The e¡ect of growth in continuous culture at various input C:Nratios on the expression of proteins involved in the transport

and metabolism of methanol and short-chain amidesby Methylophilus methylotrophus

James Mills 1, Jaqueline A. Greenwood, Colin W. Jones *Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK

Received 12 January 1998; revised 2 February 1998; accepted 2 February 1998

Abstract

Physiological regulation of proteins involved in the transport and metabolism of methanol and short-chain amides byMethylophilus methylotrophus was investigated following growth in continuous culture at various input C:N ratios. Theconcentrations of the methanol porin and methanol dehydrogenase were highest during C-limited growth (C:N6 4.6), butdeclined gradually as a function of the increasing C:N ratio and were lowest during N-limited growth (C:Ns 16.3). Incontrast, the concentrations of the amide-urea porin, the amide-urea binding protein, formamidase and acetamidase (togetherwith formamidase and acetamidase activities) were lowest during C-limited growth, but increased sharply as a function of theC:N ratio and were highest during dual CN-limited and N-limited growth (C:N 4.6^16.3). The results are discussed in terms ofthe physiological and biochemical requirements of growth at varying C:N ratios. z 1998 Federation of European Micro-biological Societies. Published by Elsevier Science B.V.

Keywords: Methylophilus methylotrophus ; Methanol and amide transport; Methanol and amide metabolism; Dual substrate-limited growth

1. Introduction

The methylotrophic bacterium Methylophilusmethylotrophus grows readily on methanol as asource of carbon, and on a wide range of compoundsincluding ammonia, short-chain amides and urea as

sources of nitrogen. Methanol enters the periplasmby simple di¡usion or via a methanol porin, depend-ing on the methanol concentration, and is then oxi-dised to formaldehyde by a PQQ-linked methanoldehydrogenase [1^4]. In contrast, short-chain amidessuch as formamide and acetamide enter the peri-plasm by simple di¡usion or via an amide-ureaporin, depending on the amide concentration, andare then transported into the cytoplasm via a bindingprotein-dependent, active transport system prior tobeing hydrolysed to ammonia and the correspondingcarboxylic acid by formamidase (formamide amido-

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 7 1 - 8

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* Corresponding author. Tel. : +44 (116) 2523458;Fax: +44 (116) 2523369; E-mail: cwj1@le.ac.uk

1 Present address: Cantab Pharmaceuticals Research Ltd,184 Cambridge Science Park, Milton Road,Cambridge CB4 4GN, UK.

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hydrolase; EC 3.5.1.49) and acetamidase (acylamideamidohydrolase; EC 3.5.1.4) respectively. All ofthese proteins have been puri¢ed and characterised[4^8,10,11], and most of the genes encoding themhave been cloned and sequenced [9^12].

In order to integrate carbon (methanol) and nitro-gen (amide) utilisation successfully, M. methylotro-phus must be able to regulate very precisely the con-centrations of the appropriate transport proteins andmetabolic enzymes. This paper describes the expres-sion of these proteins during growth in continuousculture at various input C:N ratios.

2. Materials and methods

2.1. Growth of M. methylotrophus

M. methylotrophus (NCIMB 10515) was grown incontinuous culture (dilution rate (D) 0.1 h31) at37³C, pH 7.0 in methanol-acetamide-salts mediumessentially as described previously [5], except thatthe medium contained a ¢xed concentration of acet-amide (0.53 g l31) and increasing concentrations ofmethanol (1.0^6.5 g l31) in order to produce C-lim-ited, dual CN-limited and N-limited growth condi-tions (C:N ratios 3.0^19.3). C:N ratios were calcu-lated on the basis that none of the carbon present inacetamide could be used for cell growth. The pres-ence of methanol or ammonia (produced by hydrol-ysis of acetamide) in culture supernatants was deter-mined as described previously [5,6].

2.2. SDS-PAGE and Western blotting

Cultures grown at di¡erent C:N ratios were har-vested and samples of identical cell mass were sub-jected to discontinuous SDS-PAGE as described pre-viously [5,7]. SDS-polyacrylamide gels were stainedfor protein with Kenacid blue R and scanned using

an Epson £atbed scanner; information was subse-quently digitised and analysed using Adobe Photo-shop and NIH Image packages respectively. The to-tal density of each stained protein band (e.g. themethanol dehydrogenase K-subunit; Mr 60 000) de-termined in this way was taken to represent the con-centration of the protein expressed in arbitrary units.Alternatively, unstained SDS-polyacrylamide gelswere Western blotted and then probed with antiserato the methanol porin, the amide-urea porin, theamide-urea binding protein, formamidase and acet-amidase as described previously [4,10^12].

2.3. Enzyme activities

Washed cells were prepared and assayed for for-mamidase and acetamidase activities as describedpreviously [5,9].

3. Results

M. methylotrophus was grown in continuous cul-ture (D, 0.1 h31) at seven di¡erent input C:N ratiosin the range 3.0^19.3. Analysis of culture superna-tants identi¢ed three nutrient-limited conditions, viz.C-limited (C:N ratio6 4.6; no detectable methanol,high concentration of ammonia), dual CN-limited(C:N ratio 4.6^16.3; no detectable methanol or am-monia) and N-limited (C:N ratios 16.3; no detect-able ammonia, high concentration of methanol). Celldensity increased slightly as a function of the increas-ing C:N ratio during C-limited growth, but remainedessentially constant during dual CN-limited and N-limited growth (Fig. 1a).

SDS-PAGE and image analysis showed that theconcentration of methanol dehydrogenase (measuredas the K-subunit; Mr 60 000) was highest during C-limited growth, but gradually declined as a functionof the increasing C:N ratio during dual CN-limited

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6Fig. 1. Variations in cell density, the concentrations of selected nutrients and cell proteins, and the activities of selected enzymes duringthe growth of M. methylotrophus in continuous culture at di¡erent input C:N ratios. M. methylotrophus was grown in continuous culture(D, 0.1 h31) with methanol as the carbon source and acetamide as the nitrogen source. Cell density and residual nutrient concentrationswere determined as described in Section 2. Washed cell samples were assayed for amidase activities or were subjected to SDS-PAGE,stained for protein with Kenacid blue R and quantitated by image analysis as described in Section 2. a: Cell density (R), ammonia (F),methanol (b). b: Methanol dehydrogenase (K-subunit ; Mdh-K) (E), methanol porin (a). c: Formamidase (O), formamidase activity (R),acetamidase (P), acetamidase activity (S), amide-urea porin+amide-urea binding protein (Mr 40 000 proteins) (7).

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growth and reached a minimum during N-limitedgrowth (Figs. 1b and 2a). The L-subunit (Mr 8500;not shown) behaved similarly, but its small size pre-cluded accurate quantitation. The concentration ofthe methanol porin (Mr 38 000) followed a virtuallyidentical pattern (Figs. 1b and 2a), which was re-£ected semi-quantitatively in Western blots usingantiserum to the porin (Fig. 2b).

In contrast, SDS-PAGE and image analysisshowed that the concentrations of formamidase (sub-unit Mr 51 000 by SDS-PAGE, cf. 44 438 by genesequencing and 44 481 by electrospray mass spec-trometry) and acetamidase (subunit Mr 38 000), to-

gether with the combined concentrations of theamide-urea porin (Mr 40 000 by SDS-PAGE, 39 204by gene sequencing) and the amide-urea binding pro-tein (Mr 40 000 by SDS-PAGE, 41 870 by gene se-quencing), were all lowest during C-limited growthbut increased during the later stages of C-limitedgrowth and the early stages of dual CN-limitedgrowth (C:N ratio 3.0^7.5), and then either remainedessentially constant or decreased slightly (Figs. 1cand 2a). These changes were con¢rmed and extendedsemi-quantitatively by Western blotting using anti-sera to formamidase, acetamidase, the amide-ureaporin and the amide-urea binding protein (Fig. 2c^

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Fig. 2. Variations in the concentrations of selected cell proteins during the growth of M. methylotrophus in continuous culture at di¡erentinput C:N ratios. M. methylotrophus was grown in continuous culture (D, 0.1 h31) with methanol as the carbon source and acetamide asthe nitrogen source. Washed cell samples were subjected to SDS-PAGE and either stained for protein with Kenacid blue R (a) or Westernblotted and developed with antiserum to the methanol porin (b), formamidase (c), acetamidase (d), the amide-urea porin (e) or theamide-urea binding protein (f) as described in Section 2. Lanes: 1, Mr standards; 2, C:N 3.0; 3, C:N 4.6; 4, C:N 7.5; 5, C:N 10.4;6, C:N 13.4; 7, C:N 16.3; 8, C:N 19.3. Mdh-K, K-subunit of methanol dehydrogenase; Mr 40 000 proteins, amide-urea porin+amide-ureabinding protein.

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f), which also showed that the porin and the bindingprotein behaved in an essentially identical manner.In addition, the changes in formamidase and acet-amidase concentrations were closely re£ected in theactivities of these enzymes (Fig. 1c).

4. Discussion

The results described above show that M. methylo-trophus, like several other species of bacteria, can begrown in dual CN-limited continuous culture (see[13]), and that this can be used to investigate in detailthe expression of proteins involved in the utilisationof methanol and short-chain amides.

The high expression of the methanol porin andmethanol dehydrogenase during C-limited growthclearly re£ects the need of the organism to maintainthe rate of methanol metabolism demanded by theimposed dilution rate (D, 0.1 h31) when the metha-nol concentration in the growth medium is extremelylow (see also [4,6]). Conversely, both proteins aremuch less-strongly expressed during N-limitedgrowth, when methanol is present at high concentra-tion. Under this latter condition, methanol probablytraverses the outer membrane mainly by simple dif-fusion and thus becomes available in the periplasmat relatively high concentration; lower concentra-tions of the methanol porin and methanol dehydro-genase are therefore needed to maintain the requiredrate of methanol metabolism compared with growthunder methanol limitation. This pattern of expres-sion, together with the virtually identical decline inthe concentrations of the two proteins as a functionof the increasing C:N ratio during dual CN-limitedgrowth, indicates that the expression of these pro-teins is probably controlled in concert, possibly viacarbon-regulated promoters of the type found up-stream of other mox genes (see [2]).

Similarly, the high expression of the amide-ureaporin, the amide-urea binding protein, formamidaseand acetamidase during N-limited growth re£ects theneed of the organism to maintain the required rate ofammonia production in the cytoplasm when theamide concentration in the growth medium is ex-tremely low. All four proteins are strongly repressedduring C-limited growth, presumably by the highconcentration of ammonia resulting from hydrolysis

of the excess acetamide [5,9,11,12]. Under this lattercondition, short-chain amides probably traverse theouter and inner membranes mainly by simple di¡u-sion and become available in the cytoplasm at rela-tively high concentrations; lower concentrations ofthe appropriate transport proteins and hydrolasesare therefore needed to maintain the required ratesof amide metabolism compared with growth underamide limitation. The very similar patterns of expres-sion of these four proteins suggest that they may besubject to similar genetic regulation. Indeed, thegenes encoding formamidase (fmdA), the amide-urea porin (fmdC) and the amide-urea binding pro-tein (fmdD) are all preceded by putative nitrogen-regulated promoters (although fmdA is also precededby a putative carbon-regulated promoter) [9^12]. Theacetamidase gene from M. methylotrophus has notyet been sequenced.

The observation that expression of the proteinsinvolved in methanol transport and metabolismchange gradually over a wide range of C:N ratios,whereas expression of the proteins involved in amidetransport and metabolism change more sharply overa much narrower range of C:N ratios, is of somesigni¢cance since it indicates that during dual CN-limited growth the organism is much more sensitiveto the limited availability of acetamide than to thelimited availability of methanol. This is probably be-cause methanol dehydrogenase has a Km for metha-nol that is approximately two orders of magnitudelower than that of acetamidase for acetamide (or offormamidase for formamide) [1,5,7,9,10].

The detection of this di¡erential regulation of thetransport and metabolism of methanol and short-chain amides reinforces the usefulness of employingdual CN-limited chemostat culture at varying C:Nratios to investigate enzyme expression at a physio-logical level (see also [13]), since it could clearly nothave been achieved using simple C-limited and N-limited cultures.

Acknowledgments

The authors wish to thank Dr M.A. Carver foruseful discussions, and ZENECA BioProductsfor the award of a BBSRC-CASE studentship toJ.M.

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[2] Goodwin, P.M. and Anthony, C. (1995) The biosynthesis ofperiplasmic electron transport proteins in methylotrophic bac-teria. Microbiology 141, 1051^1064.

[3] Xia, Z.-X., Dai, W.-W., He, Y.N., White, S.A., Boyd, G.D.,Matthews, F.S. and Davidson, V.L. (1996) Methanol dehy-drogenase structure. In: Microbial Growth on C1 Compounds(Lidstrom, M.E. and Tabita, F.R., Eds.), pp. 220^226.Kluwer, Dordrecht.

[4] Greenwood, J.A., Mills, J. and Jones, C.W. (1997) Puri¢ca-tion, properties and physiological regulation of a putative out-er-membrane porin for methanol in Methylophilus methylotro-phus. FEMS Microbiol. Lett. 153, 167^171.

[5] Silman, N.J., Carver, M.A. and Jones, C.W. (1989) Physiol-ogy of amidase production by Methylophilus methylotrophus :isolation of hyperactive strains using continuous culture.J. Gen. Microbiol. 135, 3153^3164.

[6] Greenwood, J.A. and Jones, C.W. (1986) Environmentalregulation of the methanol oxidase system of Methylophilusmethylotrophus. J. Gen. Microbiol. 132, 1247^1256.

[7] Silman, N.J., Carver, M.A. and Jones, C.W. (1991) Directedevolution of amidase in Methylophilus methylotrophus : puri¢-cation and properties of amidases from wild-type and mutantstrains. J. Gen. Microbiol. 137, 169^178.

[8] O'Hara, B.P., Wilson, S.A., Wyborn, N.R., Jones, C.W. andPearl, L.H. (1994) Crystallisation, preliminary x-ray analysisand secondary structure determination of formamidase fromMethylophilus methylotrophus. Protein Peptide Lett. 1, 202^205.

[9] Wyborn, N.R., Scherr, D.J. and Jones, C.W. (1994) Puri¢ca-tion, properties and heterologous expression of formamidasefrom Methylophilus methylotrophus. Microbiology 140, 191^195.

[10] Wyborn, N.R., Mills, J., Williams, S.G. and Jones, C.W.(1996) Molecular characterisation of formamidase from Me-thylophilus methylotrophus. Eur. J. Biochem. 240, 314^322.

[11] Mills, J., Wyborn, N.R., Greenwood J.A., Williams, S.G. andJones, C.W. (1997) An outer-membrane porin inducible byshort-chain amides and urea in the methylotrophic bacteriumMethylophilus methylotrophus. Microbiology 143, 2373^2379.

[12] Mills, J., Wyborn, N.R., Greenwood J.A., Williams, S.G. andJones, C.W. (1998) Characterisation of a binding protein-de-pendent, active transport system for short-chain amides andurea in the methylotrophic bacterium Methylophilus methylo-trophus. Eur. J. Biochem. 251, 45^53.

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