799 1442 2085 2728 3371 4014

Mass (m/z)

1672.2

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1[BP = 1352.6, 1672]

1352.6488

842.4994 1307.69182211.1003

1638.8818 1993.9966

1475.7728 3101.62331851.94341179.6062 1487.7643861.0512 2284.1765 2705.1604

1614.79211296.7186845.0720 1039.5979 2225.1248

1368.6412 3340.73731746.8998 2960.43162233.07861036.5364 1966.0560 2717.09451533.8057 2510.1594 3312.25832968.4570

Protein Analysis by Two-Dimensional Gel Electrophoresis of Carbon Monoxide and Methanol-inducible Proteins in

Mycobacterium sp. strain JC1 DSM 3803

JINHO HEO AND YOUNG M. KIMDepartment of Biology, Yonsei University, Seoul 120-749, Korea

ABSTRACTABSTRACT

Mycobacterium sp. strain JC1 DSM 3803 is a bacterium that is able to grow on carbon monoxide (CO) or methanol as a sole source of carbon and energy. In this study, the proteomics tools, such as two-dimensional gel electrophoresis and MALDI-TOF, were used for analysis of CO- or methanol-induced proteins in Mycobacterium sp. strain JC1. The two-dimensional image of proteins in Mycobacterium sp. strain JC1 grown on glucose was used as a control for the spot detection and the comparison of protein expression. Among the spots detected reproducibly after silver staining, eight CO-inducible and five methanol-inducible spots were identified by MALDI-TOF mass spectrometry with peptide mass fingerprinting or N-terminal amino acid sequencing. CutB and CutC, the subunits of CO dehydrogenase, FixA and FixB, the subunits of electron transfer flavoprotein, glyceraldehyde-3-phosphate dehydrogenase, and heat shock protein 65 were increased in Mycobacterium sp. strain JC1 grown on CO. Interestingly, methanol:N,N–dimethyl–4–nitrosoaniline oxidoreductase, the key enzyme for methanol dissimilation, was also slightly increased in cells grown on CO. Methanol:N,N–dimethyl–4–nitrosoaniline oxidoreductase, dihydroxyacetone synthase, 3-hexulose-6-phosphate synthase, and glyceraldehyde-3-phosphate dehydrogenase were increased in cells grown on methanol.

INTRODUCTIONINTRODUCTION The facultatively chemolithotrophic bacterium Mycobacterium sp. strain JC1 DSM 3803 is the first mycobacterium reported that is able to grow aerobically on carbon monoxide (CO) and on methanol as sole carbon and energy sources [1,2]. It has been reported that Mycobacterium sp. strain JC1 grown on CO has carbon monoxide dehydrogenase and grown on methanol possesses methanol:N,N-dimethyl-4-nitrosoaniline oxidoreductase (MNO), the key enzyme for methanol dissimilation, respectively. However, protein profile for the overall C1 compounds assimilation has not yet been performed.

The aim of the present study was to identify proteins involved in assimilation of C1 compounds in methanol or CO supplemented medium. In this study, we used the 2-DE/mass spectrometry (MS)-based proteomic analysis to profile the differentially expressed proteins.

MATERIALS AND METHODSMATERIALS AND METHODSStrains and cultivation conditions. Mycobacterium sp. strain JC1 (DSM 3803) was used in this study. Cells were cultivated at 37°C under CO chemolithoautotrophy with a gas mixture of 30% CO-70% air in standard mineral base (SMB) medium (SMB-CO). For methylotrophic growth, cells were grown at 37°C in SMB medium supplemented with 1% (v/v) methanol (SMB-MeOH). As a control, cells were cultivated at 37°C in SMB medium supplemented with 0.2% (w/v) glucose (SMB-glucose).  Cell extract preparation. The cells were harvested at the late-exponential-growth phase, washed twice with 3 mM 3-[N-morpholino]propanesulfonic acid (MOPS) buffer (pH 7.5), and then resuspended in the same buffer. Cell extracts were made by disruption by sonication for 15 min at 30 intervals, with an intensity of 60 (Sonics and Materials, Inc., Newtown, Conn.). The cell debris was removed by centrifugation at 15,000 x g for 30 min at 4°C, and the resulting supernatant was then centrifugated at 100,000 x g for 90 min. The soluble supernatant was used for two-dimensional gel analysis.  Two-dimensional electrophoresis. 2-DE was performed using precast IPG strips (pH4-7 linear, 18 cm, Amersham Pharmacia Biotechnology Inc.) in the first dimension, isoelectric focusing (IEF). Briefly, 150 ug proteins were diluted to a total volume of 350 ul with the buffer [8 M urea, 2 % CHAPS, 0.5 % IPG buffer 3-10, 20 mM DTT and a trace of bromophenol blue]. After loaded on IPG strips, IEF was carried out according to the following protocol: 12 hours of rehydration at 0 V; 2 hours at 100 V; 2 hour at 200 V; 2 hour at 400 V; 2 hours at 600 V; 6 hours at 1000V and 12 hours at 3500 V. The current was limited to 50 mA per gel. After IEF separation, the strips were immediately equilibrated with equilibration solution [50 mM Tris-HCl pH6.8, 6 M urea, 30 % glycerol, 2 % SDS, 0.01 %. BPB and 2 mM tributyl phosphine

RESULTSRESULTS

REFERENCESREFERENCES

1. Cho, J.W., H.S. Yim, and Y.M. Kim. 1985. Kor. J. Microbiol. 23:1-8. 2. Ro, Y.T., J.G. Seo, J. Lee, D. Kim, I.K. Chung, T.U. Kim, and Y.M. Kim. 1997. J. Microbiol. 35:30-39.3.

Molecular Microbiology Lab., Yonsei University

CONCLUSIONSCONCLUSIONS

1. We analyzed 4 CO-inducible proteins, 3 methanol-inducible proteins, and 5 constitutively expressed proteins as well.

2. .

(TBP)]. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 1 mm thick, 12.5 % SDS-PAGE gels. The strips were held in place with 0.5 % agarose dissolved in SDS/Tris running buffer. Electrophoresis was carried out at constant power (10 mA/gel for 60 min and 25 mA/gel for 12 hours) and temperature (20 ℃) using Ettan Dalt Six Electrophoresis Unit (Amersham Pharmacia Biotechnology Inc.). Gels were stained with silver nitrate according to the silver-staining method (3).

Mass spectrometry. Coomassie-stained proteins were excised from gel, and digested with 10ng/ ㎕ trypsin in 25mM ammonium bicarbonate. Mass spectrometry analyses were preformed on 4700 Proteomics Analyzer (Applied Biosystems, Foster City, California, USA). MALDI-TOF MS data and tandem MS data were searched via the Mascot search engine.

N-terminal amino acid sequencing. 2-DE gels were transferred to PVDF membrane, and target protein spots were selected. Amino acid sequencing were performed on Procise cLC Protein Sequencing System (Applied Biosystems).

CO Glucose

pH4 7

HSP 70

HSP 65

AtpD

MNO

CutB

CutC

FixA

FixB

Wag 31

HSP 60

GAPDH

No.matchedpeptides

Sequencecoverage

(%)

6 Heat shock protein 65 (Fragment) Mycobacterium sp. graecum DL049 6 19%

10,12 methanol:NDMA oxidoreductase(MNO) Mycobacterium sp. strain JC1 15 46%

17-20 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Mycobacterium tuberculosis (strain H37RV) 4 26%

24 FixB (electron transfer flavoprotein families ETF-alpha) Mycobacteria N/A N/A

29 FixA (electron transfer flavoprotein families ETF-beta) Mycobacteria N/A N/A

31 CutB Mycobacterium sp. strain JC1 8 44%

40 CutC Mycobacterium sp. strain JC1 3 26%

21 Wag31 protein Mycobacterium tuberculosis (strain H37RV) 4 13%

1 Chaperone protein dnaK (HSP 70) Mycobacterium paratuberculosis 7 22%

5 60 kDa chaperonin 2 (Protein Cpn60-2) (groEL protein 2) (HPS 65) Mycobacterium bovis 8 22%

7 AtpD protein Mycobacterium tuberculosis (strain H37RV) 6 17%

Spotno.

Protein Species

MALDI massmapping

No.matchedpeptides

Sequencecoverage

(%)

6 dihydroxyacetone synthase (DHAS) Mycobacterium sp. strain JC1 6 19%

10,12 methanol:NDMA oxidoreductase(MNO) Mycobacterium sp. strain JC1 15 46%

17-20 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Mycobacterium tuberculosis (strain H37RV) 4 26%

29 3-hexulose-6-phosphate synthase (HPS) Mycobacteria N/A N/A

21 Wag31 protein Mycobacterium tuberculosis (strain H37RV) 4 13%

24 FixB (electron transfer flavoprotein families ETF-alpha) Mycobacterium leprae N/A N/A

Spotno.

Protein Species

MALDI massmapping

pH4 7

Methanol

Wag 31

DHAS

MNO

GAPDH

HPS

FixB

Glucose

78.0 387.6 697.2 1006.8 1316.4 1626.0

Mass (m/z)

2719.6

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 MS/MS Precursor 2404.19 Spec #1[BP = 876.2, 2720]

876.2228

112.2996

175.5622

129.3867 618.87601246.2500391.3283262.8912 675.9757

400.3652316.0643 789.1516561.7959490.5931171.5479 991.2618658.9208 1062.2722289.0002 386.2914 818.0710502.6530 573.7848 730.0939 934.2265 1375.0038143.4433 1175.2604 1474.8491

S A G V A G GIL S D A

IL A E V

24

CO Glucose

24

CO Glucose

31 31

805.0 1320.8 1836.6 2352.4 2868.2 3384.0

Mass (m/z)

3296.7

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1[BP = 877.0, 3297]

877.0496

861.0737

893.0215

1674.8578

1163.6024

1611.8029845.0954

2016.0297

1800.83081396.70461168.5708855.05062497.28322002.9756870.0227

1731.8806 2007.05801412.6993850.0656 1088.05652211.10231253.6853824.2164 2652.28391455.70681094.0636 1747.8719 2041.9521 2382.15192226.1113

11

CO Glucose

11