JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.5.1810–1812.2001

Mar. 2001, p. 1810–1812 Vol. 183, No. 5

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Expression of Individual Copies of Methylococcus capsulatusBath Particulate Methane Monooxygenase Genes

SERGEI STOLYAR,1 MARION FRANKE,1 AND MARY E. LIDSTROM1,2*

Departments of Chemical Engineering1 and Microbiology,2 University ofWashington, Seattle, Washington 98195

Received 19 June 2000/Accepted 28 November 2000

The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) inMethylococcus 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 altersdepending on the copper levels available for growth.

Methanotrophic bacteria oxidize their growth substrate meth-ane to methanol via the methane monooxygenase (MMO). Twotypes of MMO are known, the particulate MMO (pMMO) andthe soluble MMO (sMMO) (3). The genes encoding the threesubunits of the pMMO (pmoCAB) are found in multiple copiesin methanotrophs (2, 9, 11). In Methylococcus capsulatus Bath,a type I g-proteobacterial methanotroph (3), two completecopies of pmoCAB and a third copy of pmoC are present (11).Mutant analysis has shown that neither copy of pmoCAB isessential but that copy 2 is more important than copy 1 forgrowth and whole-cell methane oxidation (11). The role of thethird copy of pmoC is unknown, but it may be essential (11).

In order to assess the relative expression of each set of pmogenes under different growth conditions, we have carried out astudy of transcripts in pmoC1 and pmoC2 mutants and com-pared these results to expression from promoter-reporter genefusions in strains containing wild-type pmoC1 and pmoC2.

Escherichia coli strains DH5a, DH5a MCR (Bethesda Re-search Laboratories, Inc.), Inva and Top10 (Invitrogen), andS17-1 (10) were grown in Luria-Bertani medium in the pres-ence of appropriate antibiotics as described previously (8).M. capsulatus Bath wild-type and mutant strains (MCK60 andMCK62) (11) were grown as described previously (11).

Transcript analysis. RNA blots were analyzed for insertionmutations of pmoC1 and pmoC2 with probes for pmoC, pmoA,and pmoB (Fig. 1). RNA was isolated from cells using thePerfect RNA total RNA isolation kit (Eppendorf-5 Prime,Inc., Boulder, Colo.). RNA blots were made, and hybridizationwas carried out at 55°C as described previously (8). The mem-branes were washed twice with 0.53 SSC (13 SSC is 0.15 MNaCl plus 0.015 M sodium citrate) at 55°C. Hybridizationprobes were generated from PCR products labeled with arandom-primed labeling kit (Boehringer Mannheim, Indianap-olis, Ind.). The PCR products were generated from the follow-ing primers: pmoC1 and pmoC2, css2F (59-CCTGTGGGTGCGGTGGTAC-39) and css9R (59-GCCTTCGTCCACGGCTTC-39); pmoA1 and pmoA2, ass1F (59-CTGGGACTTCTGGTCGGACTG-39) and mb661 (59-CCGG[A,C]GCAACGTC[C,T

]TTACC-39); pmoB1 and pmoB2, bss1F (59-CCGCCGTGGCAGCGACCGCC-39) and ESSR (59-CCTTGAACGTCTAAATCCAGC-39). These probes are specific to pmo genes inthis strain (9, 11). A transcript pattern similar to that previ-ously reported for the wild type was detected, including afull-length pmoCAB transcript and less-distinct smaller tran-scripts (Fig. 1). Since the ratios of the smaller mRNAs to thefull-length pmoCAB transcript were roughly proportional,we focused on the pmoCAB transcript as an indicator ofpMMO transcription. Figure 1 shows that each gene copywas transcriptionally active and that each was transcribed asa pmoCAB operon.

Cells grown under conditions in which expression of solubleMMO occurs contained low but detectable levels of pmoCABtranscripts, mainly copy 2 (Fig. 1). At a copper concentrationoptimal for growth (5 mM), copy 2 transcripts were dominant.However, at a concentration near the upper limit for growthbut not inhibitory (50 mM), an increase in the steady-state levelof transcripts was found for both copies, and copy 1 transcriptswere 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 andpmoC2 were determined by primer extension experiments, us-ing ThermoScript cDNA and 10 mg of total RNA. Primerswere labeled with [g-32P]ATP (6,000 Ci/mmol) (NEN) usingT4 polynucleotide kinase (Boehringer Mannheim). Radioac-tive sequencing reactions for primer extension analyses werecarried out using the T7 Sequenase kit (Amersham PharmaciaBiotech, Piscataway, N.J.). Primers for the reverse transcrip-tion and for sequencing reactions were css21R (59-CCTAAAGTGATGGTTGAC-39) for pmoC1 and css222R (59-CCAACTGTTATATCGATGTG-39) for pmoC2. The mRNA startpoint of pmoC1 was detected 135 bp upstream of the transla-tion start site, at a single A preceded by 210 (TAGACT) and235 (TTGACA) boxes separated by 16 bp (Fig. 2). ThemRNA start point of pmoC2 was detected 132 bp upstream ofthe translation start site, also at a single A preceded by 210and 235 sequences identical to those for pmoC1 (Fig. 2).These sequences are located in both cases in a region of con-served sequences, flanked by sequences that are not conservedbetween the two copies. Both putative promoters demonstratehigh similarity to the E. coli s70 210 and 235 promoter con-sensus (4). Immediately upstream of each promoter sequence

* Corresponding author. Mailing address: Department of ChemicalEngineering, Box 351750, University of Washington, Seattle, WA98195. Phone: (206) 616-5282. Fax: (206) 616-5721. E-mail: lidstrom@u.washington.edu.

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is a highly conserved AT-rich region (CCTGCGTCAAAATCt/aCTCAg/tATTTTTC). This conserved sequence is a candi-date for a regulatory sequence or an upstream promoter ele-ment (7). Transcription start sites were determined to be thesame for both operons under conditions with and withoutcopper added to the growth medium (data not shown). Apromoter for one of the copies of pmoCAB of the type II,a-proteobacterial methanotroph Methylocystis strain M hasbeen mapped, and it also resembled an E. coli s70 promoter(6). However, it was different from the sequences that wereport here and did not contain the AT-rich region.

Chromosomal reporter gene fusions for pmoC1 and pmoC2.Promoter-reporter (xylE) transcriptional fusions were gener-ated in the chromosome for pmoC1 and pmoC2 using a newintegrative vector, pMFX1. This vector contains the xylE genefrom 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-xylEtranscriptional fusions into the chromosome at the site of thepromoter fragment, as single-crossover insertions generating afused gene followed by an intact gene with the native pro-moter. A 1,548-bp fragment containing the pmoC1 promoterregion and a 443-bp fragment containing the pmoC2 promoterregion were used to generate the chromosomal insertionstrains, designated MCX13-2 and MCX215, respectively. Eachconstruct contained a promoter fragment that had the same39 end (25 bp of the 59 region of pmoC), and the remainderwas upstream DNA. These constructions were transferredto M. capsulatus Bath by conjugation as described previously(12) and were selected on kanamycin (50 mg/liter). DiagnosticPCR of chromosomal DNA (11) confirmed the expected con-structions. These mutants grew at the same rate as the wildtype. XylE (catechol dioxygenase) activities (5) were deter-mined in crude extracts of the mutants in 100 mM phosphate

FIG. 1. Northern blot analysis of pmo mRNAs from pmoC mutants. Probes are pmoA (A), pmoB (B), pmoC (C), and 16S rDNA(D). TotalRNAs from MCK60 (pmoC1 mutant) (lanes 1 to 3) and MCK62 (pmoC2 mutant) (lanes 4 to 6) were grown on medium without copper added(lanes 1 and 4), with 5 mM copper (lanes 2 and 5), or with 50 mM copper (lanes 3 and 6). Each lane contains 10 mg of RNA. The arrowheadindicates the transcripts of pmoCAB.

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

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buffer, which were obtained by passing cells through a Frenchpressure cell at 1.2 3 108 Pa followed by centrifugation for 10min at approximately 15,000 3 g, or in whole cells permeabil-ized by treatment with 2% (vol/vol) toluene for 30 min (Table1). The protein concentration was assessed spectrophotometri-cally (13).

These results show that the copy 2 promoter is expressed atabout twice the rate of the copy 1 promoter under normalgrowth conditions and that promoter activity of both pmoCcopies in mid- to late exponential growth (12 to 24 h) is higherin cells grown under normal copper conditions than in cellsgrown with no added copper. However, for each fusion theamount of reporter activity from cells grown in the absence ofadded copper was similar to the activity in cells grown withadded copper until later in growth, when a 1.5- to 2-fold dif-ference was observed. These results are qualitatively similar tothose of the Northern blot experiments (Fig. 1).

Taken together, the results presented here show that bothpmoCAB clusters are transcribed as operons from similar pro-moters but that transcription of copy 2 dominates under mostgrowth conditions tested. However, it appears that transcrip-tion of copy 1 increases to a level comparable to that of copy2 during growth with high copper levels. Since the strains withmutations of both copies show only minor growth defects (11),it seems likely that the two nearly identical copies of pmoCABin this methanotroph are functionally redundant under theconditions tested, although it is possible that they play differentroles in natural habitats.

Nucleotide sequence accession numbers. The GenBankaccession number for the fragment upstream of pmoC1 isL40804, and those for the fragment upstream of pmoC2 areU94337 and AF273026.

This work was funded by a grant from NSF (MCB 9630645).We thank R. Meima for helpful discussions and assistance with

primer extension analysis.

REFERENCES

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2. Gilbert, B., I. R. McDonald, R. Finch, G. P. Stafford, A. K. Nielsen, and J. C.Murrell. 2000. Molecular analysis of the pmo (particulate methane mono-oxygenase) operons from two type II methanotrophs. Appl. Environ. Micro-biol. 66:966–975.

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6. Nielsen, A. K., K. Gerdes, and J. C. Murrell. 1997. Copper-dependentreciprocal transcriptional regulation of methane monooxygenase genes inMethylococcus capsulatus and Methylosinus trichosporium. Mol. Microbiol.25:399–409.

7. Ross, W., K. K. Gosink, J. Salomon, K. Igarashi, C. Zou, A. Ishihama, K.Severinov, and R. L. Gourse. 1993. A third recognition element in bacterialpromoters: DNA binding by the alpha subunit of RNA polymerase. Science262:1407–1413.

8. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

9. Semrau, J. D., A. Chistoserdov, J. Lebron, A. Costello, J. J. Davagnino,J. Kenna, A. J. Holmes, R. Finch, J. C. Murrell, and M. E. Lidstrom. 1995.Particulate methane monooxygenase genes in methanotrophs. J. Bacteriol.177:3071–3079.

10. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilizationsystem for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:37–45.

11. Stolyar, S., A. M. Costello, T. L. Peeples, and M. E. Lidstrom. 1999. Role ofmultiple gene copies in particulate methane monooxygenase activity in themethane-oxidizing bacterium Methylococcus capsulatus Bath. Microbiology145:1235–1244.

12. Stolyar, S. M., V. A. Romanovskaya, and Y. R Malashenko. 1995. Search forsystems of genetic exchange in methane-oxidizing bacteria. Microbiologya64:686–691.

13. Whitaker, J. R., and P. E. Granum. 1980. An absolute method for proteindetermination based on difference in absorbance at 235 and 280 nm. Anal.Biochem. 109:156–159.

14. Xu, H. H., M. Viebahn, and R. S. Hanson. 1993. Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacte-rium Methylobacterium organophilum XX. J. Gen. Microbiol. 139:743–752.

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

Strain Promoter Copper inmediuma

XylE activity (nmol/min/mg of protein)b

after growth for the following time (h):

0 2 4 6 12 24

MCX13-2 pC1 2 1.0 8.0 13.0 13.5 32.0 65.01 1.0 4.1 7.0 13.7 52.0 106.0

MCX215 pC2 2 1.0 19.0 20.9 58.0 60.0 75.01 1.0 18.1 18.2 41.0 75.0 131.0

a M. capsulatus strains were grown in either copper-depleted medium (2) ormedium containing 20 mM copper (1).

b Experiments were replicated at least twice; each data point is the mean fromat least three replicate assays, and the results agreed by 615%.

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