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Applied and Environmental Microbiology, January 2006, p. 276-283, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.276-283.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification, Mutagenesis, and Transcriptional Analysis of the Methanesulfonate Transport Operon of Methylosulfonomonas methylovora

Mohammed Jamshad,1 Paolo De Marco,2 Catarina C. Pacheco,2 Timea Hanczar,2,3 and J. Colin Murrell1*

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom,1 Cell and Applied Microbiology, IBMC, Universidade do Porto R. Campo Alegre, 823, 4150-180 Porto, Portugal,2 Department of Biotechnology, University of Szeged, Temesvari krt. 62, 6726 Szeged, Hungary3

Received 3 June 2005/ Accepted 23 September 2005


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ABSTRACT
 
Recently identified genes located downstream (3') of the msmEF (transport encoding) gene cluster, msmGH, and located 5' of the structural genes for methanesulfonate monooxygenase (MSAMO) are described from Methylosulfonomonas methylovora. Sequence analysis of the derived polypeptide sequences encoded by these genes revealed a high degree of identity to ABC-type transporters. MsmE showed similarity to a putative periplasmic substrate binding protein, MsmF resembled an integral membrane-associated protein, and MsmG was a putative ATP-binding enzyme. MsmH was thought to be the cognate permease component of the sulfonate transport system. The close association of these putative transport genes to the MSAMO structural genes msmABCD suggested a role for these genes in transport of methanesulfonic acid (MSA) into M. methylovora. msmEFGH and msmABCD constituted two operons for the coordinated expression of MSAMO and the MSA transporter systems. Reverse-transcription-PCR analysis of msmABCD and msmEFGH revealed differential expression of these genes during growth on MSA and methanol. The msmEFGH operon was constitutively expressed, whereas MSA induced expression of msmABCD. A mutant defective in msmE had considerably slower growth rates than the wild type, thus supporting the proposed role of MsmE in the transport of MSA into M. methylovora.


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INTRODUCTION
 
Methanesulfonic acid (CH3SO3H; MSA), an important naturally occurring sulfonate, accumulates in the frozen layers of snow in Antarctica (51) and Greenland (67), but it cannot be detected anywhere else in the environment. Due to its stability and lack of further photochemical oxidation of methanesulfonicacid, microorganisms have been implicated in the oxidation of MSA. A novel methylotroph, Methylosulfonomonas methylovora strain M2, isolated from garden soil (3) used MSA as both a carbon and sulfur source, and recently, newly isolated MSA utilizers from terrestrial and aquatic habitats, including Hyphomicrobium, Methylobacterium, and Pedomicrobium species were shown to contain an inducible multicomponent MSA monooxygenase (MSAMO), enabling the metabolism of MSA as a carbon and energy source (4).

The characterization of MSA utilization as a sole carbon and energy source by Methylosulfonomonas methylovora strain M2 (3, 25) revealed the initial step to be attack of the carbon atom by activated oxygen, yielding the unstable intermediate hydroxymethanesulfonate, which spontaneously decomposed to formaldehyde and sulfite (20). The unique, multicomponent MSAMO enzyme was a cytoplasmic enzyme induced in M. methylovora strain M2 when grown on MSA as sole carbon and energy source (25). MSAMO could also be induced when MSA was the sole sulfur source. The formaldehyde produced was assimilated into cell biomass via the serine pathway (25) or dissimilated via formate to carbon dioxide and water to yield reducing equivalents for energy generation and biosynthesis.

The structural genes for MSAMO were clustered on the chromosome of M. methylovora strain M2 on a 7.5-kb SphI DNA restriction fragment (11). Sequence analysis (11) revealed the presence of seven open reading frames (ORFs) (Fig. 1A), in which msmABCD encode the large ({alpha}) and small (ß) subunit hydroxylase components, the ferredoxin, and reductase, respectively. msmEF encoded components of a putative MSA transport system of the ABC-type superfamily. Closely associated with the msmABCD genes was an incomplete ORF, orfX, with sequence similarity (55 to 60%) to transcriptional activators of the LysR-type family (57).


Figure 1
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  		FIG. 1. (A) Physical map of the msmHGFEABCD region. Rectangles indicate the designated genes. Triangles mark the two putative promoters. The arrows indicate proposed transcripts. (B) Transcriptional analysis of the msm operons. The direction of the arrow indicates the direction of the RT reaction, with the 5' positions of the primer pair given above. The blank boxes containing a cross represent the lack of positive amplification. (M) RNA from MSA-grown cells; (Me) total RNA from methanol-grown M. methylovora strain M2 cells. The numbers refer to the gene sequence in the updated sequence (GenBank accession number AF091716).

Characterization of the MSAMO (19, 46) enzyme revealed the existence of a two-component hydroxylase (43 and 23 kDa), a ferredoxin (16 kDa), and a reductase (38 kDa) component, all of which were necessary for MSAMO activity (26). The molecular mass of the native hydroxylase was indicative of an {alpha}3ß3 structure, and its oxidized spectrum suggested the presence of a Rieske [2Fe-2S] center, similar to hydroxylases from several hydroxylating dioxygenases which have iron sulfur centers as cofactors (11, 35). A unique extended spacer region between the two conserved histidine residues of the Rieske-iron center motif of the {alpha} subunit of the MSAMO was ubiquitous in all MSA-utilizing organisms containing the MSA monooxygenase (4, 11) and may confer substrate specificity toward MSA in the MSAMO enzyme system (4). The ferredoxin component of MSAMO (MsmC) was a small acidic electron transfer protein containing a Rieske [2Fe-2S] center, with spectral characteristics similar to those of other proteins containing Rieske [2Fe-2S] centers. The reductase component of M. methylovora (MsmD) possessed significant identity (conserved flavin adenine dinucleotide and NAD-binding motifs and putative plant type [2Fe-2S] binding motifs) to the reductase components of known oxygenases, which possess chloroplast-like [2Fe-2S] centers (35).

The aim of this study was to complete the cloning and sequencing of the msmEF gene cluster and also to investigate the role of these gene products in the uptake and transport of MSA into M. methylovora strain M2, since under physiological conditions (28) the sulfonate moiety of MSA is charged, thus active transport is necessary to transport MSA to the cytoplasmic enzyme, MSAMO. We also report on the transcriptional regulation of the msm operons in response to various carbon sources under defined growth conditions.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions.
The methylotroph Methylosulfonomonas methylovora strain M2 was routinely grown and maintained on mineral salts medium (MinE) adapted from that described by Owens and Keddie (41) containing the following (per liter of distilled water): K2HPO4, 1.2 g; KH2PO4, 0.624 g; CaCl2 · 2H2O, 0.05 g; MgSO4 · 7H2O, 0.165 g; (NH4)2SO4, 0.5 g. Phosphates were autoclaved separately. Two milliliters of trace elements solution (61), previously filter sterilized, was added per liter. In reporter gene experiments, phosphate buffer was used at 4x strength (45.6 mM) to avoid acid limitation and to obtain growth to higher cell densities. Antibiotics were used at the following final concentrations: chloramphenicol, 50 mg/liter; tetracycline, 10 mg/liter; trimethoprim, 20 mg/liter.

Carbon substrates were added as filter-sterilized solutions to a final concentration of 15 mM, unless otherwise stated. Methanol was added after media had been autoclaved and cooled, from a filter-sterilized stock.

For solid media, DIFCO Bacto agar was added to 15 g per liter prior to sterilization. All medium solutions were autoclaved for 15 min at 121°C unless stated otherwise.

Escherichia coli strains used in this study were grown in Luria-Bertani broth (LB) in the presence of appropriate antibiotics to ensure plasmid maintenance as described by Sambrook et al. (52). SOB and SOC media used in transformation procedures were prepared as described by Hanahan (15). The plasmids used in this study are described in Table 1. For solid complex media, 1.5% (wt/vol) of DIFCO Bacto agar was added to LB prior to sterilization by autoclaving. When required, IPTG (isopropyl-ß-D-thiogalactopyranoside; Calbiochem) at a final concentration of 24 µg/ml and/or X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; NBL Gene Science) at 50 µg/ml was added to solid media.


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  		TABLE 1. Description of plasmids used in this study

DNA manipulations.
Standard molecular genetic manipulations, including plasmid isolation, E. coli electroporation, restriction enzyme digestion, and Southern hybridization, were performed according to the method of Sambrook et al. (52). Plasmids were transferred between E. coli and M. methylovora by conjugative transfer (33). The rapid extraction of chromosomal DNA from M. methylovora was carried out by the method of Oakley and Murrell (40). Plasmid DNA was prepared as outlined by Sambrook et al. (52).

Construction of clone libraries of M. methylovora strain M2 DNA.
Genomic DNA libraries were constructed in the multicopy vectors pUC18 and pUC19 (New England Biolabs). The vectors were digested with the appropriate restriction enzyme and then subjected to 5' phosphate removal by treatment with calf intestine alkaline phosphatase (Boehringer Mannheim) to prevent self-ligation of the vector. Genomic DNA from M. methylovora was digested to completion with the same (or compatible) restriction enzyme and then size fractionated through an agarose gel. The desired DNA fragments were cut out of the gel and extracted using the GENECLEAN kit II (Bio 101, California) according to the manufacturer's instructions. These fragments were then ligated into the vector using T4 DNA ligase (BRL) in accordance with the manufacturers' instructions and subsequently used to transform chemically competent cells (E. coli XL1-Blue [Stratagene] or TOP10 [Invitrogen Corporation]).

DNA sequencing and analysis.
DNA sequencing was carried out using DyeDeoxy terminators (PE Applied Biosystems, United Kingdom) by the University of Warwick Sequencing Facility, with Perkin-Elmer ABI 373A and 377 automated sequencers. Dimethyl sulfoxide (DMSO) to a final concentration of 5% (vol/vol) was added to cycle sequencing reaction mixtures when sequencing GC-rich regions or problematic templates. Analysis of DNA sequences and homology searches were carried out with standard DNA sequencing programs and the BLAST server of the National Center for Biotechnology Information using the BLAST algorithm (1, 2).

PCR.
PCR was performed in 50-µl reaction mixtures in 0.5-ml microcentrifuge tubes using a Hybaid Touchdown thermal cycling system. Taq polymerase (Gibco BRL) was used. After an initial denaturation step of 94°C for 5 min, Taq polymerase was added. Then 30 cycles of 94°C for 1 min, annealing temperature (varied with primer) for 1 min, and 72°C for 1 min were performed, followed by a final extension of 10 min at 72°C. Reaction products were checked for size and purity on 1% (wt/vol) agarose gels. Twenty-five microliters of 2x stock DMSO-betaine (2.6 M betaine, 2.6% [vol/vol] DMSO) was added when problems were encountered during PCR amplification.

Isolation of total RNA.
Total RNA from M. methylovora growing exponentially in batch cultures (optical density at 540 nm [OD540], 0.25 to 0.3) on MSA (15 mM) or methanol (15 mM) was extracted using a method based on that of Nielsen et al. (38). RNA preparations (1 to 5 µl) were examined using 1.2% (wt/vol) agarose gels. DNA was removed from RNA samples using RQ1 DNase (Promega) according to the manufacturer's instructions. The RNA was quantified by measuring the A260 and A280 using a DU-70 spectrophotometer (Beckman). One A260 unit is 40 µg/ml RNA.

RT-PCR.
Fifty picomoles of primer was added to 1 µg of DNA-free RNA and water to a final volume of 5 µl, heated for 10 min at 65°C, and quickly cooled on ice. To this mixture, 4 µl of 5x Expand reverse transcription (RT) buffer, 2 µl 100 mM dithiothreitol, 2 µl of each deoxynucleoside triphosphate (10 mM), and 1 µl of Expand reverse transcriptase (40 units/µl) (Roche) were added. After incubation at 42°C for 60 min, the mixture was put on ice to stop the reaction. The cDNA transcripts generated were directly used in standard PCRs (Table 2 shows positions and sequences of primers). Negative controls for PCR were RNA only and H2O. M. methylovora DNA as a template was used as a positive control and to optimize the conditions for PCR analysis. The products of the reactions were visualized on Tris-borate-EDTA-agarose gels.


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  		TABLE 2. List of primers used in the reporter gene fusion construction and RT-PCR analysis to amplify the intergenic regions between the msm genesa

Reporter gene experiments with the msmA-E promoter region.
The intergenic region between msmABCD and msmEFGH was amplified by PCR (using Pfu polymerase; Fermentas, Lithuania) using primers ProA and ProE (Table 2), and the resulting amplicon was cut with HindIII and ligated into the HindIII site of broad-host-range expression vector pPR9TT (53) in either orientation, yielding constructs pTIMI (start of msmA in frame with lacZ) and pIMIT (start of msmE in frame with lacZ). Primers msmPA and msmPE (Table 2) were used to amplify a slightly smaller region corresponding exactly to the intergenic region. This second amplicon was ligated into the HindIII site of plasmid pCM130 (34), yielding constructs pCM130PA and pCM130PE. The constructs were confirmed by restriction analysis and by sequencing of amplicons generated by PCR using vector-based external primers. Transconjugants for enzyme assays were grown in the presence of the appropriate antibiotic to mid-exponential phase before harvesting. Cell extracts were obtained by sonication of cells in a Branson Sonifier 250 (4 cycles of 30 s at 50% duty, output 3) and centrifugation at 16,000 x g for 20 min. The protein content of extracts was quantified by the BCA protein assay kit (Pierce) using bovine serum albumin (BSA) as a standard. ß-Galactosidase activity was measured in cell extracts by following the method of Miller (36). Catechol 2,3-dioxygenase (C2,3O) activity was quantified by following the method of Kunz and Chapman (31).

Construction of an insertion mutant of msmE.
Plasmid pDM5 containing the MSAMO structural and MSA transport-encoding genes carried on a 7.5-kb SphI DNA restriction fragment was digested with SphI and EcoRI, yielding three fragments of 2.16 kb, 2.30 kb, and 3.03 kb. The 2.30-kb fragment containing msmE and msmF was isolated from an agarose gel (GENECLEAN kit II; Bio 101, California) and subcloned into pUC19 (New England Biolabs) to give plasmid pMJ1. The resultant ampicillin-resistant clones were analyzed by restriction mapping and PCR using the MSME FOR and MSMF RT (Table 2) primers (397-bp product) to confirm the presence of msmE. A 926-bp SalI restriction fragment internal to the msmE gene was then excised and replaced with a gentamicin resistance cassette (889 bp) from plasmid p34S-Gm (12), carried on a SalI DNA restriction fragment. The resulting plasmid was designated pMJ2. In the final step, the construct containing the mutated gene was subcloned into the narrow-host-range suicide vector pK18mobsacB (Tn5 Kmr selectable marker, the Bacillus subtilis sacB gene as a counterselectable marker, and RP4 mob machinery) (56) and introduced into M. methylovora by conjugation. The protocol was based on that of Martin and Murrell (33). A 10-ml overnight culture of E. coli strain S17.1 (59) donor cells was collected by filtration on a sterile 0.2-µm nitrocellulose filter (Millipore type GS) using 47-mm Millipore filter units. This was then washed with 50 ml of sterile MinE-10 mM phosphate solution. Fifty milliliters of the recipient, M. methylovora strain M2 (OD540 ~0.3), was collected on the same filter as the donor strain and washed again. The filter was then incubated for 24 h at 30°C on MinE agar containing 15 mM formate, 10 mM phosphate, and 0.02% (wt/vol) Proteose peptone. Cells were retrieved by vigorously washing the filters in 1 ml of sterile MinE–phosphate medium. Dilutions of this cell suspension were spread onto appropriate selective media and incubated at 30°C until colonies formed (typically 7 to 10 days). Transconjugants were selected on plates containing gentamicin at 5 µg/ml and then screened on MinE–methanol plates containing 5 µg/ml of gentamicin and 10 µg/ml of kanamycin to select for the loss of the vector. Ten gentamicin-resistant colonies were isolated, and two exhibited sensitivity to kanamycin and resistance to sucrose (10% wt/vol), suggesting loss of the vector-borne antibiotic resistance marker and counterselection cassette via a double-crossover recombination event. Southern hybridization, DNA sequencing (data not shown), and the ability of M. methylovora to grow on MSA (Fig. 2) were parameters used in the further examination of these two putative mutant colonies, designated mutant 6 and mutant 7.


Figure 2
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  		FIG. 2. Growth curve of wild-type (WT) M. methylovora strain M2 and the msmE insertion mutant 7. The OD540 was monitored using a Beckman DU-70 spectrophotometer. Cells were grown with MSA (15 mM) as the sole carbon and energy source. For the negative control, water replaced the carbon source.

Nucleotide sequence accession number.
The newly acquired msmFGH sequence was submitted to GenBank and used to revise the original pDM5 msm sequence (11). The GenBank accession number for the revised pDM5 sequence is AF091716.


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RESULTS
 
Cloning and sequence analysis of the putative MSA transport system genes.
To obtain the complete sequence of the putative components of the MSA transport system from Methylosulfonomonas methylovora, Southern blotting was used to identify fragments in the chromosome containing flanking msm genes. This approach used a random-priming labeled 397-bp PCR-derived msmEF hybridization probe, spanning both msmE and msmF from M. methylovora, and identified an ~5.5-kb SstI fragment containing putative MSA transport genes. This SstI fragment was cloned into SstI-cut pUC19 to generate the recombinant plasmid pMJ386 and then sequenced.

The forward primer MSM E FOR and the reverse primer MSMF RT (Table 2) were used to amplify a 397-bp msmEF fragment from the recombinant plasmid pMJ386. Direct partial sequencing of this amplification product produced sequence similarity to the msmA gene of M. methylovora strain M2. Sequencing of the recombinant plasmid pMJ386 yielded the DNA sequence previously not present on plasmid pDM5 (11). Analysis of this sequence revealed four ORFs, two of which, msmE and msmF (incomplete), had been described previously (11) and two which were unidentified, designated msmG and msmH (Fig. 1A). All were divergently transcribed from the MSAMO structural gene cluster msmABCD. The four components were proposed to constitute an MSA/sulfonate transport system belonging to the ABC-type superfamily of transporters. Sequence alignment analysis of these open reading frames with other related transport system components (Table 3) further supported the proposed role of the products of these genes in the transport of MSA into M. methylovora.


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  		TABLE 3. Comparison of the amino acid sequences of MsmE, MsmF, MsmG, and MsmH with other related proteins in the SwissProt protein databasea

Sequence analysis of msmE.
Analysis of msmE from M. methylovora (10, 11) suggested that the translated open reading frame encoded a 385-amino-acid protein, with a theoretical isoelectric point (pI) of 9.8 and calculated molecular mass of 41,669 Da. This closely resembled the mass of a polypeptide observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MSA-grown cells of M. methylovora (data not shown). The presence of a hydrophobic leader sequence was previously noted (10), and further evidence for this leader sequence was provided by the prediction of a possible cleavage site near the N terminus, between residues 19 and 20 (GenBank accession no. AF091716) by the SignalP WWW server (39; this study), thus targeting MsmE to the periplasmic space. The MsmE protein revealed significant identity with other periplasmic substrate binding proteins, in particular, several components of organosulfonate uptake systems. For example, the taurine-binding periplasmic constituent of Escherichia coli (5, 65) also contains a putative leader peptide, as does the atsR-encoded sulfate ester transport protein of Pseudomonas aeruginosa PAO1 (21).

Representative BLAST scoring sequences (Table 3) reveal between 23 and 27% identity (38 to 52% similarity) between MsmE and other known periplasmic substrate binding proteins. These proteins vary in size and substrate specificity and, as a consequence, little sequence identity was detected between these binding proteins. However, a common feature of such proteins studied, in particular, the (Sbp) sulfate binding protein of Salmonella enterica serovar Typhimurium (42), implicated hydrogen bonding for the interaction between the substrate and binding protein. As a result, conformational changes in the protein facilitated substrate retention (32, 50) and subsequent interaction with the associated integral membrane component which was a highly specific reaction not observed in the absence of bound substrate (16, 43). Further evidence for the involvement of MsmE in MSA translocation in M. methylovora was gained by disruption of msmE by marker exchange mutagenesis, which resulted in a significant decrease in growth by comparison to wild-type M. methylovora cells (Fig. 2).

Sequence analysis of msmF.
The msmF gene sequence revealed that it encoded a 301-amino-acid polypeptide with a calculated molecular mass of 33,248 Da and a theoretical pI of 9.4. The predicted sequence had 33 to 39% identity (52 to 54% similarity) to membrane-associated components from several microorganisms, including E. coli, Pseudomonas spp., Mesorhizobium loti, Brucella melitensis, and Methanopyrus kandleri AV19 (Table 3). Further characterization of MsmF using the HMMTOP transmembrane topology prediction server (62) and the PSORT server employing the detection method of Klein et al. (29) identified six probable, highly hydrophobic membrane-spanning regions, each of 15 to 25 amino acids. Sequence conservation between the transmembrane domains of different ABC-type transporters is typically low (16, 47).

Sequence analysis of msmG.
msmG, located 3' of msmF, encoded a 287-amino-acid polypeptide with a molecular mass of 31,369 Da and a theoretical pI of 6.1 (22). Analysis of the protein sequence revealed a high degree of relatedness to ATP-binding ABC-type components (Table 3), ranging between 37 and 45% identity and 56 and 65% similarity. The likely location of this protein within the cell, its hydrophobicity, and its membrane-spanning domains were determined using the PSORT program, version 6.4 (WWW version). Association with the bacterial inner membrane was implied, a common trait among such components of the ATP-binding ABC-type transporters (8, 16).

Sequence alignment analysis of MsmG with the other ATP-binding homologues (data not shown) listed in Table 3 revealed highly conserved characteristic motifs for this type of protein (54, 66). The Walker A motif (GPSGCGKT) of MsmG revealed good identity to this consensus sequence in representatives of the ATP-binding ABC-type family of proteins (GXXGXGKS/T). The Walker B motif (VLLLDEPY) closely matched the canonical sequence hhhhDEPF (16, 23, 58). The invariant residues lysine in the Walker A motif and aspartate in the Walker B motif were conserved in MsmG. These residues are known to be critical for nucleotide (Mg ATP) binding and phosphoryl transfer in related proteins (6).

Sequence analysis of msmH.
msmH was incomplete, and the translated protein sequence revealed a polypeptide of 163 amino acids (17,711 Da). The theoretical pI was 10.3. The available sequence of MsmH revealed 35 to 44% identity (55 to 68% similarity) to related ABC-type transporter permease proteins (Table 3). The presence of three hydrophobic membrane-spanning domains was predicted by HMMTOP and PSORT computational analysis. It was clear that the C terminus of the MsmH protein was absent, since the high-scoring homologues were typically between 110 and 130 residues longer and three further transmembrane regions are expected in the terminal 100 to 150 residues, thus completing the six characteristic hydrophobic membrane-spanning elements. In both the MsmF and MsmH, a consensus motif similar to the EAA loop (EAA- - -G- - - - - - - - -I-LP) (7) was identified, approximately 80 to 100 residues from their C termini. This motif is conserved between hydrophobic membrane-spanning proteins occurring on a hydrophilic loop exposed to the cytosol. However, Saurin et al. (55) reported conservation of the primary glutamate residue (E) at position 1 in 50% of EAA sequences, whereas 75% conservation was observed for the alanine residue (A) at position 3 of the EAA consensus sequence. Mutational analysis of this conserved region within MalF and MalG (integral membrane-associated components) of the E. coli maltose transport system (37) revealed that this motif was important in facilitating the interaction between the integral membrane-spanning permease domains and the ATP-binding components of the ABC transport complex.

Transcriptional analysis of the msmEFGH cluster in M. methylovora.
RT-PCR analysis of total RNA from an MSA-grown culture of M. methylovora revealed no product for the region between msmE and msmA (Fig. 1B, M, lane 4), which confirmed our previous assumption that the two clusters were divergently transcribed from this promoter region. RT-PCR products were observed for the intergenic regions spanning the msmABCD region (Fig. 1B, M, lanes 5, 6, and 7) and the msmEFGH region (Fig. 1B, M, lanes 1, 2, and 3). Amplification of transcripts for the regions between msmE-msmF, msmF-msmG, and msmG-msmH (Fig. 1B, Me, lanes 1, 2, and 3) indicate that these genes are linked and cotranscribed as a polycistronic mRNA. The detection of transcript for msmEFGH, also in methanol-grown cells, indicated that methanol failed to repress expression of these genes, which are thus actively transcribed even in the absence of MSA.

Again with methanol-grown cells, no RT-PCR transcript was detected between msmE and msmA (Fig. 1B, Me, lane 4), confirming that msmEFGH and msmABCD were divergently transcribed from two separate promoters, located within the 427-nucleotide region between msmE and msmA. RT-PCR failed to generate transcripts for the regions between msmA-msmB, msmB-msmC, and msmC-msmD when methanol was the growth substrate (Fig. 1B, Me, lanes 5, 6, and 7), which correlates well with the observations of Davey (9) that expression of MSAMO occurred only during growth on MSA and not on alternative carbon sources such as methanol.

Reporter gene expression with the msmA-E promoter region.
To examine the differential expression from the msmE and msmA promoters in response to methanol or MSA, reporter gene fusions were constructed. ß-Galactosidase activity was detectable in E. coli transformed with plasmid pTIMI (data not shown). However, transconjugant strains of M. methylovora with either plasmid pTIMI or pIMIT grown on MSA showed no activity. DNA from transconjugants was extracted by plasmid miniprep, and since plasmid yields were too low to be visible on gels, they were tested by PCR using vector-based primers and restriction analysis. To ascertain the presence of a fully correct construct in transconjugant clones, E. coli DH5{alpha} was back transformed with miniprep extracts. Plasmids retrieved from E. coli transformants were positive after PCR amplification with specific primers, but their restriction profiles were aberrant, suggesting that constructs pTIMI and pIMIT were unstable in M. methylovora and underwent rearrangement.

XylE-reporter plasmid constructs pCM130 (empty vector) (34), pCM130PA (intergenic region cloned in the msmABCD orientation), and pCM130PE (intergenic region cloned in the msmEFGH orientation) were then conjugated into M. methylovora and proved to be stable. C2,3O activity was measured after growth on four carbon sources. In clones harboring pCM130PA and pCM130PE, when grown on MSA, average activities were amplified approximately 53 and 588 times, respectively, compared to those seen after growth on methanol (Table 4). After growth on methylamine or formate, the activities were very low with all constructs (not shown).


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  		TABLE 4. C2,3O activities in transconjugant clones of M. methylovora strain M2a

Phenotypic characterization of the msmE mutant.
Further evidence for the involvement of the MsmE protein in MSA transport in M. methylovora was gained by disruption of msmE by marker exchange mutagenesis. A knockout plasmid was constructed and transferred to M. methylovora by conjugation. The msmE gene of M. methylovora was disrupted by replacement of a central region (926 bp) of the gene with a gentamicin resistance cassette (Gmr) from p34S-Gm. This mutated version of msmE was transferred into pK18-mobsac vector, which could then be transferred into E. coli S17-1 before mating with M. methylovora. The Bacillus subtilis sacB counterselectable marker system was used. The product of this sucrose-inducible gene, levansucrase, proved lethal when cells were exposed to sucrose, thus making the discrimination between single- and double-crossover recombination events possible. Transconjugants exhibiting loss of the vector backbone and the sacB counterselection cassette via a double-crossover recombination event were phenotypically characterized, and significant differences in growth were observed between the mutant and wild type (Fig. 2). Southern hybridization analysis with the 889-bp gentamicin resistance cassette probe (data not shown) confirmed the rare double-crossover homologous recombination event in mutant 7, and it was therefore selected for further analysis. Direct partial sequencing of the disrupted msmE gene amplified by PCR from mutant 7 confirmed the sequence of the gentamicin resistance cassette flanked by the msmE gene sequence (data not shown) and that the phenotypic changes observed in mutant 7 were due to a disrupted msmE gene.

The growth curves of mutant 7 (Fig. 2) revealed that the growth rate of mutant 7 (0.012 h–1) was clearly less than the growth rate of the wild type on MSA (0.114 h–1), and the final optical density was approximately 35% of that of the wild type. The fact that the mutant grew at all was interesting, suggesting that the cell still managed to take up MSA but to a lesser degree than the wild type. From other experimental data (not shown here), it was clear that reduction in the concentration of MSA from 15 mM to 0.5 mM affected expression of the MSAMO in wild-type M. methylovora, with a concomitant decrease in biomass. Growth of the mutant on MSA could have been a consequence of MSA transport via a nonspecific (low affinity) transport system. The existence of a second periplasmic MSA-binding protein cannot be ruled out, since, for example, two periplasmic binding proteins were observed for the sulfate/thiosulfate transport complex (Sbp-CysP) of Escherichia coli K-12 (63) involved in the binding of sulfate and thiosulfate, respectively.


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DISCUSSION
 
ABC transport systems usually comprise of three components: a periplasmic substrate binding protein, an integral membrane-associated component, and an ATP-binding protein (16, 17, 27). De Marco et al. (11) identified two putative components of such a system in M. methylovora. msmE and msmF revealed a high degree of identity to periplasmic substrate-binding and membrane-associated components, respectively (Table 3). In the case of the putative MSA transport system of M. methylovora, we propose that the newly identified MsmF and MsmH polypeptides constitute the membrane-associated permease in which each polypeptide possesses six hydrophobic transmembrane regions. These elements are proposed to form a ring-like opening, based on the structural analysis of the eukaryotic drug resistance P-glycoprotein (47, 48, 49) that allows substrate transport across the biological membrane. The msmG-encoded ATP-binding component of this system would function as a homodimer to couple ATP hydrolysis to substrate transport. Intergenic transcripts for msmABCD and msmEFGH were observed when MSA served as a carbon source, suggesting the coordinated expression of msmABCD and msmEFGH (Fig. 1).

The genetic organization of the cluster resembled several well-studied systems involved in import and export of a variety of compounds (14, 27). Most of the existing research was performed with E. coli and S. enterica serovar Typhimurium in which, for example, the histidine (HisJQMP) (18, 30) and maltose (MalEFGK) (7) import systems were comprised of a periplasmic substrate-binding protein HisJ/MalE, the dimeric membrane-associated permease HisMQ/MalFG, and the homodimeric ATP-binding subunits HisP/MalK. The sulfate/thiosulfate transport complex (Sbp-CysPTWA) of Escherichia coli K-12 (60) also revealed similar organization, whereby cysTW encoded the membrane components, CysA was the ATP-hydrolyzing complex, and CysP and Sbp were responsible for thiosulfate and sulfate binding in the periplasmic space, respectively.

When total RNA from methanol-grown M. methylovora served as a template, no transcripts were detected for msmABCD. However, msmEFGH was still transcribed (Fig. 1), suggesting that induction of the MSAMO in M. methylovora occurs only during growth with MSA and that a basal level of transcription of genes encoding the putative MSA transport system remains during growth on other substrates. In this way, cells would be constitutively primed to actively respond to the presence of MSA in the environment.

The clustering of msmEFGH and msmABCD (Fig. 1) and their functional relationship suggested that these eight genes constitute two operons for the coordinated expression of MSAMO and the MSA transporter system.

The intergenic DNA region between msmE and msmA consisted of two divergent (putative) promoters, directing expression of MSAMO structural genes (msmABCD) and putative MSA transport system genes (msmEFGH). Reporter gene fusion analysis in M. methylovora with the msmE-msmA intergenic region revealed highly MSA-dependent reporter gene activity with either orientation of the msmA-E promoter region, which correlated well with the RT-PCR analysis.

Genetic organization similar to that found for msm genes of M. methylovora was seen in E. coli and P. aeruginosa PAO1 in operons encoding sulfonate sulfur utilization systems (44, 45). Structural genes encoding the desulfonating oxygenases were flanked by the components responsible for sulfonate uptake. Expression and mutagenesis studies confirmed the role of these genes in the transport of sulfonates (44, 63, 65). In E. coli, in-frame deletion mutagenesis of the tauABC and ssuABC genes (in which tauA and ssuA, tauB and ssuB, and tauC and ssuC express the periplasmic substrate binding protein, ATP-binding enzyme, and a integral membrane-associated protein, respectively) (13) resulted in transport mutants unable to utilize taurine and aliphatic sulfonates, respectively, thus confirming the role of these genes.

This work is the first report of the use of marker exchange mutagenesis for gene inactivation in M. methylovora. Growth of the msmE::Gm mutant on MSA was much slower than that of the wild type. Inactivation of msmE will also result in deleterious polar affects on the expression of all downstream genes in the msmFGH operon. Since msmABCD are cotranscribed, this ultimately implicates the putative gene products in the transport of MSA. Similar findings were observed in E. coli and Bacillus subtilis carrying disrupted periplasmic binding protein encoding genes (64).


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ACKNOWLEDGMENTS
 
This work was supported by a BBSRC Committee Studentship to M.J.

We thank P. M. Santos and C. Marx for kind gifts of vectors pPR9TT and pCM130, respectively. Our gratitude goes to P. Moradas-Ferreira for support and to D. Kelly for advice throughout this project.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: 44 24 76 523553. Fax: 44 24 76 523568. E-mail: J.C.Murrell{at}warwick.ac.uk Back


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Applied and Environmental Microbiology, January 2006, p. 276-283, Vol. 72, No. 1
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