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J Bacteriol. Mar 2001; 183(5): 1810–1812.
PMCID: PMC95072
Note

Expression of Individual Copies of Methylococcus capsulatus Bath Particulate Methane Monooxygenase Genes

Abstract

The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth.

Methanotrophic bacteria oxidize their growth substrate methane to methanol via the methane monooxygenase (MMO). Two types of MMO are known, the particulate MMO (pMMO) and the soluble MMO (sMMO) (3). The genes encoding the three subunits of the pMMO (pmoCAB) are found in multiple copies in methanotrophs (2, 9, 11). In Methylococcus capsulatus Bath, a type I γ-proteobacterial methanotroph (3), two complete copies of pmoCAB and a third copy of pmoC are present (11). Mutant analysis has shown that neither copy of pmoCAB is essential but that copy 2 is more important than copy 1 for growth and whole-cell methane oxidation (11). The role of the third copy of pmoC is unknown, but it may be essential (11).

In order to assess the relative expression of each set of pmo genes under different growth conditions, we have carried out a study of transcripts in pmoC1 and pmoC2 mutants and compared these results to expression from promoter-reporter gene fusions in strains containing wild-type pmoC1 and pmoC2.

Escherichia coli strains DH5α, DH5α MCR (Bethesda Research Laboratories, Inc.), Invα and Top10 (Invitrogen), and S17-1 (10) were grown in Luria-Bertani medium in the presence of appropriate antibiotics as described previously (8). M. capsulatus Bath wild-type and mutant strains (MCK60 and MCK62) (11) were grown as described previously (11).

Transcript analysis.

RNA blots were analyzed for insertion mutations of pmoC1 and pmoC2 with probes for pmoC, pmoA, and pmoB (Fig. (Fig.1).1). RNA was isolated from cells using the Perfect RNA total RNA isolation kit (Eppendorf-5 Prime, Inc., Boulder, Colo.). RNA blots were made, and hybridization was carried out at 55°C as described previously (8). The membranes were washed twice with 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 55°C. Hybridization probes were generated from PCR products labeled with a random-primed labeling kit (Boehringer Mannheim, Indianapolis, Ind.). The PCR products were generated from the following primers: pmoC1 and pmoC2, css2F (5′-CCTGTGGGTGCGGTGGTAC-3′) and css9R (5′-GCCTTCGTCCACGGCTTC-3′); pmoA1 and pmoA2, ass1F (5′-CTGGGACTTCTGGTCGGACTG-3′) and mb661 (5′-CCGG[A,C]GCAACGTC[C,T]TTACC-3′); pmoB1 and pmoB2, bss1F (5′-CCGCCGTGGCAGCGACCGCC-3′) and ESSR (5′-CCTTGAACGTCTAAATCCAGC-3′). These probes are specific to pmo genes in this strain (9, 11). A transcript pattern similar to that previously reported for the wild type was detected, including a full-length pmoCAB transcript and less-distinct smaller transcripts (Fig. (Fig.1).1). Since the ratios of the smaller mRNAs to the full-length pmoCAB transcript were roughly proportional, we focused on the pmoCAB transcript as an indicator of pMMO transcription. Figure Figure11 shows that each gene copy was transcriptionally active and that each was transcribed as a pmoCAB operon.

FIG. 1
Northern blot analysis of pmo mRNAs from pmoC mutants. Probes are pmoA (A), pmoB (B), pmoC (C), and 16S rDNA(D). Total RNAs from MCK60 (pmoC1 mutant) (lanes 1 to 3) and MCK62 (pmoC2 mutant) (lanes 4 to 6) were grown on medium without copper added (lanes ...

Cells grown under conditions in which expression of soluble MMO occurs contained low but detectable levels of pmoCAB transcripts, mainly copy 2 (Fig. (Fig.1).1). At a copper concentration optimal for growth (5 μM), copy 2 transcripts were dominant. However, at a concentration near the upper limit for growth but not inhibitory (50 μM), an increase in the steady-state level of transcripts was found for both copies, and copy 1 transcripts were present at levels similar to those of copy 2 transcripts.

Transcription start site mapping for pmoC1 and pmoC2.

The locations of the transcription start sites of pmoC1 and pmoC2 were determined by primer extension experiments, using ThermoScript cDNA and 10 μg of total RNA. Primers were labeled with [γ-32P]ATP (6,000 Ci/mmol) (NEN) using T4 polynucleotide kinase (Boehringer Mannheim). Radioactive sequencing reactions for primer extension analyses were carried out using the T7 Sequenase kit (Amersham Pharmacia Biotech, Piscataway, N.J.). Primers for the reverse transcription and for sequencing reactions were css21R (5′-CCTAAAGTGATGGTTGAC-3′) for pmoC1 and css222R (5′-CCAACTGTTATATCGATGTG-3′) for pmoC2. The mRNA start point of pmoC1 was detected 135 bp upstream of the translation start site, at a single A preceded by −10 (TAGACT) and −35 (TTGACA) boxes separated by 16 bp (Fig. (Fig.2).2). The mRNA start point of pmoC2 was detected 132 bp upstream of the translation start site, also at a single A preceded by −10 and −35 sequences identical to those for pmoC1 (Fig. (Fig.2).2). These sequences are located in both cases in a region of conserved sequences, flanked by sequences that are not conserved between the two copies. Both putative promoters demonstrate high similarity to the E. coli ς70 −10 and −35 promoter consensus (4). Immediately upstream of each promoter sequence is a highly conserved AT-rich region (CCTGCGTCAAAATCt/aCTCAg/tATTTTTC). This conserved sequence is a candidate for a regulatory sequence or an upstream promoter element (7). Transcription start sites were determined to be the same for both operons under conditions with and without copper added to the growth medium (data not shown). A promoter for one of the copies of pmoCAB of the type II, α-proteobacterial methanotroph Methylocystis strain M has been mapped, and it also resembled an E. coli ς70 promoter (6). However, it was different from the sequences that we report here and did not contain the AT-rich region.

FIG. 2
pmoC1 and pmoC2 transcription start sites. Primer extension analysis for pmoC1 (A) and pmoC2 (B) is shown. RNA was isolated from M. capsulatus cells grown for 2 h in batch culture without CuSO4 (fourth lanes) or with 20 μM CuSO4 added (fifth lanes). ...

Chromosomal reporter gene fusions for pmoC1 and pmoC2.

Promoter-reporter (xylE) transcriptional fusions were generated in the chromosome for pmoC1 and pmoC2 using a new integrative vector, pMFX1. This vector contains the xylE gene from pHX200 (14) inserted into the KpnI sites of pAYC61 (1), with the Kmr gene from pUC4K replacing the Apr gene of pAYC61. This vector can be used to insert promoter-xylE transcriptional fusions into the chromosome at the site of the promoter fragment, as single-crossover insertions generating a fused gene followed by an intact gene with the native promoter. A 1,548-bp fragment containing the pmoC1 promoter region and a 443-bp fragment containing the pmoC2 promoter region were used to generate the chromosomal insertion strains, designated MCX13-2 and MCX215, respectively. Each construct contained a promoter fragment that had the same 3′ end (25 bp of the 5′ region of pmoC), and the remainder was upstream DNA. These constructions were transferred to M. capsulatus Bath by conjugation as described previously (12) and were selected on kanamycin (50 mg/liter). Diagnostic PCR of chromosomal DNA (11) confirmed the expected constructions. These mutants grew at the same rate as the wild type. XylE (catechol dioxygenase) activities (5) were determined in crude extracts of the mutants in 100 mM phosphate buffer, which were obtained by passing cells through a French pressure cell at 1.2 × 108 Pa followed by centrifugation for 10 min at approximately 15,000 × g, or in whole cells permeabilized by treatment with 2% (vol/vol) toluene for 30 min (Table (Table1).1). The protein concentration was assessed spectrophotometrically (13).

TABLE 1
XylE activity of transcriptional fusions of pmoC in M. capsulatus Bath cells grown under different conditions

These results show that the copy 2 promoter is expressed at about twice the rate of the copy 1 promoter under normal growth conditions and that promoter activity of both pmoC copies in mid- to late exponential growth (12 to 24 h) is higher in cells grown under normal copper conditions than in cells grown with no added copper. However, for each fusion the amount of reporter activity from cells grown in the absence of added copper was similar to the activity in cells grown with added copper until later in growth, when a 1.5- to 2-fold difference was observed. These results are qualitatively similar to those of the Northern blot experiments (Fig. (Fig.11).

Taken together, the results presented here show that both pmoCAB clusters are transcribed as operons from similar promoters but that transcription of copy 2 dominates under most growth conditions tested. However, it appears that transcription of copy 1 increases to a level comparable to that of copy 2 during growth with high copper levels. Since the strains with mutations of both copies show only minor growth defects (11), it seems likely that the two nearly identical copies of pmoCAB in this methanotroph are functionally redundant under the conditions tested, although it is possible that they play different roles in natural habitats.

Acknowledgments

This work was funded by a grant from NSF (MCB 9630645).

We thank R. Meima for helpful discussions and assistance with primer extension analysis.

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