J. Basic Microbiol. 35 (1995) 4,217-227

(Laboratoire de GCnCtique MolCculaire des Microorganismes et des Interactions Cellulaires, CNRS URA 1486, Institut National des Ciences AppliquCes, F-69621 Villeurbanne Cedes, France and ' Bereich Biochemie, Fakultat fur Biowissenschaften, Pharmazie und Psychologie, Universitat Leipzig, D-04 103 Leipzig, FRG)

The fix Escherichia coli region contains four genes related to carnitine metabolism KNUT EICHLER", ANNE BUCHET, FABIENNE BOURGIS, HANS-PETER KLEBER' and MARIE-ANDR~E MANDRAND-BERTHELOT

(Received 27 March 19957Accepted 4 April 1995)

Anaerobic carnitine metabolism in Escherichia coli was recently shown to involve six genes organized in the cai operon and located at the first minute on the chromosome. The DNA sequence lying at the 5' end of the cai locus was further investigated. It contains four open reading frames organized as an operon. In vivo overexpression of this DNA region revealed four polypeptides with apparent molecular masses of 27, 33, 45 and 6 kDa. These proteins displayed significant amino acid sequence homologies with polypeptides encoded by thefixABCX operons from Azorhizobium caulinodans and Rhizobium meliloti. The four ORFs were thus namedfixABCX. The first two gene products were also found to share a high degree of sequence similarity with the subunits /3 and a, respectively, of mammalian electron transfer flavoproteins, suggesting a role for these proteins in a redox reaction. A singly polycistronic 5 kb mRNA transcript was detected in Northern blots under anaerobic conditions in the presence of DL-carnitine. Expression of afixA-ZacZ transcriptional fusion was induced by L(-)-carnitine and crotonobetaine but not by D(+)-carnitine, y-butyrobetaine, glycinebetaine and choline as found previously for the carnitine pathway. Similarly, thefix operon was repressed by glucose and nitrate. Moreover, expression of thefix operon was induced by the global regulatory proteins CRP and FNR and repressed by the histone-like protein H-NS. All these regulatory proteins have been shown also to control expression of carnitine enzymes. Results from Northern blots and lacZ fusion studies indicate a common regulation of expression offix and cai operons, which implies a physiological linkage between these two loci.

The facultative anaerobe Escherichia coli synthesizes a number of respiratory chains under anaerobic conditions (STEWART 1988). Depending on the availability of alternative electron acceptors (in order of decreasing theoretical energy yield : nitrate, trimethylamine-N-oxide (TMAO) and fumarate), the cell can produced one or more of the terminal reductases of the electron transport pathway. In the absence of these electron acceptors, anaerobic growth of E. coli can be significantly stimulated in a complex medium supplemented by carnitine or its dehydration product crotonobetaine (SEIM et al. 1982). Since crotonobetaine is then reduced to y-butyrobetaine, it has been postulated that crotonobetaine serves as terminal electron acceptor.

Owing to its crucial role in P-oxidation of fatty acids in mammals, and to the following increasing demand for this compound in medicine, considerable work has been dedicated to the synthesis of the physiological L(-)-carnithe enantiomer by microbiological methods (JUNG et al. 1993). Recently, we characterized at the first minute on the E. coli chromosome the complex caiTABCDE operon encoding six proteins required for carnitine metabolism (EICHLER et al. 1994a). One of the proteins, encoded by the caiB gene, has been unambigously identified

*) Present address: c/o Hoffmann-LaRoche AG, Bau 64/40, Grenzacherstr. 124, CH-4002 Basel, Switzerland

218 K. EICHLER et al.

as carnitine dehydratase, based on the perfect identity of the N-terminal sequence of the purified enzyme with that derived from the nucleotide sequence (EICHLER et al. 1994~). According to the large number of proteins involved, the carnitine pathway appears to be much more complicated than the two-step reaction sequence previously described. Apart from CaiA which is thought to support the crotonobetaine reductase activity of the system, CaiT is suggested to be the transport system for carnitine or related trimethylammonium compounds. CaiC is suggested to be a crotonobetainekarnitine CoA ligase, CaiD a carnitine racemase and CaiE a protein required for the synthesis of the still unknown cofactor necessary for carnitine dehydratse, carnitine reductase (EICHLER et al. 1994b) and carnitine racemase activities (EICH- LER et al. 1994a).

Transcription of the cai operon is induced during anaerobic growth in the presence of carnitine. The activator of carbon catabolic operons CRP is required for its induction. In addition, the histone-like H-NS protein as well as the G' factor required for activation of stationary-phase genes exert a repressive effect on carnitine metabolism (EICHLER et al. 1994 a).

In the course of our study dealing with genetic characterization of the E. coli cai operon (EICHLER et al. 1994c), we detected four distinct ORFs which are located upstream of cai. The four ORFs are transcribed in the opposite direction to cai and bear significant similarities with thefixABCX nitrogen fixation genes of diazotrophic bacteria (EARL et al. 1987, ARIGONI et al. 1991). This report describes their structural organization and shows that they share a common regulatory pattern with the carnitine pathway. The function of these genes seems to be of primary importance for the electron transfer processes of the cell and is thus intimately correlated to carnitine metabolism.

Materials and methods

Bacterial strains, plasmids and media: E. coli NM 522 (supE rhi-l A (lac-proAB) A (mcr-hsdM)S ( r y r - J [F' proAB' lucP lucZAMIS]) (Stratagene) was used for the propagation of plasmids and E. coli K38 (HfrCphoAlpit-10 tonA22 ompF627 relA1 spoTl A+) (CHANG and COHNE 1978) for overexpression of FixABCX. NM.522 was also used as recipient for construction of mutant MAMlOl by transduction of Acrp4.5 using the nearby cysG98 : : TN5 insertion. MAMIOO (hns : : TnlO), MAMI02 (rpoN: : TnlO) and MAM103 (~$61 : : TnlOfir22) were derivates of strain NM 522. Plasmids pUCl8/19 (Boehringer) were used as the cloning and sequencing vectors. Overexpression was carried out withthe help of plasmids pT7-5 and pT7-6, described by TABOR and RICHARDSON (1985). The monocopy plasmid pJEL250 (VALENTIN-HANSEN et al. 1986) was used for studies of /3-galactosidase transcriptional fusions with the fix operon, since direct integration into the chromosome has been shown to be lethal (unpublished data). An 1.3 kb EcoRVIKpnI fragment harbouring thefix promoter region and the N-terminal part offixA was fused to the lacZ gene of pJEL250 and transformed into NM522 and derivatives. Bacteria were grown aerobically in L-broth at 30°, 37" or 42 "C, as indicated, or on plates with Luria broth containing 1.5% (wlv) agar. Anaerobic growth took place at 30 "C in tightly stoppered 250 ml bottles filled almost to the top with buffered (pH 7.5) rich medium TYEP without glucose (BEGG et ul. 1977) supplemented with 2 pM ammonium molybdate as describedprevioulsly (Wu and MANDRAD-BERTHELOT 1986). The minimal medium used was M9 (MANIATIS et al. 1982). When required antibodies were added at the following final concentrations : ampicillin SO pg/ml, kanamycin 20 pg/ml and rifampicin 200 pg/ml.

DNA manipulation: The following procedures were carried out using standard methods described by MANIATIS et al. (1982): preparation of plasmid DNA, agarose gel electrophoresis, DNA restriction and ligation, bacterial transformation in the presence of calcium chloride and Northern blotting.

DNA sequence analysis : DNA restriction fragments encompassing the entire region of interest were subcloned into pUC18/19. Both strands of the region were sequenced using the T7 sequencing kit from Pharmacia according to the chain termination procedure (SANGER et al. 1977). Computer analysis of DNA and amino acid sequences was performed with Mac Molly Tetra from Soft Gene GmbH. The Swiss-Prot and NBRF protein databases were searched using the algorithm FASTA of PEARSON and LIPMAN (1988).

Escherichiu colifixABCX operon 219

Northern analysis: RNA was prepared from log-phase cultures harboring pUC19 with the 5.0 kb KpnI-Suu3A fragment encoding thefi locus according to the procedure of SHIMOTSU et ul. (1986). Cells were grown under anaerobic conditions in the presence or absence of 20 mM DL-carnitine or in the presence of 100 mM nitrate. The probes used were internal fragments of thefixB andfiwC genes, excised from agarose gel and then labelled with the DIG dUTP. The hybridization was done in 50% (v/v) formamide, 5 xSSC, 1 % (w/v) blocking reagent (Boehringer Mannheim), 0,l % N-laurylsarcosine, 0.02% SDS at 55 "C overnight. The membrane was washed with 0.1 xSSC/O. 1 % SDS at 72 "C for 2x 15 min. Detection was carried out by the DIG Luminescent Detection Kit (Boehringer Mannheim).

Overexpression of Fix proteins : Overexpression was conducted using the in vivo amplification system of TABOR and RICHARDSON (1985). Plasmids pT7-5 and pT7-6, which contain the T7010 promoter upstream of the same polylinker placed in both directions, were used. Thus, hybrid plasmids pT7-5KE50 and pT7-6KE5 1 (Fig. 1) were created by insertion of the 5.0 kb KpnI-Suu3Afragment in both orientations into pT7-5 and pT7-6. These two plasmids were transformed into strain K38 harbouring the compatible plasmid pGP1-2 containing the gene coding for T7 RNA polymerase under the control of the heat inducible dp, promoter. The proteins specifically expressed from the cloned chromosomal region of E. coli 044 K74 were labelled with [3SS]methionine/cysteine (1000 Ci/mmol, NEN DuPont). Cells were then lysed in 60 mM Tris-HC1 (pH 6.8); 1 % SDS; 1 % 2mercaptoethanol by heating for 5 min at 95 "C and finally, loaded onto SDS-polyacrylamide gels as described by LAEMMLI (1970).

Enzyme assay: /3-Galactosidase activity was determined on cells harvested at the end of the exponential growth phase. Enzyme activity was assayed and specific activity was calculated as described by MILLER (1972).

Results and discussion

Cloning and expression of the upstream region of the cai locus

The previously described genomic library of E. coli strain 044 K74 was used to isolate colonies which harbour the region surrounding the cai locus, by screening with three different degene- rate oligonucleotide probes derived from the N-terminal sequence of the purified carnitine dehydratase (EICHLER et al. 1994~) . The entire cloned region of the E. coli chromosome comprised about 18 kb. A 5.0 kb KpnI-Sau3A fragment was further subcloned for overexpres- sion, subcloning and sequencing.

Overexpression was conducted following the in vivo amplification system of TABOR and RICHARDSON (1985). Plasmids pT7-5 and pT7-6 containing the T7010 promoter upstream of a polylinker placed in both directions were used. Thus, hybrid plasmids pT7-5KE50 and pT7-6KE5 1 were created by insertion of the 5.0 kb KpnI-Sau3A fragment in both orientations. The proteins specifically expressed from the cloned chromosomal region of E. coli 044 K74 were labelled with [35S]methionine/cysteine (1000 Ci/mmol, NEN DuPont), and separated on SDS-polyacrylamide gels, as described by LAEMMLI (1970). Three O m s were expressed from pT7-6KE51 which carries the 5' end of the cai locus in the KpnI-Sau3A orientation (Fig. 1, lane 1). Polypeptides with approximate molecular masses of 27 kDa, 33 kDa and 45 kDa were detected on the autoradiograph. The smallest polypeptide of less than 6 kDa (Fig. 1, lane 6) might correspond to the gene product of the fourth ORE Expression of the same insert cloned in the opposite direction in pT7-5KE50 revealed a truncated polypeptide of 25 kDa encoded by the first gene of the cai locus (Fig. 1, lanes 2 and 5).

Sequence analysis

The nucleotide sequence of a 4.2 kb KpnI-NruI fragment located upstream of the cai locus was determined. The EMBL accession number for the nucleotide sequence reported in this paper is X71977. Four ORFs encoding polypeptides of 268, 313, 427 and 95 aa, with predicted

220 K. EICHLER et al.

Fig. 1 Overexpression of four E. coli ORFs located upstream of the cai locus. Gene expression was induced, using the T7 RNA polymerase-promoter system of TABOR and RICHARDSON (1985).Proteins were labelled with ~-['~S]methionine/cysteine, after heat induction at 42 "C and addition of nfampicin. The labelled polypeptides were separated on (a) a 12.5% and (b) a 17.5% polyacrylamide gel containing 0.1 % SDS. Visualisation was carried out by autoradiography. The radioactivity of each sample loaded onto the gel was approximately lo6 cpm. Positions of molecular mass markers are shown in kilodaltons. Lanes: 1, pT7-6KE51 showing three major polypeptides (FixC, FixB and FixA from top to bottom); 2 and 5, pT7-5KE50 showing a truncated polypeptide of 25 kDa encoded by the cai locus; 3 and 4, pT7-5 without insert; 6, pT7-6KE51 showing an additional smaller than 6 kDa protein which might be FixX

molecular masses of 27,161 Da, 3 3 3 18 Da, 45,629 Da and 10,480 Da respectively, were found in this sequence. The orientation and the length of gene products of the first three ORFs were in good agreement with the in vivo results. There are three possible start codons for the first ORF but a putative SD-sequence (GGAGA) is only observed 5 nt upstream of the third ATG, which we chose as the probable start codon of ORF- 1. From nucleotide 173 to 188 a perfectly conserved CRP consensus sequence (TGTGA-N,-TCACA ; DE CROMBRUGGHE et al. 1984) was previously identified (EICHLER et al. 1994a). Two inverted repeats (indicated by arrows in Fig. 2) were found 130 nt upstream of the proposed start codon of ORF-1. 230 and 336 nt upstream of this start codon two nucleotide sequences were located which are similar to the -24/-12 (GG/GC) proposed promoter region of the fxABCX operons of Rhizobium meliloti (EARL et al. 1987) and Azorhizobium caulinodans (KAMINSKY et al. 1988) and of the nitrogen fixation (nif) genes from a number of free-living bacteria (for reviews see KUSTU et al. 1989, THONY and HENNECKE 1989). Hence it is anticipated that this sequence is recognized by the alternative RNA polymerase containing d4. At a distance of 74 nt from the first putative -24/- 12 promoter an upstream activator sequence (USA) TGT-N,,-ACA was detected, which is known to be necessary for NifA dependent activation of transcription of Bradyrhizobium japonicum nifgenes (ALVAREZ-MORALES et al. 1986). The E. coli glnG (ntrC) gene product, which closely resembles NifA and regulates the glnA structural gene for glutamine synthetase (MAGASANIK 1993) is a likely activator protein for this sequence. In addition, the nucleotide sequence in the possible upstream regulatory region exhibits short homopolymeric dA and dT stretches, which

Escherichia colifixABCX operon

-24/ -12 UAS ATGGTATTC AATAAXTGT A T T C m G A T T G G T G T C U TTTTTGTTTC GGGTGAATAG TACCCATAAG TTATTCGACA T A A G W A ACCACAGTGT AAAAACAAAG CCCACTTATC

-24 / AGAACGTTTT TCCGTTAATT TTGGTTATTA ATCAGTTTGT TATGTTCTTT TGTGETAAA TCTTGCAAAA AGGCAATTAA AACCAATAAT TAGTCAAACA ATACAAGAAA ACACCCATTT

CRP

-----I-___ -------- > ----------- -12 ------- -> AAAATAXAT CTGACTTTCA ATATTGGTGA TCTATAAAAC AATATTGAAA ATTTCTTTTT TTTTATCGTA GACTGAAAGT TATAACCACT AGATATTTTG TTATAACTTT TAAAGAAAAA

<============ <============= GCTACGCCAT GTTTTCAATA TTGGTGAGGA ACTTAACAAT ATTGAAAGTT GGATTTATCT CGATGCGGTA CAAAAGTTAT AACCACTCCT TGAATTGTTA TAACTTTCAA CCTAAATAGA

GCGTGTGACA TTTTCAATAT TGGTGATTAA AGTTTTATTT CCAAATTAAA GGGCGTGATA CGCACACTGT AAAAGTTATA ACCACTAATT TCAAAATAAA GGTTTAATTT CCCGCACTAT

f i x A

TCTGTAATTA ACACCACCGA TATGAACGAC GTTTCCTTCA TGATTTCTG m T G C A A T G AGACATTAAT TGTGGTGGCT ATACTTGCTG CAAAGGAAGT ACTAAAGACC TCTACGT

SD M

Fig. 2

22 1

210

270

330

390

450

510

Nucleotide sequence of the possible regulatory region of the& locus. The putative ribosome binding site (SD), the CRP consensus, the upstream activator sequence (TGT-N,,-ACA) (ALVAREZ-MORALES et al. 1986) and -24/- 12 (GG/GC) proposed promoter regions ( KUSTU et al. 1989, mow and HENNECKE 1989) are underlined. Two inverted repeats located upstream are indicated by arrows. The nucleotide sequence reported here has been deposited at the EMBL under accession number X71977

can act as curved DNA sequences binding to the E. coli histone-like protein H-NS (TANAKA et al. 199 1). Additional studies will be required to elucidate the real biological function of these regulatory elements. No terminator sequence was detected downstream of the last ORE

With the derived polypeptide sequences from the four ORFs NBRL and Swiss-Prot data bases were searched for homologies using the method of PEARSON and LIPMAN (1988). Surprisingly, the four E. coli proteins (27; 33 ; 45 and 10 kDa) bear significant similarities with thefixA (29.1 %, 28.6% identity),fzB (28.4%, 29.5% identity)fixC (36.8%, 35.7% identity) and fixX (40.0%, 42.2% identity) gene products from the diazotrophs A. caulinodans and R. meliloti respectively, that are involved in nitrogen fixation (ARIGONI et al. 1991, EARL et al. 1987). No precise biochemical function has yet been assigned to thefixABCX gene products. It has been proposed by EARL et al. (1987) that the related operon codes for a nitrogenase-speci- fic electron transport system which is unique to bacteria that fix nitrogen aerobically. Since the facultative anaerobic bacterium E. coli is not known to be able to fix nitrogen, we can exclude the involvement of these gene products in nitrogen fixation. As previously reported, we observed a remarkable similarity between the first and the second ORFs (FixA and B homolo- gues) and the /3 (26.4% identity) and Q (33.3% identity) subunits of mammalian electron transfer flavoproteins (ETFs), respectively. Therefore, according to the hypothesis of ARIGONI et al. (1991), it might be possible that the FixA and FixB homologues act as electron transfer proteins in a redox process.

As observed for FixC in R. meliloti (EARL et al. 1987), its 45 kDa E. coli counterpart was shown to contain a putative signal sequence, following the criteria defined by WATSON (1984). Neverthelesse, further investigation is required to determined whether the signal sequence is cleaved and whether the FixC homologous protein is located in the cytoplasmic membrane or in the periplasmic space.

I6 J . Basic Microbiol. 35 (19934

222 K. EICHLER et al.

The fourth ORF was identified as encoding a Fix-like polypeptide on the basis of amino acid comparisons with other previously establishedfixx sequences. At the C-terminal part of the protein a group of four cysteins, three of which are arranged in a C-x-x-C-x-x-C cluster typical of ferredoxins (BRUSCHI and GUERLESQUIN 1988) is similar to those already identified in rhizobia. Consequently, this supports the hypothesis that FixX could be a 3Fe-3S cluster protein able to transfer electrons.

In the framework of a systematic E. coli sequencing project the fix locus was already sequenced by YURA et al. (1992). Apart from the sequence and the homology with thefix ABCX operon from rhizobia no additional information about function or regulation was provided. A comparison between the bothfix nucleic acid sequences revealed an identity of more than 95 %. Only a few changes in the entire nucleotide sequence were observed. ThefixA andfixX genes were absolutely identical in length. A large discrepancy was detected for the size offixB which reaches 313 codons in our estimation against 76 codons to 193 in that of YURA et al. (1992). This could be explained by a single change from T, in the presumed start codon ATG we determined forfixB, to C occunring in the sequence reported by YURA et al. (1992). The ATG previously reported forfixC is not identical with our finding. A deletion of 7 nucleotides occurs in the N-terminal part of the sequence published by YURA et al. (1992). The potential start codon is thus moved upsteam including a frameshift and the resulting ORF (fix0 appears 3 nucleotides longer than the sequence presented in this work. In addition, the comparison of the derived total amino acid sequence revealed a high degree of identity apart from few conservative amino acid exchanges and the smaller FixB protein derived from the sequence of YURA et al. (1992). Discrepancy between both sequences may be due to the different strains from which the DNA was obtained. The E. coli strain 044 K74 used in this study was isolated from the intestine of a rat after a carnitine rich diet and selected for its high activity of carnitine metabolism. On the contrary, the K12 strain W3110 used by YURA et al. (1992) is a typical laboratory strain which has undergone several rounds of mutagenesis.

Northern analysis

In order to determine whether E. colifix-like genes are transcribed as an operon, Northern analysis was carried out. mRNA was extracted from log-phase anaerobic cultures of E. coli NM522 harboring pUC19 with the 5 kb KpnIISau3A fragment grown either in the presence or absence of 20 mM DL-carnitine or in the presence of 100 mM nitrate (Fig. 3). Using DNA probes derived fromfixB (Fig. 3) and fucC (data not shown), a mRNA species around 5 kb was only detected in the culture induced by carnitine (Fig. 3, lane 1). This result suggests that fix-like genes in E. coli are organized in an operon and are related to the metabolism of carnitine.

Transcriptional regulation of the fix operon

To confirm the suggested role of thefix operon in the carnitine pathway we decided to study the pattern of its expression in relation to the known regulation of carnitine metabolism (JUNG et al. 1987). Transcriptional regulation studies were performed using the pAB30 plasmid carrying afixA-lacZ operon fusion on the low copy number vector pJEL250 (VALENTIN-HAN- SEN etal. 1986), since direct integration into the chromosome was not reliable. Indeed, chromosomalfix mutants obtained by cassette insertion from plasmid fusions showed a normal growth rate under aerobic conditions but displayed a poor growth rate under anaerobiosis and a high level of mortality even in rich media. In addition, after a few subcultures, mutants became unstable and were subject to a high frequency of chromosome rearrangements as observed by Southern blotting (data not shown). The high mortality as well as the rearrangements of the chromosome could be an indication that fuc-like gene products play a crucial role in the cell, possibly in electron transfer processes.

Escherichia colifixABCX operon 223

Fig. 3 Luminograph of a Northern blot from thefix locus. RNA was extracted as described by SHIMOTSU et al. (1986) from E. coli NM522 harboring pUCl9 with the 5 kb KpnVSau3A fragment grown anaerobically in Luria-Broth in the presence of 20 mM DL-Carnitine (lane I), 100 mM nitrate (lane 2 ) and without supplement (lane 3). Total RNA (50 pg) was size fractionated on 1.2 % (w/v) agarose gel containing 1.1 % (v/v) formaldehyde, blotted onto positively charged nylon membrane (Boehringer Mannheim) and probed with a 1.2 kb EcoRV fragment harbouringfixB labelled with DIG dUTP

To determine whether the fa operon was subject to effectors of carnitine metabolism, cells were grown under anaerobic conditions in the presence of different trimethylammonium compounds (Table 1). In agreement with results from the Northern blot experiment, thefix operon was transcribed when carnitine was present either as the L-enantiomer or as the DL-carnitine racemic mixture at double the concentration. Crotonobetaine was also able to induce the transcription at the same level. On the contrary, D(+)-carnitine and y-butyrobetaine, although involved in this pathway, had no inducing effect. Other betaines like glycinebetaine and choline had no effect.

As observed above by Northern blotting, thefix operon is repressed under aerobic conditions even in presence of the previously mentioned inducers @-galactosidase activity : 35 nmol o-nitrophenol liberated m i d (mg bacterial dry weight)-'). We then tested other compounds for their ability to inhibit the pAB30 fusion expression when cells were grown under anaerobic conditions in a medium supplemented with 20 mM D,L-carnitine. Results are shown in Table 2. Glucose, a general catabolic repressor, severely reduces the transcription of the f i x operon. Expression levels in the presence of various electron acceptors were dependent on their nature : nitrate totally inhibited the expression, whereas trimethylamine-N-oxide had a partial effect and fumarate did not affect thefix operon expression at all. SEIM et al. (1982) have postulated for crotonobetaine a possible function as a terminal electron acceptor under anaerobic condi- tions. This hypothesis could explain the fact that the carnitine pathway is repressed when a more energetically favourable molecule, like nitrate, is available. Transcription of thefix operon seems to follow a similar behaviour. Moreover at equimolar concentrations, the other betaines (glycinebetaine and choline) did not affect induction of the f ix operon by D,L-carnitine. Since

I h*

224 K. EICHLER et al.

Table 1 Expression of the fixA-lacZ fusion in the presence of different trimethylammonium compounds

Trimethylammonium Concentration /3-galactosidase activity' compound' ( w )

pJEL250 pAB30 (fi)cA-lacZ)

D,L-camitine L-camitine Crotonobetaine D-carnitine y-butyrobetaine Glycinebetaine Choline

- 20 10 10 10 10 10 10

20 30 25 30 ND3 ND ND ND

30 5500 6000 5000

20 25 20 25

I Strain NM522 carrying either the monocopy vector pJE1250 or the hybrid plasmid pAB30 was grown under anaerobic conditions at 30 "C in presence of different trimethylammonium compounds as indicated.

' /3-galactosidase activity was determined as described in Methods and expressed in nmol o-nitrophe- no1 liberated min? (mg bacterial dry weight)-'. Data represent the mean of three independent experi- ments. ND: not determined.

this pattern of induction was in perfect agreement with that already described for carnitine metabolism (JUNG et al. 1987) the fix operon is thought likely to play a role in carnitine metabolism (EICHLER 1993).

It has previously been shown that carnitine metabolism is controlled via the CAMP receptor protein CRP, and that it is altered by the histone-like H-NS protein (EICHLER et al. 1994a). To examine the possible involvement of these general regulators in the f i x operon expression, pAB30 was introduced into crp and hns mutants. B-galactosidase activities measured after anaerobic cultivation in presence of 20 m~ DL-carnitine are given in Table 3. In the hns mutant, a 3-fold increase of thefix operon expression was observed. Examination of the DNAregulatory sequence 5', tofixA (Fig. 2) reveals the presence of curved DNA regions characterized as dA and dT rich stretches. They may function as regulatory regions to which the histone-like H-NS

Table 2 Influence of various growth conditions on thefix operon induction

Addition' Concentration /?-galactosidase activity of thefixA-lacZ

None - 5500 Glucose 0.4% 300 Nitrate 40 70 Trimethylmine-N-oxide 40 2000 Fumarate 40 5000 y -butyrobetaine 10 4500 Glycinebetaine 10 5400 Choline 10 5300

' Strain NM522 harbouring hybrid plasmid pAB30 was grown anaerobically at 30 "C in presence of 20 mM DL-camitine as inducer and the tested substances at the indicated concentrations.

' /3-galactosidase activity was determined as described in Methods and expressed in nmol o-nitrophenol liberated min-' (mg bacterial dry weight)-'. Representative results of three separate experiments are shown.

(mM) fusion'

Escherichia colifirABCX operon 225

Table 3 Expression of thefix4-lacZ fusion in various genetic backgrounds.

Strain' relevant genotype 8-galactosidase activity* NM522 MAM 100 MAMlOl MAM 102 MAM 103

Wild-type hns CrP rpoN fnr

5500 18 000

120 500 80

' Different mutant strains derived from NM522 harbouring the pAB30 plasmid were grown anaerobically at 30 "C in presence of 20 m~ DL-carnitine as inducer.

* P-galactosidase activity was determined as described in Methods and expressed in nmol o-nitrophenol liberated min-' (mg bacterial dry weight)-'. Representative results of three separate experiments are shown.

protein binds in vivo and acts as a repressor. In the crp mutant, the expression level of the pAB30 fusion was dramatically reduced. Activation by the CRP protein could occur through its binding to the perfectly conserved CRP binding site located in the region upstream of the f ix operon (Fig. 2). This could be further correlated with the repression observed in the presence of glucose (Table 2) and confirms that transcription is controlled via the CAMP level. These results support the conclusion that the f i x operon shares common regulatory features with carnitine metabolism. Examination of the promoter sequence showing a -24/- 12 box specific for the binding of RNA polymerase containing the alternative d4 factor (Fig. I) prompted us to analyse the influence of an rpoNmutation. A significant decrease of& expression was indeed observed in an rpoN mutant, lacking the alternative RNA polymerase d4 factor required for the transcription of nitrogen fixation genes of free living or symbiotic bacteria (KUSTU et al. 1989). However, the non-negligible residual activity measured in this mutant strongly suggests that transcription of the f i operon could initiate from another site. Finally, as fa operon expression is totally repressed under aerobic conditions (see above) we analyzed the expression of the fix-lacZ fusion in an f i r background. The f i r gene encodes a general regulator which activates transcription of several anaerobic respiratory operons (GUEST 1992). Introduction of the fusion into anfnr mutant led to the complete loss of /3-galactosidase activity. However, the promoter region does not contain an FNR binding site suggesting that the transcription of the f ix operon is not directly regulated by this activator.

Conclusion

Our data demonstrate that fa homologous genes and the cai locus implicated in carnitine metabolism are located in close proximity on the E. coli chromosome. It has been recently shown in R. meliloti that the genes encoding functions essential to the catabolism of betaines, including carnitine, lie adjacent tofix genes in the symbiotic region of the megaplasmid pSym (GOLDMANN et al. 1991). As the same localisation is also found for E. coli, we would suggest that these functions were associated during evolution and that the Fix counterparts have a functional relationship with the proteins encoded by the cai locus, which is exemplified by induction of transcription of thefa operon in the presence of L(-)-carnitine and crotonobetaine under anaerobic conditions. Both operons are dependent on general regulatory proteins (H-NS, CRP, FNR) (EICHLER et al. 1994a). The Fix proteins could play a role in the transfer of electrons to crotonobetaine reductase CaiA (EICHLER et al. 1994b) encoded by the cai operon. Crotono- betaine reductase would then convert crotonobetaine to y-butyrobetaine (EICHLER et al. 1994a). This putative function remains still to be elucidated.

226 K. EICHLER et al.

Acknowledgements

We are grateful to S. TABOR for generously providing bacterial and plasmid strains. We also thank L. F. Wu, PH. LEJEUNE and W. NASSER for helpful discussions and scientific advice and V. JAMES for reading the manuscript. We acknowledge the support of this work by grants of the Centre National de la Recherche Scientifique and the Direction de la Recherche et des Etudes Doctorales. This work was also supported by grants from the Deutschen Akademischen Austauschdienst and the Ministkre des Affaires EtrangBres (Procope grant no 92 636).

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