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1 Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, OR 97331, USA
2 Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA
3 Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA
Correspondence
Luis A. Sayavedra-Soto
sayavedl{at}science.oregonstate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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54-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C2–C5 n-alkanes and showed little to no growth on C6–C8 n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the ‘P. butanovora’ bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C2–C8 carbon sources. In acetate- or lactate-grown cells, C2–C8 n-alcohols failed to induce β-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced 
45 % of the β-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C2–C5 n-alcohols induced β-galactosidase activity, whereas C7 and C8 n-alcohols did not. BmoR may act as a 54-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. 
C6). The bmoG-deficient mutant did not grow on C2–C8 n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.
The GenBank/EMBL/DDBJ accession number for the sequence of the bmo operon and its adjacent genes of ‘Pseudomonas butanovora’ is AY093933.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Beauvais & Lippard, 2005; Blevins & Perry, 1972; Brazeau et al., 2001; Hamamura et al., 2001; Marín et al., 2001, 2003; van Beilen & Funhoff, 2007; Yuste et al., 1998), little is known about the bacterial regulatory processes that tightly control the expression of the metabolic pathways involved in the utilization of the alkane and the metabolites that result from its oxidation. Our studies on ‘Pseudomonas butanovora’, a Gram-negative β-proteobacterium that is closely related to the genus Thauera (Anzai et al., 2000) and is capable of growth on C2–C9 n-alkanes (Takahashi et al., 1980), are helping us to understand how alkane-utilizing bacteria adapt to use the available growth substrate. For example, we have documented the effects of n-alkane chain length on the physiology of ‘P. butanovora’ and on the expression of its butane monooxygenase (BMO), the enzyme that initiates the oxidation of the n-alkane (Doughty et al., 2006, 2007; Halsey et al., 2006). Our studies have revealed that BMO is induced by the primary alcohols produced by BMO activity rather than by the alkane substrates (Doughty et al., 2005; Sayavedra-Soto et al., 2005), and that the expression of BMO is subjected to feedback repression by propionate, a product of odd-, not even-, chain-length alkane metabolism (Doughty et al., 2006). Propionate repression of BMO is released following the induction of propionate consumption, suggesting that BMO expression and alkane oxidation are closely coupled to the abilities of BMO to generate products and of the metabolic pathways to consume them (Doughty et al., 2006, 2007). We have also shown that the BMO structural genes are transcribed as a single polycistronic mRNA and that a putative 54-binding motif is present at the 5' end of the BMO operon (Sluis et al., 2002).
In this manuscript, we extend our observations to the molecular mechanisms involved in the regulation of BMO expression. We show that a gene (bmoR) located upstream of the BMO operon encodes a peptide (BmoR) similar to 54-transcriptional regulators. BmoR was more necessary for growth on C6–C8 alkanes than for shorter C2–C5 alkanes. Furthermore, we present evidence that a gene (bmoG) at the 3' end of the BMO operon encodes a peptide (BmoG) similar to GroEL chaperonins. BmoG is necessary for the expression of BMO activity, but not for the transcription of the BMO mRNA. The genes for BMO, BmoR and BmoG have nucleotide sequence similarities to genes involved in methane oxidation in the methanotrophic bacteria Methylococcus capsulatus Bath (Csaki et al., 2003), Methylosinus trichosporium Ob3b (Stafford et al., 2003) and Methylocella silvestris BL2 (Theisen et al., 2005). Thus, the significance of our work is discussed in the context of the regulation of BMO expression as affected by the presence of C2–C8 n-alkanes and n-alcohols, and compared to the composition and function of genes that are associated with methane metabolism.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A genomic library of ‘P. butanovora’ was constructed with partially MboI-digested DNA, cosmid SuperCos 1 and Gipapack III Gold packaging extract, as directed by the manufacturer (Stratagene). The library was screened for cosmids containing bmoX. Colonies of positive clones were grown in Luria–Bertani medium for cosmid DNA isolation. Cosmid DNA was prepared by alkaline lysis and used directly as template for DNA sequencing with custom-made primers. DNA sequence determination was performed by the Center for Genomic Research and Biocomputing (CGRB) Core Laboratories at Oregon State University. DNA sequence was assembled using in-house CGRB bioinformatics resources and analysed by BLAST at http://www.ncbi.nlm.nih.gov/BLAST/. The nucleotide sequence of the bmo operon and its adjacent genes has the accession number AY093933 and can be retrieved at http://www.ncbi.nlm.nih.gov/.
Mutant strain construction.
Gene inactivation was performed by electroporation, as described previously (Doughty et al., 2005; Sluis et al., 2002; Vangnai et al., 2002), with plasmid constructs containing ORF1, bmoR or bmoG interrupted by a kanamycin (Kanr) or gentamicin (Genr) cassette to inactivate the corresponding gene and confer resistance to the antibiotic. Plasmids pRkan6 and pRgm8 were used for the ‘P. butanovora’ BmoR-deficient mutant strains R6 and R8-X : : lacZ, respectively. The plasmids were constructed using PCR-amplified bmoR that included upstream and downstream DNA (primers C1.2-F5 and C1.2-R3). The amplified bmoR was cloned into pGEM-T (Promega) for disruption with the Kanr cassette (to form pRkan6) and into pGEM-T
PstI for disruption with the Genr cassette (to form pRgm8). A similar approach was used to produce the ORF1-deficient mutant ‘P. butanovora’ ORF1, with the pertinent primers and plasmids (primers C1.2-F7 and C1.2-R5, and plasmids pORF1kan4 and pORF1gm9). The bmoG deletion–insertion mutant ‘P. butanovora’ G2 was constructed by amplifying two PCR fragments separately, one for the N terminus of bmoG, including a flanking upstream region (primers koBmoGF and BAMbmoGR), and another for the C terminus, including a flanking downstream region (primers BAMbmoGF and koBmoGR). The amplified products were ligated to the Kanr cassette and cloned into pGEM-T Easy (Promega) to form pGkan2. Mutant strains were corroborated by Southern hybridization (Fig. 1). Axenic cultures were started using single colonies obtained by streaking on lactate plates. The main features of the bacterial strains and plasmids are described in Table 1. The nucleotide sequences of the primers used for PCR DNA amplification are in Table 2.
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; Hamamura et al., 1997). BMO activity in whole cells was routinely measured using ethene-dependent ethene oxide (EtO) production and expressed in nmol min–1 (mg protein)–1, as described previously (Doughty et al., 2007; Hamamura et al., 1997). Protein concentrations were determined using the micro Biuret assay, as described by Gornall et al. (1949).
When n-alkanes served as growth substrates, 2 mmol (200 µM aqueous concentration) of the respective n-alkane was added to each vial. When lactate, acetate, propionate, butyrate or pentanoate served as the C source for growth, acid concentrations were balanced to 12 mM carbon equivalents (4.0, 6.0, 4.0, 3.0 and 2.4 mM, respectively), which was sufficient to support growth of ‘P. butanovora’ to OD600 0.6. Growth on C2–C8 n-alkanes of wild-type ‘P. butanovora’, and mutant strains ‘P. butanovora’ R6 (bmoR inactivated) and ‘P. butanovora’ G2 (bmoG inactivated), was monitored for 7 days. The cell densities (OD600), exponentially transformed, were used to calculate doubling times for each strain on each n-alkane substrate.
Determination of product-dependent and -independent activities in the bmoX reporter strains.
‘P. butanovora’ bmoX : : lacZ : : kan (X : : lacZ) is a bmoX reporter strain that is kanamycin-resistant and expresses β-galactosidase (lacZ) activity as controlled by the bmoX promoter. This strain, since it lacks BMO, is unable to grow on C2–C9 n-alkanes (Sayavedra-Soto et al., 2005). The bmoX reporter ‘P. butanovora’, and mutants ‘P. butanovora’ R8-X : : lacZ (bmoR inactivated in the bmoX reporter strain) and ‘P. butanovora’ ORF1-X : : lacZ (ORF1 inactivated in the bmoX reporter strain), were grown on organic acids under the same conditions as the wild-type strain. Induction assays were performed in 10 ml serum vials with 1 ml cell suspension (0.5 OD600 or 0.25 mg protein ml–1) with the pertinent inducer (1 mM). At the end of a 2 h induction period, the β-galactosidase activity was determined and expressed in Miller units, as described previously (Doughty et al., 2005).
RNA extraction and RT-PCR.
Total RNA was extracted from cells (2x109) harvested in stationary growth phase using an Aurum Total RNA Mini Kit (Bio-Rad) as directed by the manufacturer, with minor modifications. Namely, the lysozyme treatment was omitted and a second DNase I treatment was added to ensure no DNA contamination. RNA concentrations and purity were determined using a NanoDrop ND-1000 UV-Vis Spectrophotometer. Synthesis of total cDNA was performed via random hexamers using an iScript cDNA Synthesis Kit (Bio-Rad) with 800 ng total RNA as template. For the RT-PCR reactions, primers BmoXF1 and BmoXR1 were used for bmoX, primers BmoCF1 and BmoCR1 were used for bmoC, and primers BmoGrt-F and BmoGrt-R were used for bmoG. The primer nucleotide sequences are shown in Table 2
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The most upstream ORF, named ORF1, is similar to genes that encode transcriptional regulators of the GntR family, with the highest identity to a gene in the β-proteobacterium Nitrosospira multiformis (accession no. YP_412321; 52 % identity and 69 % positives in the encoded amino acid sequence). The next ORF begins 460 bp downstream of ORF1 and ends 3013 bp upstream of bmoX. This ORF is 2010 bp in length and is in the same orientation as the bmo operon. Since the putative encoded product shares sequence similarity to 54-transcriptional regulators, the gene was named bmoR. The product of bmoR (BmoR) is predicted to contain all essential domains of a functional 54-transcriptional regulator, including the activator-interaction domain, the ATPase domain, and the C-terminal helix–turn–helix DNA-binding domain (Rappas et al., 2007; Schumacher et al., 2006). The N terminus of BmoR has similarities to the N terminus of the essential transcriptional regulator AcoR of acetoin metabolism in Ralstonia eutropha H16 (Kruger & Steinbuchel, 1992). BmoR shares greatest similarity to the transcriptional activator of action/glycerol metabolism from Burkholderia fungorum strain LB400 (accession no. ZP_00284962; 45 % identity and 61 % positives in the encoded amino acid sequence). BmoR shares only 34–36 % amino acid sequence identity to the MmoR of the methanotrophs. A putative 70 motif was identified in the promoter region of bmoR, suggesting that it is transcribed separately from ORF1 (Fig. 1). The levels of mRNA of BmoR were determined by RT-PCR in cells in the exponential phase of growth. Higher levels were observed in cells grown on butane than in cells grown on lactate (data not shown).
ORFs 3 and 4 appear to form an operon that ends 524 bp upstream of bmoX (Fig. 1). These ORFs were identified as a putative IstAB operon based on sequence similarities to a transposase from Azoarcus sp. EbN1 (accession no. CAI08254; IstA and IstB show 58 and 69 % identity, and 68 and 82 % positives in the encoded amino acid sequences, respectively). The mRNAs of ORFs 3 and 4 were at similar levels in lactate-grown cells and in cells after n-butanol induction experiments. These genes were not selected for further study and their role in ‘P. butanovora’ remains to be determined.
Another ORF located 345 bp downstream of bmoC in the BMO operon (Fig. 1) was named bmoG based on the similarity to the gene encoding MmoG in methanotrophs (31–32 % amino acid similarity). However, the product of bmoG (BmoG) is most similar to a chaperonin GroEL protein from the 
-proteobacterium Raoultella planticola (accession no. O66212; 34 % identity and 52 % positives in the encoded amino acid sequence). The gene for BmoG is 1707 bp in length and has a putative 70 promoter that suggests transcription separate from that of the BMO operon. A controlling inverted repeat for chaperonin expression (CIRCE) motif was identified in the promoter region, which is thought to be the binding site for the typical regulator of chaperonin genes, the HrcA protein (Lund, 2001). RT-PCR confirmed the presence of bmoG mRNA and that bmoG was transcribed separately from bmoC. In addition, possible terminator sequences could be inferred within 200 nt downstream of the bmoC stop codon (Zuker, 2003). The mRNA of bmoG was detected at similar levels during exponential growth on either lactate or butane, and was detected at higher levels in the stationary phase of butane-grown cells.
Analysis of mutants in ORF1, bmoR and bmoG
Mutant strains of ‘P. butanovora’ were constructed in which ORF1, bmoR or bmoG was insertionally inactivated. Southern hybridization corroborated that the insertion of the antibiotic cassettes occurred only at the desired location (Fig. 1b). All strains were selected on lactate agar plates containing the appropriate antibiotic. Single colonies were tested for growth on C2–C8 n-alkanes in liquid medium (Fig. 2, Table 3). The mutant strain with ORF1 inactivated grew as well as the wild-type strain on C2–C8 n-alkanes (data not shown). The mutant strain ‘P. butanovora’ R6 (bmoR inactivated) grew on C2–C5 alkanes more slowly than the wild-type after an extended lag phase of about 60 h, and did not grow on n-alkanes C6–C8 within 120 h (Fig. 2, Table 3). The mutant strain ‘P. butanovora’ G2 (bmoG inactivated) did not grow on any of the n-alkanes tested (C2–C8). These data suggested that bmoR and bmoG were involved in n-alkane metabolism and that bmoR was necessary for optimal growth, more so on C6–C8 than on C2–C5 n-alkanes.
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). Induction of product-independent BMO activity has been previously characterized in ‘P. butanovora’ and occurs upon entry into stationary phase after complete consumption of non-alkane-carbon growth sources (Sayavedra-Soto et al., 2005). Here, the β-galactosidase activities of ‘P. butanovora’ X : : lacZ, R8-X : : lacZ and ORF1-X : : lacZ were monitored during growth on lactate and upon entry into stationary phase (Fig. 3). All three strains of ‘P. butanovora’ expressed negligible β-galactosidase activity during the lag and exponential phases of the growth curve. Upon entering stationary phase, however, β-galactosidase activity increased in the three mutant strains, albeit to a lower extent in the R8-X : : lacZ mutant than in either the X : : lacZ or the ORF1-X : : lacZ strain (Fig. 3).
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). These results were intriguing, since the ‘P. butanovora’ R6 strain grew moderately well on C2–C5 alkanes after a 60 h lag period (Fig. 2, Table 3). To examine this phenomenon in more detail, we proceeded to grow ‘P. butanovora’ strain R8-X : : lacZ on acetate, propionate, butyrate or pentanoate, as described in Methods, and assayed the cells for their ability to induce β-galactosidase activity in response to 1-butanol. Interestingly, 1-butanol significantly induced β-galactosidase expression in the R8-X : : lacZ strain following growth on propionate, butyrate or pentanoate, but not on acetate or lactate (Fig. 4e). We proceeded to examine the range of alcohols that could act as inducers of β-galactosidase activity in the R8-X : : lacZ mutant following growth on propionate (Fig. 4f). In these cells, C2–C5 n-alcohols significantly induced β-galactosidase activity (90 Miller units), although in the case of C3–C5, to a lower level than that observed in the reporter strain with a functional bmoR (300–375 Miller units; Fig. 4c). The C7 and C8 chain length alcohols completely failed to induce β-galactosidase activity in the R8-X : : lacZ mutant (Fig. 4f). A possible explanation for the above results is that a second BMO regulatory system is expressed following growth on C3–C5 organic acids.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2006, 2007; Sayavedra-Soto et al., 2005). Here, we extended our studies to the characterization of two ORFs adjacent to the bmo operon, bmoR and bmoG. The protein encoded by bmoR is similar to 54-transcriptional regulators. Our data indicated that BmoR was necessary for the optimal expression of BMO in response to C2–C8 n-alcohols, regardless of the substrate that ‘P. butanovora’ used for growth. The expression of BMO in response to C2–C5 n-alcohols was less affected by the lack of BmoR after growth on C3 or C5 fatty acids than after growth on lactate or acetate (Fig. 4e, f).
The regulation of 54-dependent promoters is typically tightly controlled, and transcription is initiated in response to a specific signal (De Carlo et al., 2006; Tucker et al., 2006; Xie et al., 2006). In this connection, BMO regulation in ‘P. butanovora’ displayed two phenotypes atypical of 54-dependent promoters. First, BMO was induced in response to C starvation, as well as by the alcohol and aldehyde products of n-alkane oxidation. To our knowledge, toluene ortho-monooxygenase in Pseudomonas stutzeri OX1 is the only other known 54-dependent promoter that responds to either C starvation or the presence of an inducer molecule, toluene, via a 54-transcriptional activator, TouR (Solera et al., 2004). Since BMO induction by C limitation persisted following the deletion of bmoR, product-dependent and -independent induction of BMO must occur through separate molecular mechanisms. Second, most 54-dependent promoters respond exclusively to specific signal molecules, but the BMO 54-dependent promoter responds to a broad range of physiological substrates (i.e. n-alcohols C2–C8). This is similar to the 54-promoter Pu of Pseudomonas putida for the utilization of aromatic hydrocarbons (Abril et al., 1989; Velazquez et al., 2006). These observations make the promoter of BMO an interesting model for further investigation of the regulation of short-chain-length n-alkane utilization at the molecular level. Our data also suggested the presence of multiple transcriptional regulators in ‘P. butanovora’ with differing sensitivities to alcohols of various chain lengths. Identification of cis-acting promoter elements (upstream binding sequences for transcriptional regulators) may provide clues to determine the mechanisms by which multiple transcriptional regulators contribute to BMO expression.
The alcohol chain length-dependent expression of bmoX in the BmoR-deficient mutant was reminiscent of the bmoX expression patterns observed with different chain-length n-alkanes (Doughty et al., 2006). Propionate was both a potent repressor of BMO transcription and an inhibitor of BMO activity (Doughty et al., 2007) that persisted until propionate catabolism was induced (Doughty et al., 2006). It is intriguing to consider the extension of our model of organic acid repression of BMO to attempt to explain the situation described in this manuscript, whereby alcohols >C6 would not induce BMO in the bmoR-deficient mutant grown on propionate (Fig. 4f). BmoR might also be involved in the physiological adaptation to consume the products of long-chain-length n-alkanes (>C6) by a feedback mechanism (Fig. 5). The absence of BmoR would then prevent the optimal transformation of >C6 fatty acids, which then might still accumulate and repress BMO if the system for β-oxidation of fatty acids >C6 is not induced in cells grown on fatty acids C6. There is a precedent for carbon chain length-dependent regulation of fatty acid oxidation in bacteria in several recent papers in the literature. For example, there are two sets of β-oxidation genes in P. putida, one of which activates the formation of fatty acid CoA thioesters of chain lengths >C4 quite slowly, and is only activated when the other system is incapacitated (Olivera et al., 2001). In E. coli, a fatty acid acyl-CoA synthetase (encoded by fadK) with much higher activity toward shorter-chain-length fatty acids than toward longer ones (i.e. >C10) has been discovered (Campbell et al., 2003; Morgan-Kiss & Cronan, 2004). This contrasts with the traditional acyl-CoA synthetase FadD in E. coli, which has poor activity toward fatty acids <C10 (Campbell et al., 2003). Similarly, in Mycobacterium avium, a medium-chain-length acyl-CoA synthetase with no activity toward chain lengths <C5 and low activity >C13 has been identified (Morsczeck et al., 2001).
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54-transcriptional regulator of BMO may be considered in terms of the phylogenetic similarities between ‘P. butanovora’ and the Azoarcus strains in the β-proteobacteria (Anzai et al., 2000). In the genomes of Azoarcus BH72 (GenBank accession no. AM406670) and Azoarcus sp. EbN1 (GenBank accession no. CR555306) there are 54-transcriptional regulators very similar to BmoR (E scores 4e-59) that are located adjacent to putative alcohol (e.g. azo2845 in Azoarcus BH72 or ebA3654 in Azoarcus sp. EbN1) and aldehyde dehydrogenase genes (e.g. azo2931 in Azoarcus BH72 or ebA4625 in Azoarcus sp. EbN1). In addition, the genome of Azoarcus sp. BH72 contains genes for a putative multi-component aromatic/alkene monooxygenase (azo1219–azo1222) with 30 % predicted amino acid sequence identity to BMO. Genes that encode a putative 54-transcriptional regulator (azo1225) and a putative GroEL chaperonin (azo1223) are present immediately adjacent to the 3' end of the putative monooxygenase of Azoarcus sp. BH72. ‘P. butanovora’ also has genes that encode 54-transcriptional regulators adjacent to alcohol dehydrogenases and aldehyde dehydrogenases (D. J. Arp, unpublished results; Vangnai et al., 2002). Due to the similarities of BmoR to the 54-transcriptional regulators of Azoarcus strains, perhaps the absolute requirement for BmoR to facilitate induction of BMO by alcohols C6–C8 (Fig. 4f) reflects an evolutionary history that is more closely associated with the metabolism of more complex or longer-chain alcohols and fatty acids than with the oxidation and metabolism of relatively short-chain n-alkanes.
It is interesting that a chaperonin is required in both ‘P. butanovora’ and methanotrophic bacteria to produce an active monooxygenase. In the well-characterized model systems described for Methylococcus capsulatus Bath or Methylosinus trichosporium Ob3b, inactivation of the gene encoding MmoG does not produce sMMO or its mRNA (Csaki et al., 2003; Stafford et al., 2003). We were able to provide circumstantial evidence that in ‘P. butanovora’ BmoG is also involved in the production of active BMO, although BmoG is not required to produce BMO mRNA. Biochemically, MmoG and BmoG are similar in that both are longer than most GroEL proteins, by about 40 aa for MmoG and 30 aa for BmoG, and neither contains the GGM repeats on the C terminal that are typical of many GroEL proteins. The significance of the CIRCE motif located upstream of bmoG but not in mmoG in some methanotrophs remains to be determined. Future research on the expression of active BMO in heterologous hosts should take into account the inclusion of bmoG.
Our current understanding of the components involved in the regulation of BMO is summarized in Fig. 5. Two elements are necessary for the full induction of BMO activity by n-alkanes: an alcohol that results from the oxidation of the alkane (Sayavedra-Soto et al., 2005), and the proper assembly of BMO, likely through the assistance of BmoG. These, of course, will not occur without the low levels of BMO activity that develop upon carbon starvation, activity that primes the further production of BMO (Sayavedra-Soto et al., 2005). We also know that downstream metabolites that result from the oxidation of the alkane affect the levels of BMO (Doughty et al., 2006, 2007). However, elusive still are the regulatory elements of product-independent activity and the exact mechanisms and alternative regulators for the expression–repression by which downstream metabolites control the production of BMO activity (Fig. 5).
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ACKNOWLEDGEMENTS |
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Edited by: J. A. Vorholt
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Received 27 August 2007;
revised 4 October 2007;
accepted 13 October 2007.
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) and the R6 mutant strain of ‘P. butanovora’, in which bmoR was insertionally inactivated (
). The inoculum was taken from cells growing exponentially on lactate.
