Uptake of Carbon Monoxide and Hydrogen at Environmentally Relevant Concentrations by Mycobacteria.

1. Uptake of Carbon Monoxide and Hydrogen at Environmentally Relevant Concentrations by Mycobacteria.

2. Abstract • Mycobacterium bovis BCG, M. gordonae, and M. marinum ->oxidize CO • M. smegmatis -> consume H2 • M. bovis BCG, M.gordonae, M. smegmatis, and M. tuberculosis H37Ra oxidize CO (environmentally relevant concentrations [<50 ppm]) • M. gordonae and M. smegmatis-> oxidizeH2 (environmentally relevant concentrations [10 ppm]) • M. avium or M. microti,-> CO was not cosumed!! • M. smegmatis and M. gordonae oxidized CO under suboxic (10 and 1% atmospheric oxygen) and anoxic conditions in the presence of nitrate • Phylogenetic analyses of coxL PCR products indicated that mycobacterial sequences form a subclade distinct from that of other bacterial coxL, with limited differentiation among fast- and slow-growing strains.

3. Dissimilatory nitrate reduction +2e- + 2H+ NO2- +H2O Nitrate(NO3-)

4. Denitrification Nitrite reductase Nitrate reductase Nitric oxide(NO) Nitrite(NO2-) Nitrate(NO3-) Nitric oxide reductase Dinitrogen(N2) Nitrous oxide(N2O) Nitrous oxide reductase

5. Introduction • Mycobacteria colonize soil and aquatic environments as well as various vertebrate hosts.They have been classified as slow or fast growing based on maximal growth rates in culture. • Slow- and fast-growing strains promote survival during nutrient starvation, when preferred substrates are otherwise lacking, or when conditions lead to imbalanced growth. • Mycobacteria experience hypoxic and anoxic conditions with or without nutrient limitation,which also must elicit adaptive responses. • H2 and CO found in the atmosphere at concentrations of about 0.5 to 0.6 ppm and 0.1 to 0.5 ppm, and also found within lung or respiratory gases at concentrations of about 5 to 50 ppm and 2 to 5 ppm.

6. Introduction • Several fast- and slow-growing mycobacteria (e.g., M. smegmatis and M. tuberculosis H37Ra) grow lithotrophically by using high CO concentrations. • Slow growth on 100-ppm CO by Mycobacterium sp. strain JC1 • In contrast, growth on high H2 and CO2 concentrations has been reported for only one mycobacterial strain to date, M. gordonae • Purpose of this study • The ability of mycobacteria to consume these gases at environmentally relevant concentrations (oxic, suboxic, and anoxic conditions). • Obtaining phylogenetic relationships between mycobacteria and other CO oxidizers.

7. MATERIALS AND METHODS • 1.Culture growth • M. gordonae, M. marinum, M. microti, and M. tuberculosis H37Ra were obtained from the American Type Culture Collection. • M. avium, M. bovis BCG, and M. smegmatis were obtained from M. Glickam (Memorial Sloan-Kettering Cancer Center) • Cultures were grown in serum bottles (60 or 160 cm3) containing one of three media: Middlebrook 7H9 with oleic acid-albumin-dextrose-catalase (OADC) supplement (4.5 ml), Middlebrook 7H9 without OADC supplement (4.5 ml), or a pyruvate (25 mM), yeast extract (0.05%),mineral salts medium (PYE) (9.5 ml). • After sealing with butyl rubber stoppers and crimp caps, media were inoculated with 0.5-ml volumes of suitable cell suspensions. Cultures were incubated at 37°C (30°C for M. marinum and M. smegmatis) with shaking at 125 rpm

8. Oleic Acid-albumin-Dextrose-Catalase (OADC) • Oleic Acid served as a source of carbon • The albumin acts as a protective agent by binding free fatty acids, which may be toxic to Mycobacterium species • Dextrose is an energy source; • Catalase destroys toxic peroxides that may be present in the medium

9. MATERIALS AND METHODS 2.CO and H2 utilization. • M. smegmatis and M. gordonae were grown aerobically with PYE and Middlebrook 7H9 with OADC. • Bottle headspaces contained ambient air, nitrogen with either 0.2 or 2% oxygen (suboxic treatments), or nitrogen only (anaerobic treatments) • CO uptake by M. bovis BCG, M. marinum, M. microti, and M. tuberculosis H37Ra was assessed by growing cultures in Middlebrook 7H9 with or without OADC supplement as described above. CO was added to culture headspaces at concentrations up to 1% 2 to 3 weeks postinoculation. • CO concentrations were determined by gas chromatography

10. Gas chromatography • 이동상(移動相)에 기체를 사용하여, 혼합기체시료를 그 성분기체의 열전도율의 차를 이용하여 검출 ·정량하는 기기분석법 • 고정상(固定相)에 흡착성이 있는 고체의 분말 미립자를 사용하는 기체-고체크로마토그래피와, 적당한 비활성 고체분말의 표면에 비휘발성 액체를 보유시킨 기체-액체크로마토그래피로 나뉜다. 전자는 보통 끓는점 400 ℃ 정도까지의 유기화합물 전반에 걸쳐 분석을 할 수 있고, 후자는 무기화합물의 기체 및 끓는점이 낮은 탄화수소의 분석에 적합하다. 나선모양으로 감은 금속관(column이라 한다)에 활성탄 ·실리카겔 ·실리콘 ·그리스를 삼투시킨 규조토 등을 충전하고, 여기에 분석하고자 하는 시료를 흡착시킨 다음 수소 ·헬륨 등의 기체(carrier라 한다)를 통과시키면 컬럼의 다른 끝에서 시료의 성분기체가 흡착성이 작은 성분부터 차례로 단리(單離)되어 나온다. 이때, 컬럼에 들어가기 전의 캐리어기체와 컬럼에서 나온 기체의 열전도율을 비교하여 검출한다

11. MATERIALS AND METHODS • 3.PCR analyses • primersOMPf(5GGCGGCTT[C/T]GG[C/G]AA[C/G]AAGGT-3) and O/Br(5[C/T]TCGA[T/C]GATCATCGG[A/G]TTGA-3) • Positive controls (CO oxidizer), Oligotropha carboxidovorans • negative controls (no CO oxidation has been observed in vivo), Lutibacterium anuloederans • Amino acid sequences were deduced from partial coxL sequences by using ExPAsy and were aligned with other coxL sequences by using Clustal X, with manual adjustments as necessary. The resulting alignments were analyzed by using PAUP* 4.0b (Sinauer Associates, Inc.,Sunderland, Mass.) to determine phylogenetic relationships among taxa. After excluding gapped positions, 367 residues were subjected to a neighbor-joining algorithm (1,000 bootstrap replicates) for tree construction.

12. Results

13. Growth of M. smegmatis in mineral salts plus various carbonand energy sources Energy source & Carbon source Carbon source Energy source Energy source & Carbon source

14. CO uptake by M. smegmatis under various incubation conditions with killed controls • air (○), • 10% air (●), • 1% air (□), • anoxic plus nitrate (■), • anoxic (△), autoclaved(▲).

15. H2 uptake by M. smegmatis under varied incubation conditions • air (○), • 1% air (●), • anoxic(□), • anoxic plus nitrate (■),

16. H2 uptake by M. smegmatis grown with pyruvate-mineral salts or Middlebrook 7H9 with OADC • pyruvate-mineral salts (○) • Middlebrook 7H9+OADC (●). Middlebrook 7H9+OADC PYE

17. ‘CO uptake by M. gordonae grown with pyruvate-mineral salts under various incubation conditions • air (○), • 1% air (●), • anoxic(□), • anoxic plus nitrate (■),

18. CO uptake by M. marinum (E) and M. microti (F) grown with Middlebrook 7H9 plus OADC. • M. marinum (○) • M. microti (●)

19. PCR products from genomic extracts of selected mycobacteriausing coxL primers

20. CO uptake by M. tuberculosis H37Ra (squares) and M.bovis BCG (circles) under various growth conditions • M. tuberculosis H37Ra (squares) M. bovis BCG (circles) • Middlebrook 7H9, (open symbols); Middlebrook 7H9 plus OADC, (closed symbols) • M. tuberculosis H37Ra grown with Middlebrook 7H9 and incubated with about 50 ppm CO (△).

21. PCR products from genomic extracts of selected mycobacteria using coxL primers. Mycobactrial CO-DH

22. Discussion • M. bovis BCG and M. tuberculosis H37Ra consume high and environmentally relevant CO concentrations more slowly than other strains when grown with Middlebrook 7H9 containing or lacking OADC supplement. M. marinum uses CO similarly, albeit with faster uptake rates. M. gordonae and M. smegmatis CO utilization appears more sensitive to culture conditions. ㅡ>these organisms encompass a range of responses to CO, H2, and culture conditions that provide a framework for understanding activity by pathogenic M. tuberculosis. Results for M. microti yields a coxL PCR product, the sequence of which is virtually identical to that of the coxL PCR product from M.tuberculosis H37Ra ->Assuming M. microti contains a complete cox operon, lack of CO uptake indicates that its controls of expression differ from controls for other mycobacteria.