Cloning and expression of the Mycobacterium fortuitum Superoxide dismutase gene

FEMS Microbiology

Letters I34 (1995) 273-278

Cloning and expression of the Mycobacterium fort&urn superoxide dismutase gene M. Carmen Menkndez, Pilar Domenech Dept. Medicina Prel~entil,a.

Facultad

de Medicina,

Received 7 September

Unicersidad

‘, Jesus Prieto 2, Maria J. Garcia Autcinoma,

1995; revised 17 October

Arzobispo

Morcillo

1995; accepted 20 October

4, 2802%Madrid.

*

Spain

1995

Abstract In this paper

of the superoxide dismutase (sod) gene from dismutase activity with its identity being confirmed by expression in M. aurum. The amino acid sequence was found to be similar to that of superoxide dismutases of several other origins. A region downstream of the sod gene also showed similarities to the corresponding sequences of the two main mycobacterial pathogens: M. leprae and M. tuberculosis. Analysis of enzymatic activity showed this enzyme in M. fortuitum required manganese as cofactor. Mycohacterium

Kewords:

we report

fortuitum.

Mycobacteria;

the cloning,

sequencing

and expression

A single gene was found to code for superoxide

Mywbacterium

fortuitum;

Superoxide

1. Introduction The majority of human infections attributed to fast growing mycobacteria are caused by members of the Mycohacterium fortuitum complex [l I. M. fortuiturn can be grown easily in vitro and has been used as a model to study the intracellular killing of mycobacteria [2]. The determination of molecular similarities and differences between major pathogenic mycobacteria such as h4. tuberculosis or M. leprae and opportunistic mycobacteria such as M. fortuitum

* Corresponding author. Tel.: + 34 (1) 397 5440; Fax: + 34 (I) 397 5353; E-mail: [email protected]. ’ Present address: Dept. Biotecnologia. C.I.B. Smithkline Beecham. Santiago Grisolia s/n, 28760-Madrid, Spain. ’ Present address: E.M.B.L., Postfach 102209, Meyerhofstrasse I, 6900 Heidelberg, Germany. Federation of European Microbiological SSDl 037% 1097(95)00419-X

Societies

dismutase;

Gene expression

could lead to a better understanding of the molecular processes underlying mycobacterial infectivity and virulence. Mycobacteria are able to multiply whilst resident within macrophages, but activated macrophjages can inhibit this intracellular proliferation. Oxidative killing is one of the mechanisms by which macrophages control intracellular proliferation of bacteria. However, several genes have been identified, mostly in Escherichia coli, that appear to play a role at protecting against oxidative damage; those encoding for superoxide dismutases and hydroperoxidases [3]. Moreover, it has been shown that lsuperoxide dismutase has a high rate of transcription in phagocytosed M. auium bacilli [4]. Superoxide dismutases (SODS, EC I. 15.1. I ) are a family of antioxidant enzymes which catalyse the dismutation of the superoxide anion to hydrogen peroxide [S].

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M.C. Mene’ndez et al. / FEMS Microbiology

There are several kinds of SODS among organisms. Those depend upon the metal cofactor carried which may be copper/zinc, iron or manganese. The genes have previously identified, cloned and sequenced for Fe-SOD in M. tuberculosis (a slow grower) [6l and for Mn-SOD in M. leprae (which is non-cultivable in vitro) [7]. In addition, the crystal structure of M. tuberculosis Fe-SOD has recently been published 181. Within the genus A4ycobacterium, SOD has been identified as a major protein antigen [9], and the highly conserved amino acid sequences within the SOD has been used recently as a basis for the detection and identification of mycobacteria by polymerase chain reaction (PCR) [lo]. We report here the cloning, sequencing and expression of the superoxide dismutase gene of IV. fortuitum. This enzyme is shown to be homologous with SODS of the two major mycobacterial pathogens: M. leprae and M. tuberculosis.

Letter-s 134 (1995) 273-278

2.3. Southern blots and hybridizations

2. Materials and methods

Mycobacterial and plasmid DNAs were digested with suitable restriction enzymes as recomended by the manufacturers, DNA fragments were separated by electrophoresis on 0.8% (w/v) agarose gels. Southern blots and hybridizations were performed as previously described [ 121. Colony blots were prepared using standard techniques [ 131. A 500-bp fragment of sodA gene from genomic DNA of M. tuberculosis 79500 [ 141 was amplified by PCR, with oligonucleotides: SOD3.1, 5’-ACA TCT CGG GTC AGA TCA ACG AGC; and SOD4, 5’-GAC GTT CTT GTA CTG CAG GTA G, labelled, and used as a probe. PCR reaction conditions consisted of 30 cycles of 40 s at 94°C 30 s at 58°C and 1 min at 72°C with subsequent 10 min extension at 72°C. The kanamycin resistance gene from Tn 903 (Pharmacia) was also labelled and used as a probe. Probes were labelled using the Prime-a-Gene System (Promega) and [ a- 32P]dCTP (Amersham) according to the manufacturer’s protocol.

2. I. Bacterial

2.4. DNA sequencing

strains and plasmids

M. fortuitum ATCC 6841 (American Type Culture Collection, Type strain), M. aurum Ll [l 11, M. gadium ATCC 27726 (Type strain) and M. smegmatis mc’155 (W. Jacobs, Albert Einstein College of Medicine, NY) were maintained on slopes of Lowenstein-Jensen medium and stored at 4°C. The vectors pUCl8 and pUC19 were used for cloning and sequencing mycobacterial DNA. The shuttle vector pRR3 was kindly provided by B. Gicquel (Institut Pasteur, Paris, France) 1111. E. coli DH5 was used as host for cloning mycobacterial DNA. E. coli strains were grown on LB medium supplemented with ampicillin (100 pug ml-’ 1 and kanamycin (40 pg ml ’ ) as required. 2.2. DNA isolation and manipulation Genomic DNA was prepared from mycobacteria as previously described [ 121. Concentrations of DNA in each sample were quantified by ultraviolet absorbance at 260 nm and stored in ethanol at - 20°C. Standard protocols were used for cloning, plasmid isolation and restriction enzyme analysis [ 131.

and computer analysis

Plasmid sequences were determined using sequenase version 2.0 according to the manufacturers’ protocol (United States Biochemicals) using [a35SldATP (Amersham). Both direct and reverse universa1 pUC primers were used as well as synthetic oligonucleotides (CBM, Cantoblanco, Madrid, Spain). Both strands were sequenced independently. Amino acid sequences were determined and aligned to closely related SODS using the GCG package program (version 8). 2.5. SOD activity in non-denaturing-PAGE Non-denaturing polyacrylamide gel electrophoresis (ND-PAGE; 10% W/V) was carried out [ 131 on samples containing 50- 100 pug of protein extracts prepared by ultrasonic disruption of the bacteria [61. Gels were stained for SOD activity as previously described [6]; SOD activity was visualised as a white band against a blue background. Discrimination between Fe-SOD and Mn-SOD activity was made by inhibition studies of Fe-SOD protein extracts by treatment with hydrogen peroxide (20-200 mM for 2

M.C. Menthdez

h at room temperature) ND-PAGE gels 1151. 2.6. Expes.sion

before

et al. / FEMS Microbiology

running

of the M. fortuitum

samples

in

sodA

A 1.36-kb SmaI fragment containing the M. fortuitum sod gene was cloned into the single ScaI site of the mycobacterial shuttle vector pRR3 [l 11. The new plasmid, designated pRMFV, was electroporated [6] into M. smegmatis mc’ 155 and M. aurum Ll, from which kanamycin-resistant colonies were selected and transformants tested for SOD activity.

3. Results and discussion 3.1. The sodA qf M. fortuitum A BamHI partial library (l-2 kb) of M. ,fortuiturn ATCC 6841 genomic DNA was ligated into

Letters

134 f IYY51 273-278

275

pUC 18 and, using a labelled 500-bp amplified fragment from M. tuberculosis sod gene as a probe, a plasmid clone, designated pMFV, was isalated by colony blot hybridization. The nucleotide sequence of the BamHI insert (1478 bp) from pMFV was determined and an open reading frame (dRF; 621 bp) was identified with a predicted protein molecular mass of 23 kDa (data not shown) (EMBL accession number: X709 14). Comparison of nucleotide sequences within the ORF coding region between this clone #and that recently described for the same M. fortuit#m strain [lo] showed point mutations at both extremities of the sequence (data not shown). These mutations may be explained by the fact that these authors used universal primers for mycobacterial sod gene for the amplification and subsequent partial sequencing of that gene [lo]. The protein encoded by the ORF was homologous with several iron and manganese SODS from a vari-

TATTr “ ‘GGAGTCGCGCAT' ‘P."‘-,?'r' ":

Fig. I Nucleotide sequence of the neighbour regions of the M. fiwtuitum sod gene within the pMFV insert. (A) Potential Shiple-Dalgamo sequence is overlined; AMB stands for ambar stop codon; a region homologous to the E. co/i - 35 promotor consensus sequence is overlined (__._,,.). Palindromic sequences are overlined >1 -+z : repeated sequences similar to MPTR are underlined. Sequence$ with partial identity to the REP sequences are showed in bold lower-case typeface. (B) Comparison of palindromic sequences downstream ‘from the M. ,fortuitum (M.F), M. lepme (M.L) and M. tuberculosis (M.T) sod genes. Gaps are indicated by dashes.

276

M.C. Me&dez

et al./ FEMS Microbiology

ety of bacterial and eukaryotic sources (results not shown). The primary structure suggests that SOD from M. fort&urn could be a Mn-protein by the analysis of Parker and Blake 1161. Several regulatory proteins have been described in E. coli that bind to specific sequences in the promoter region of sod4 gene (coding for Mn-SOD) [ 171. Upstream of the sodA of M. fort&urn, it was not possible to identify any target sequences similar to those described in E. coli (Fig. IA). The higher G-C genomic content of mycobacterial DNA (approx. 70%), relative to E. coli DNA could make those potential target nucleotide sequences differ when both bacteria are compared. Sequences with similarities to the repetitive extragenie palindromic (REP) consensus sequences of E. roli (GCC(G/T)GATGCG(G/A)CaC/T)) [18] and to the major polymorphic tandem repeat (MPTR) consensus sequences of M. tuberculosis (GCCGGTGTTG) [ 191 were identified downstream from the M. fort&urn sod gene (Fig. IA). In addition, sequences located downstream from the M. fortuitum sod gene were found to contain two palindromic sequences that shared strong similarities to rho-independent terminator sequences in E. coli 1201 (Fig. IA). One of these is related to another similar sequence also present in a similar location with respect to the sod gene in M. leprae [7] (Fig. 1B). Further studies are required to clarify the role that these sequences might play in gene regulation. 3.2. SOD actioiry in fast-growing

Lettetx 134 (19951273-278

Fig. 2. Non-denaturing-PAGE stained for SOD activity with and without H,O, treatment. (A) Fast growing mycobacterial species (50 mg pro& was loaded for sample). (B) M. a~r~rn transformed with pRMFV (V) or with pRR3 shuttle vector (R) (100 mg protein was loaded for sample). Ec, commercial E. coli Mnand Fe-SODS; MS, M. smegn~tis; Mf, M. jortuitum; Ma, M. owum wild-type; Mg, M gadium. M. aururn transformants grown ar different temperatures. Vl and Rf at 3U”C, V2 and R2 at 37°C.

mycnbacteria 3.3. Expression

The SOD activity assays showed a single band in each preparation with a differing electrophoretic mobility for each species of fast growing mycobacteria tested (Fig. 2A). InhibitIon of SOD activity by H,O, treatment of protein extracts (see Materials and methods) could only be observed in M. aurum extracts indicating Fe-SOD activity in this species. M. fortuitum, M. smegmatis and M. gadium seemed to require manganese as cofactor (Fig. 2A). This characterization as well as the difference in its electraphoretic mobility suggested M. a&r’Llm as a good mycobacterial host to be transformed with the M. fortuitum sodA (Fig. ZA), since both SOD activities could be easily differentiated by ND-PAGE and inhibitory treatment of protein extracts.

of the M. fortuitum

sodA

The inability to successfully obtain M. fortuitum SOD activity in E. coli transformed with plasmid pMFV prompted the testing for suitability of fast growing mycobacteria as transformant hosts. M. smegmatis mc2 155 and M. aurum Ll were found to be suitable for transformation with pRMFV (see Materials and methods). Kanamycin-resistant colonies were selected and mycobacterial protein extracts were tested for SOD activity (Fig. 2B). A high levei of expression was obtained (data of M smegmatis transformants not shown). We concluded that transcriptional and translational signals are provided by sequences from the cloned DNA fragment itself.

M.C. Me&de:

et al. / FEMS Microbiology

Transformants were tested for inhibition studies using H20, (see Materials and methods); the band having the same mobility as M. aurum SOD disappeared after treatment with hydrogen peroxide (Fig. 2B). M. aurum is an environmental mycobacteria, therefore, its optimum growth is at 30°C. This could explain the lower Fe-SOD enzyme activity in bacteria grown at 37°C compared to 30°C both wild-type M. aurum (not shown) and transformants (Fig. 2B, lanes VI and RI versus V2 and R2). To demonstrate that SOD activity was due to the presence of the recombinant plasmid (pRMFV) in the mycobacteria, DNA preparations belonging to M. aurum and M. smegmatis (transformants and wild-types) were tested by hybridizing against the SOO-bp probe from M. tuberculosis sodA and the kanamycin resistance gene probe from Tn903 (see Materials and methods). The results (Fig. 3B, C) showed the presence of both pRR3 and the M. ,fortuifum sod gene in only the recombinant but not in the wild-type bacteria.

Letters

134 f IYY5J 273-278

217

Low identity at the genomic level between genes encoding for SOD in M. tuberculosis and in M. aurum would be expected since no positive signal was obtained in M. aurum by hybridization ,with the M. tuberculosis sod gene (Fig. 3B). A similar result was also found for M. Radium (not shown),; another chromogenic fast growing mycobacteria. On’the contrary, a hybridization signal is detected in M. smegmafis (3-kb fragment, lanes MS and Sv, Fig, 3B). In conclusion, these studies indicate that M. .forfuitum SOD seems to be more related to M. leprue SOD than to M. tuberculosis SOD as amino acid sequences are more similar, both share manganese as cofactor and have rho-independent terminator-like sequences downstream of their genes. This ,result is striking if we consider the taxonomical relationships between the fast growing mycobacteria, M. ituberculosis and M. leprae. It suggests, however, that a better knowledge of genetic control and function of the M. ,fortuitum enzyme could be of assistance to understand the role of SOD in oxidative metabolism studies of the non-cultivatable M. leprae.

8 5 3

1.6

Fig. 3. Southern tuhercuhsis

blot analysis.

(A)Ethidium bromide

stain of BarnHI-digested DNAs. (B)Hybridization using a fragment of M. and methods). (C) Hybridization using the TnY03 kanamycine cassette as a probe (see kb (BRL); pR, pRR3 shuttle vector; pV, pRMFV: R, M. LIWU~ Ll transformed with pRRB: Ma. M. LI transformed with pRMFV: Ms. M. .srnegmnris mc’ 155 wild-type; Sv, M. smvpnntis mc’ 155

sod gene as a probe (see Materials

Materials and methods). L, Ladder I aurunr Ll wild-type: V, M. aurum transformed with pRMFV.

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Acknowledgements We are grateful to Dr. B. Gicquel for providing the pRR3 shuttle plasmid, and Dr. W.R. Jacobs for providing the M. smegmafis mc*155 strain. We also thank Dr. C. Moreno and Dr. I. Outschoom for helpful review of the manuscript. This work was supported by a grant from the Comision Interministerial de Ciencia y Tecnologia (Ref.: Sa191-1034; Spain).

References [II Wayne, L.C. and Sramek, H.A. (1992) Agents of newly recognized or infrequently encountered mycobacterial diseases. Clin. Microbial. Rev. 5, l-25. [21 Geertsma, M.F., Nibbering, P.H., Pos, 0. and van Furth, R. (1990) Interferon-y-activated human granulocytes kill ingested Myobacterium fortuitum more efficiently than normal granulocytes. Eur. J. Immunol. 20, 869-873. [31 Farr, S.B. and Kogoma, T. (1991) Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbial. Rev. 55, 561-585. J.E. (1994) Induction of My[41 Plum, G. and Clark-Curtiss. cobacterium avium gene expression following phagocytosis by human macrophages. Infect. Immun. 62, 476-483. 151 Fridovich, I. (1984) Superoxide dismutases. J. Biol. Chem. 264, 116 I-7764. 161Zhang, Y., Lathigra, R., Garbe, T., Catty, D. and Young, D. (1991) Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Myobacterium tuberculosis. Mol. Microbiol. 5, 38 I-39 I. H.S., Lamb, F.I., Davis, E.O., Jenner, P.J., 171 Thangaraj, Jeyakuma, L.H. and Colston. M.J. (1990) Identification, sequencing and expression of Mycobacterium leprae superoxide dismutase, a major antigen. Infect. Immun. 58, 19371942. Is1 Cooper, J.B., McIntyre, K., Badasso, M.O., Wood, S.P., Zhang, Y., Garbe, T.R. and Young, D. (I 995) X-ray stucture analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions. J. Mol. Biol. 24, 53 l544. S.H.E., Hermans. P.W.M. and [91 Young, D.B., Kaufmann,

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Thole, J.E.R. (1992) Mycobacterial protein antigens: a compilation. Mol. Microbial. 6, 133-145. [IO] Werner-Zolg, J. and Philippi-Schulz, S. (1994) The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR. J. Clin. Microbial. 32, 2801-28 I?. [I II Ranes, M.G., Rauzier, J., Lagranderie, M., Gheorghiu. M. and Gicquel, B. (1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a ‘mini’ mycobacterium-Escherichia coli shuttle vector. J. Bacterial. 172, 2793-2797. II21 Domenech, P., Menendez, M.C. and Garcia, M.J. (1994) Restriction fragment length polymorphisms of 16s rRNA genes in the differentiation of fast-growing mycobacterial species. FEMS Microbial. Lett. I 16, 19-24. [I31 Sambrook, J.. Fritsch. E.F. and Maniatis, T. (1989) Molecular Cloning: A laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1141 Zhang, Y., Garcia, M.J., Lathigra, R., Allen, B.W., Moreno. C., van Embden, J.D.A. and Young, D. (1992) Alterations in the superoxide dismutase gene of an isoniazid-resistant strain of Mycobacterium tuberculosis. Infect, Immun. 60, 2 1602165. [I51 Matsumoto, T., Terauchi, K.. Isobe. T., Matsuoke, K. and Yamakura, F. f 199 1) Iron- and manganese-containing superoxide dismutases from Methyfomonas: identity of protein moiety and aminoacid sequence. Biochemistry 30, 32103216. 1161Parker, M.W. and Blake, C.C.F. f 1988) Iron and manganese containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett. 229,377-328. iI71 Hassan, H.M. and Schrum. L.W. (1994) Roles of manganese and iron in the regulation of the biosynthesis of manganesesuperoxide dismutase in Escherichia co/i. FEMS Microbial. Rev. 14, 315-324. 1181Stem, M.J., Ames, G.F.L., Smith. N.H., Robinson, E.C. and Higgins, C.F. (1984) Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell 37, 1015-1026. iI91 Hermans, P.W., van Soolingen, D. and van Embden, J.D.A. (1992) Characterization of a major polymorphic tandem repeat in M.vcobacterium tuberculosis and its potential use in the epidemiology of Myobacterium kansasii and Mycobncrerium gordonae. J. Bacterial. 174, 4 157-4165. R.M. and Chamberlin, M.J. DOI Reynolds, R., BermudezCruz, (1992) Parameters affecting transcription termination by Escherichia coli RNA polymerase. Analysis of I3 rho-independent terminators. J. Mol. Biol. 224, 3 I-5 I