identical, 64% similar including conservativereplacements) (Fig. 6),E. coli (LivM; 27% identical, 67% simi-Ia, IIa), transmembraneA-helices (Fig. 3B) and was organised

in the cytoplasmic membrane such that both the N-and C-termini lar) andS. typhimurium(LivM; 27% identical, 66% similar)(Adams et al.,1990; Hoshino and Kose,1990; Matsubara et al.,were exposed on the periplasmic side (Kyte and Doolittle,1982;

Hofmann and Stoffel,1993; Rost et al.,1995). 1992). It should be noted, however, that BraE/LivM containedan additional 55262 amino acids at their N-terminal end com-Partial nucleotide sequencing offmdF as far as theSphI re-

striction site at the end of the insert showed that the gene con- pared with FmdF.It was concluded that FmdE and FmdF are hydrophobic,tained at least 697 bp, and was preceded by a potential ribo-

some-binding site (AGGAGA) centred ten nucleotides upstream transmembrane proteins that probably comprise part of thebinding protein-dependent amide-urea transport system inM.of the ATG start codon and103 nucleotides downstream from

the TAA stop codon offmdE. The derived amino acid sequencemethylotrophus.

50 Mills et al. (Eur. J. Biochem. 251)

Table 1. Purification of the amide-urea binding protein (mature FmdD) from M. methylotrophus. The binding protein was purified from aformamide-limited continuous culture (D 0.1 h21) of M. methylotrophusby cell fractionation, ammonium sulphate fractionation, and anion-exchangeFPLC as described in the Materials and Methods section. The concentration of the binding protein as a percentage of the total protein was determinedby SDS/PAGE and image analysis. HSS, high-speed supernatant; AS super, ammonium sulphate supernatant.

Fraction Volume [Protein] Total protein [FmdD] Total FmdD Purification Yield

ml mg ml21 mg % protein mg -fold %

Washed cells 48.4 31.0 1500.4 4.5 67.5 1.0 100.0HSS 13.0 20.0 260.0 15.3 39.8 3.4 59.065% AS super 26.5 1.04 27.6 32.4 8.9 7.2 13.2FPLC 16.3 0.55 8.9 95.0 8.5 21.1 12.6

Table 2. Effect of unlabelled compounds on the binding of [14C]ureato purified amide-urea binding protein (mature FmdD). Binding of[14C]urea to the purified binding protein was determined by equilibriumdialysis. Unlabelled compounds were present at 25-fold molar excessover [14C]urea.

Addition [14C]Urea binding

%

None 100Urea 66 2 (5)Formamide 66 3 (4)Acetamide 29618 (3)Propionamide 91 (2)Butyramide 85 (2)Formate 104 (2)Acetate 121 (2)Ammonia 100 (2)

Fig. 7. Purification of the amide-urea binding protein (maturewestern blotting showed that antiserum raised against the puri-FmdD) from M. methylotrophus. The binding protein was purified from

a formamide-limited continuous culture (D 0.1 h21) of M. methylo- fied binding protein cross-reacted strongly with the binding pro-trophusby cell fractionation, ammonium sulphate fractionation, and an-tein (mature FmdD), but not with the amide-urea porin (matureion-exchange FPLC as described in the Materials and Methods section.FmdC).Samples taken from each stage of the purification, procedure were sub-jected to SDS/PAGE and stained for protein. Lanes are as follows:1,

Physiological regulation of amide-urea binding protein ex-molecular mass standards; 2, washed cells; 3, broken cells; 4, high-pression. M. methylotrophuswas grown in continuous culturespeed supernatant fraction ; 5, 65% ammonium sulphate supernatant; 6,under various nutrient limitations with methanol as the carbonbulked anion-exchange FPLC fractions; 7, molecular mass standards.source and either ammonia, formamide, acetamide, or urea asthe nitrogen source. Washed cells were subjected to SDS/PAGEand the amide-urea binding protein was detected by westernPurification and properties of mature FmdD. Mature FmdDblotting (Fig. 8A and B). The concentration of the binding pro-was purified from a formamide-limited continuous culture ofM.tein was highest during growth under amide or urea limitationmethylotrophususing cell fractionation, ammonium sulphate(formamide/urea. acetamide@ ammonia), and was greatly di-fractionation, and anion-exchange FPLC (Table1; Fig. 7). Theminished during growth under methanol limitation (i.e. in thefinal purification factor of 21.1 indicated that the protein com-presence of a high concentration of ammonia resulting eitherprised 425% of the total cell protein. The purified proteinfrom its direct addition or from the hydrolysis of excess amideswas shown by SDS/PAGE to be.95% pure, and by aminoor urea). These results indicated that the amide-urea binding pro-acid sequencing to have the expected N-terminal sequencetein, like formamidase and the amide-urea porin (Mills et al.,(ADYPTA-). SDS/PAGE and gel-filtration chromatography1997), was induced by formamide and urea, and repressed byyielded subunit and native molecular masses of 41 kDa (cf.high concentrations of ammonia. It is likely, therefore, that ma-41870 Da from the derived amino acid sequence of matureture FmdD is the binding protein component of a transport sys-FmdD) and 45 kDa, respectively, indicating that the native pro-tem for short-chain amides and urea.tein is a monomer.

Equilibrium dialysis followed by Scatchard analysis showedthat the protein bound [14C]urea stoichiometrically (approxi- Urea transport. As washed cells of.M. methylotrophuspre-

pared from a urea-limited continuous culture exhibited not onlymately 0.8 nmol urea nmol protein21) and with high affinity (Kd

7.2µM), indicating that urea is bound strongly at a single site a high concentration of the amide-urea binding protein (andhence of the putative transport system), but also a very high(data not shown). Binding of [14C]urea was inhibited by a 25-

fold excess of unlabelled amides and urea (formamide/urea. urease activity (1.05µmol urea hydrolysed min21mg drycells21), urea uptake was measured as the rate of [14C]urea disap-acetamide@ propionamide/butyramide), but not by formate,

acetate or ammonia (Table 2), showing that mature FmdD is a pearance from the reaction mix rather than as the apparent rateof [14C]urea appearance in the cells, i.e. as a combination ofbinding protein for short-chain amides and urea, SDS/PAGE and

51Mills et al. (Eur. J. Biochem. 251)

the growing culture, and was commensurate with strong induc-tion of the transport system in cells grown under these condi-tions. [14C]urea disappearance was strongly inhibited by theuncoupling agent FCCP (20µM) and by unlabelled formamide(5 mM), neither of which was an inhibitor of, or a substrate for,partially purified urease.

The presence of a high-affinity, active transport system forurea (and short-chain amides) inM. methylotrophuswas stronglysupported by the very low steady-state concentration of urea(,50 µM) in a urea-limited continuous culture (D 0.1 h21) ofM. methylotrophus.This was122 orders of magnitude lowerthan both theKm for urea of the partially purified urease(3.8 mM) and the calculated steady-state concentration of ureain the absence of an active transport system (1.7 mM).

DISCUSSION

The work described above shows thatM. methylotrophuscontains a high-affinity, binding2protein-dependent, active-transport system for short-chain amides and urea composed atleast of a periplasmic binding protein (FmdD) and two trans-membrane proteins (FmdEF). FmdDEF closely resemble, re-Fig. 8. Physiological amide-urea binding protein (mature FmdD) ex-spectively, the periplasmic binding protein and the two trans-pression in M. methylotrophus.M. methylotrophuswas grown in con-

tinuous culture (D 0.1 h21) under various nutrient limitations. Washedmembrane proteins present in various leucine/isoleucine/valinecells were subjected to SDS/PAGE and either stained for protein (A) ortransport systems. The latter also contain an ATP-binding pro-transferred to nitrocellulose and probed with antiserum to the bindingtein, and hence are classed as ABC transporters. Thus, althoughprotein (B). Lanes are as follows:1, molecular mass standards; 2, am-the driving force for the FmdDEF system has not yet been iden-monia-limited (methanol-excess) cells; 3, methanol-limited (ammoniatified, by analogy with these other transport systems it is mostexcess) cells; 4, acetamide-limited (methanol-excess) cells ; 5, methanol-

likely to be ATP. The gene encoding the putative ATP-bindinglimited (acetamide excess) cells; 6, formamide-limited (methanol ex-protein was not found upstream offmdC(Greenwood, J. A. andcess) cells ; 7, methanol-limited (formamide excess) cells ; 8, urea-limitedJones, C. W., unpublished results) and hence is probably located(methanol excess) cells; 9, methanol-limited (urea excess) cells. Notedownstream offmdF (i.e. was not cloned in pSW1).that formamidase runs anomolously on SDS/polyacrylamide gels, giving

a subunit molecular mass of approximately 51 kDa compared with a The Fmd transport system is thus significantly different frompredicted value of only 44438 Da from the nucleotide sequence offmdA the putative amide transport system inPseudomonas aeruginosa,(Wyborn et al.,1994, 1996). Abbreviations are as follows: maturewhich is encoded by two of the genes in theami operon andFmdC, amide-urea porin; Mdh-A and Mdh-β, A- andβ-subunits of meth- appears to be comprised of a single transmembrane proteinanol dehydrogenase.

(AmiS) plus an ATP-binding protein (AmiB). Theami operonalso encodes a high-affinity amide-binding protein (AmiC)which, although closely related to FmdD, is located in the cyto-plasm rather than in the periplasm and comprises part of a twocomponent sensor-regulator system (Wilson et al.,1993; Pearlet al.,1994).

In terms of their relative abilities to bind amides, it is inter-esting to note that of the seven amino acid residues implicatedvia X-ray crystallography in the binding of acetamide by AmiC(Pearl et al.,1994), six are conserved or conservatively replacedin mature FmdD. These are Ser85 (Ser97), Tyr104 (Tyr116), andTyr150 (Tyr163), which form hydrogen bonds with the oxygenand nitrogen atoms of acetamide, and Tyr83 (Tyr95), Tyr152(Try165), and Thr233 (Ser245) that interact with the methylgroup of acetamide by Van der Waals’ forces. In contrast,Pro107, which forms a hydrogen bond with the nitrogen atomof acetamide, is replaced by Phe119. These differences are prob-ably at least partly responsible for the relatively poor binding of

Fig. 9. Transport of urea by washed cells ofM. methylotrophus. Dis- formamide by AmiC (Pearl et al.,1994) and of acetamide byappearance of [14C]urea (50µM) from a reaction mix containing washed mature FmdD. The other related periplasmic binding proteinscells ofM. methylotrophusprepared from a urea-limited continuous cul-

(BraC, LivJ, and LivK) conserve only Ser85 and Tyr152, andture (D 0.1 h21) was measured as described in the Materials and Methodsexhibit no conservative replacements.section. Additions: none (d); 20µM FCCP (j); 5 mM formamide (m).

Several proteins related to AmiS and/or AmiB which arealso possibly involved in the transport of short-chain amides orurea have recently been identified in other bacteria, namelyuptake and metabolism (see also Jahns et al.,1988). Using this

approach, the disappearance of [14C]urea (50µM) followed a AmiS2 and AmiB2 inRhodococcussp. R312, ORFP3 inM.smegmatis, and UreI and UreG inH. pylori (Cussac et al.,1992;non-linear time course (Fig. 9) that was not significantly altered

by the presence of methanol as an additional energy source. The Mahenthiralingam et al.,1993; Wilson et al.,1995; Chebrou etal.,1996). However, none of the transmembrane proteins (AmiS,initial rate of [14C]urea disappearance (>25 nmol min21 mg dry

cells21) was at least three times the rate of urea utilisation by AmiS2, UreI, ORFP3) are closely-related to FmdEF.

52 Mills et al. (Eur. J. Biochem. 251)

Goodwin, P. M. & Anthony, C. (1995) The biosynthesis of periplasmicIt is likely, therefore, that there are at least two types ofelectron transport proteins in methylotrophic bacteria,Micobiologytransport system for short chain amides and/or urea in bacteria,141, 105121064.viz. the FmdDEF type (containing a periplasmic binding protein,

Greenwood, J. A., Cornish, A. & Jones, C. W. (1990) Binding protein-two transmembrane proteins, and possibly an ATP-binding pro-dependent lactose transport inAgrobacterium radiobacter, J. Bacte-

tein, and thus probably comprising an ABC transporter), and an riol. 172, 170321710.AmiSB type (which lacks the periplasmic binding protein andHames B. D. (1981) An introduction to polyacrylamide gel electrophore-one of the transmembrane proteins, and hence may represent asis, inGel electrophoresis of proteins ; a practical approach(Hames,novel sub-family of ABC transporters; see Wilson et al.,1995). B. D. & Rickwood, D., eds) pp.1291, IRL, Oxford.

Hofmann, K. & Stoffel, W. (1993) TMbase2 a database of membrane-The high-affinity nature of urea uptake byA. eutrophus, K.spanning protein segments,Biol. Chem. Hoppe-Seyler 347, 1662pneumoniaeand B. megaterium(Jahns et al.,1988; Jahns and173.Kaltwasser,1989) would suggest that these organisms may con-

Hoshino, T. & Kose, K. (1990) Cloning, nucleotide sequences and iden-tain a urea transport system of the former type, although thetification of products of thePseudomonas aeruginosaPAO bra genesbinding protein would need to be anchored to the cytoplasmicwhich encode the high-affinity branched chain amino acid transportmembrane inB. megateriumsince this organism does not con- system,J. Bacteriol. 172, 553125539.

tain an outer membrane. Jahns, T., Zobel, A., Kleiner, D. & Kaltwasser, H. (1988) Evidence forSimple considerations of solute diffusion rates at very low carrier-mediated, energy-dependent uptake of urea in some bacteria,

external concentrations highlight the need for bacteria to synthe- Arch. Microbiol. 149, 3772383.Jahns, T. & Kaltwasser, H. (1989) Energy-dependent uptake of urea bysise appropriate outer-membrane porins and cytoplasmic mem-

Bacillus megaterium, FEMS Microbiol. Lett. 57, 13218.brane transport systems under these conditions, even for smallJones, D. T., Taylor, W. R. & Thornton, J. M. (1992) A new approachuncharged substrates that exhibit relatively high permeability co-

to protein fold recognition,Nature 358, 86289.efficients (Nikaido and Vaara,1985; Jahns et al.,1988). This isKyte, J. & Doolittle, R. F. (1982) A simple method for displaying thestrongly supported by the presence inM. methylotrophusnot

hydropathic character of a protein,J. Mol. Biol. 157, 1052132.only of a high-affinity active transport system for short-chainLandick, R. & Oxender, D. L. (1985) The complete nucleotide sequencesamides and urea, but also of an outer-membrane porin for theseof the Escherichia coliLIV-BP and LS-BP genes,J. Biol. Chem.substrates (Mills et al.,1997), both of which are maximally ex- 260, 825728261.pressed during growth under amide or urea limitation. Mahenthiralingam, E., Draper, P., Davies, E. O. & Colston, M. J. (1993)

Cloning and sequencing of the gene which encodes the highly induc-It is concluded, therefore, that the role of the active transportible acetamidase ofMycobacterium smegmatis, J. Gen. Microbiol.system for short-chain amides and urea inM. methylotrophusis139, 5752583.to catalyse the accumulative uptake of formamide, acetamide,

Matsubara, K., Ohnishi, K. & Kiritani, K. (1992) Nucleotide sequencesand urea into the cytoplasm prior to hydrolysis by their respec-and characterisation ofliv genes encoding components of the high-tive hydrolases, all of which exhibitKm values (>1.1mM; seeaffinity branched-chain amino acid transport system inSalmonellaalso Silman et al.,1991 ; Wyborn et al.,1994) that are up to typhimurium, J. Biochem.(Tokyo) 112, 932101.

two orders of magnitude higher than the steady-state externalMerrick, M. J. (1988) Nitrogen assimilation by bacteria,Soc. Gen.concentrations of these substrates during growth under amide orMicrobiol. Symp. 42, 3312361.urea limitation. Mills, J., Wyborn, N. R., Greenwood J. A., Williams, S. G. & Jones, C.

W. (1997) Molecular characterisation of an outer-membrane porininducible by short-chain amides and urea in the methylotrophic bac-The authors are indebted to Dr Mark Carver and Professor W. J.terium Methylophilus methylotrophus, Microbiology 143, 23732Brammar for useful discussions, to Dr K. Lilley for the N-terminal2379.amino acid sequencing, to Dr M. J. Sutcliffe for the optimal fold thread-

Mobley, H. L. T. & Hausinger, R. P. (1989) Microbial ureases: signifi-ing, and to Mr Paul Tyler for help with part of the work. NRW and JMcance, regulation and molecular characterisation,Microbiol. Rev. 53,were supported by Collaborative Awards in Science and Engineering852108.studentships (U.K. Science and Engineering Research Council) in col-

Mobley, H. L. T., Island, M. D. & Hausinger, R. P. (1995) Molecularlaboration with Zeneca BioProducts.biology of microbial ureases,Microbiol. Rev. 59, 4512480.

Nazos, P. M., Antonucci, T. K., Landick, R. & Oxender, D. L. (1986)Cloning and characterisation oflivH, the structural gene encoding a

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Note added in proof.The derived amino acid sequence of FmdD is alsosimilar to the nitrile hydratase regulators NhlC (27% identical; 51%similar) and NhhC (24% identical; 49% similar) fromRhodococcusrhodochrousJ1 [Komeda, H., Kobayashi, M. & Shimizu, S. (1996a)Proc. Natl Acad. Sci. USA 93, 426724272; (1996b)J. Biol. Chem. 271,15796215802] and to the acetamidase regulator ORFc (24% identical ;45% similar) from M. smegmatis[Parish, T., Mahenthiralingam, E.,Draper, P., Davis, E. O. & Colston, M. J. (1997) Microbiology 143,226722276].