The Actinobacterial mce4 Locus Encodes a Steroid Transporter
2008; Elsevier BV; Volume: 283; Issue: 51 Linguagem: Inglês
10.1074/jbc.m805496200
ISSN1083-351X
AutoresWilliam W. Mohn, Robert van der Geize, Gordon R. Stewart, Sachi Okamoto, Jie Liu, Lubbert Dijkhuizen, Lindsay D. Eltis,
Tópico(s)Caveolin-1 and cellular processes
ResumoBioinformatic analyses have suggested that Mce proteins in diverse actinobacteria are components of complex ATP-binding cassette transporter systems, comprising more than eight distinct proteins. In Mycobacterium tuberculosis, these proteins are implicated in interactions of this deadly pathogen with its human host. Here, we provide direct evidence that the Mce4 system of Rhodococcus jostii RHA1 is a steroid uptake system. Transcriptional analyses indicate that the system is encoded by an 11-gene operon, up-regulated 4.0-fold during growth on cholesterol versus on pyruvate. Growth of RHA1 on cholesterol and uptake of radiolabeled cholesterol both required expression of genes in the mce4 operon encoding two permeases plus eight additional proteins of unknown function. Cholesterol uptake was ATP-dependent and exhibited Michaelis-Menten kinetics with a Km of 0.6 ± 0.1 μm. This uptake system was also essential for growth of RHA1 on β-sitosterol, 5-α-cholestanol, and 5-α-cholestanone. Bioinformatic analysis revealed that all mce4 loci in sequenced genomes are linked to steroid metabolism genes. Thus, we predict that all Mce4 systems are steroid transporters. The transport function of the Mce4 system is consistent with proposed roles of cholesterol and its metabolism in the pathogenesis of M. tuberculosis. Bioinformatic analyses have suggested that Mce proteins in diverse actinobacteria are components of complex ATP-binding cassette transporter systems, comprising more than eight distinct proteins. In Mycobacterium tuberculosis, these proteins are implicated in interactions of this deadly pathogen with its human host. Here, we provide direct evidence that the Mce4 system of Rhodococcus jostii RHA1 is a steroid uptake system. Transcriptional analyses indicate that the system is encoded by an 11-gene operon, up-regulated 4.0-fold during growth on cholesterol versus on pyruvate. Growth of RHA1 on cholesterol and uptake of radiolabeled cholesterol both required expression of genes in the mce4 operon encoding two permeases plus eight additional proteins of unknown function. Cholesterol uptake was ATP-dependent and exhibited Michaelis-Menten kinetics with a Km of 0.6 ± 0.1 μm. This uptake system was also essential for growth of RHA1 on β-sitosterol, 5-α-cholestanol, and 5-α-cholestanone. Bioinformatic analysis revealed that all mce4 loci in sequenced genomes are linked to steroid metabolism genes. Thus, we predict that all Mce4 systems are steroid transporters. The transport function of the Mce4 system is consistent with proposed roles of cholesterol and its metabolism in the pathogenesis of M. tuberculosis. The mce genes of Mycobacterium tuberculosis have garnered much interest due to their demonstrated role in pathogenesis, although the function of these genes remains unclear. The first mce gene, now known as mce1A, was discovered when a DNA fragment cloned from M. tuberculosis into a noninvasive Escherichia coli strain enabled uptake of the latter bacterium by nonphagocytic mammalian epithelial (HeLa) cells and facilitated its phagocytosis by macrophages (1Arruda S. Bomfim G. Knights R. Huimabyron T. Riley L.W. Science. 1993; 261: 1454-1457Crossref PubMed Scopus (307) Google Scholar). A subsequent study showed that coating latex beads with the protein encoded by mce1A facilitated uptake of the beads by HeLa cells, and so the gene was designated mce for mammalian cell entry (2Chitale S. Ehrt S. Kawamura I. Fujimura T. Shimono N. Anand N. Lu S. Cohen-Gould L. Riley L.W. Cell. Microbiol. 2001; 3: 247-254Crossref PubMed Scopus (131) Google Scholar). The genome sequence of M. tuberculosis revealed four loci (mce1–4), each containing two yrbE genes followed by six mce genes (3Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 396: 190-198Crossref Scopus (52) Google Scholar). The Mce proteins are predicted to have similar structures and to be secreted (4Mitra D. Saha B. Das D. Wiker H.G. Das A.K. Tuberculosis (Edinb.). 2005; 85: 337-345Crossref PubMed Scopus (13) Google Scholar), and Mce1A was localized to the surface of M. tuberculosis cells (2Chitale S. Ehrt S. Kawamura I. Fujimura T. Shimono N. Anand N. Lu S. Cohen-Gould L. Riley L.W. Cell. Microbiol. 2001; 3: 247-254Crossref PubMed Scopus (131) Google Scholar). The capacity to facilitate uptake by HeLa cells was further narrowed to a small region of the MceA1 protein (2Chitale S. Ehrt S. Kawamura I. Fujimura T. Shimono N. Anand N. Lu S. Cohen-Gould L. Riley L.W. Cell. Microbiol. 2001; 3: 247-254Crossref PubMed Scopus (131) Google Scholar, 5Casali N. Konieczny M. Schmidt M.A. Riley L.W. Infect. Immunol. 2002; 70: 6846-6852Crossref PubMed Scopus (36) Google Scholar, 6Lu S. Tager L.A. Chitale S. Riley L.W. Anal. Biochem. 2006; 353: 7-14Crossref PubMed Scopus (23) Google Scholar). Coating beads with either Mce3A or Mce3E also facilitated such uptake (7El-Shazly S. Ahmad S. Mustafa A.S. Raja Al-Attiyah R. Krajci D. J. Med. Microbiol. 2007; 56: 1145-1151Crossref PubMed Scopus (45) Google Scholar), whereas coating beads with Mce2A failed to do so (2Chitale S. Ehrt S. Kawamura I. Fujimura T. Shimono N. Anand N. Lu S. Cohen-Gould L. Riley L.W. Cell. Microbiol. 2001; 3: 247-254Crossref PubMed Scopus (131) Google Scholar). Together, these studies suggest that some of the Mce proteins might interact with host cells and play a role in uptake of M. tuberculosis by host cells. However, direct evidence for this function of Mce proteins is lacking. Mutational studies also support the involvement of Mce proteins in pathogenesis of M. tuberculosis. One genome-wide screen suggested that disruption of mce1 and mce4 genes causes early and late growth defects, respectively, in mice (8Sassetti C.M. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12989-12994Crossref PubMed Scopus (1057) Google Scholar), and these phenotypes were confirmed with mce1D and yrbE4A deletion mutants, respectively (8Sassetti C.M. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12989-12994Crossref PubMed Scopus (1057) Google Scholar, 9Joshi S.M. Pandey A.K. Capite N. Fortune S.M. Rubin E.J. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11760-11765Crossref PubMed Scopus (133) Google Scholar, 10Pandey A.K. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4376-4380Crossref PubMed Scopus (722) Google Scholar). Another genome-wide screen suggested that disruption of yrbE4A impairs macrophage infection (11Rosas-Magallanes V. Stadthagen-Gomez G. Rauzier J. Barreiro L.B. Tailleux L. Boudou F. Griffin R. Nigou J. Jackson M. Gicquel B. Neyrolles O. Infect. Immun. 2007; 75: 504-507Crossref PubMed Scopus (57) Google Scholar), and a yrbE4A deletion mutant was severely impaired in replication in interferon γ-activated macrophages (10Pandey A.K. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4376-4380Crossref PubMed Scopus (722) Google Scholar). Disruption of any one of yrbE1B, mce2A, and mce3A genes in M. tuberculosis increased survival of intratrachaelly infected mice (12Gioffre A. Infante E. Aguilar D. de la Paz Santangelo M. Klepp L. Amadio A. Meikle V. Etchechoury I. Romano M.I. Cataldi A. Hernandez R.P. Bigi F. Microbes Infect. 2005; 7: 325-334Crossref PubMed Scopus (124) Google Scholar). Similarly, deletion of nearly entire mce3 or mce4 operons increased long term survival of mice following low dose aerosol infection, and the latter deletion also decreased bacterial burdens of the mice (13Senaratne R.H. Sidders B. Sequeira P. Saunders G. Dunphy K. Marjanovic O. Reader J.R. Lima P. Chan S. Kendall S. McFadden J. Riley L.W. J. Med. Microbiol. 2008; 57: 164Crossref PubMed Scopus (82) Google Scholar). By contrast, an mce1A deletion was hypervirulent in mice (14Shimono N. Morici L. Casali N. Cantrell S. Sidders B. Ehrt S. Riley L.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15918-15923Crossref PubMed Scopus (175) Google Scholar). Thus, mce mutant strains have phenotypes generally supporting the involvement of these genes in pathogenesis, but the mutants exhibited disparate virulence phenotypes in different assays. Analysis of genome sequences indicated that diverse actinobacteria, including members of Mycobacterium, Nocardia, Rhodococcus, and Streptomyces, harbor mce loci (15Casali N. Riley L.W. BMC Genomics. 2007; 8: 60Crossref PubMed Scopus (169) Google Scholar, 16McLeod M.M. Warren R.L. Hsiao W. W.L. Araki N. Myhre M. Fernandes C. Miyazawa D. Wong W. Lillquist A.L. Wang D. Dosanjh M. Hara H. Petrescu A. Morin R.D. Yang G. Stott J.M. Schein J.E. Shin H. Smailus D. Siddiqui A.S. Marra M.A. Jones S. J.M. Holt R. Brinkman F. S.L. Miyauchi K. Fukuda M. Davies J.E. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15582-15587Crossref PubMed Scopus (499) Google Scholar). Moreover, PCR-based assays revealed the existence of mce4 loci in 11 of 20 Mycobacterium spp. screened (17Haile Y. Caugant D.A. Bjune G. Wiker H.G. FEMS Immunol. Med. Microbiol. 2002; 33: 125-132Crossref PubMed Google Scholar). The actinobacteria with mce loci include both pathogens and free-living bacteria, strongly suggesting that the function of these genes is not limited to virulence. Bioinformatic analyses (15Casali N. Riley L.W. BMC Genomics. 2007; 8: 60Crossref PubMed Scopus (169) Google Scholar, 18Tekaia F. Gordon S.V. Garnier T. Brosch R. Barrell B.G. Cole S.T. Tuber. Lung Dis. 1999; 79: 329-342Abstract Full Text PDF PubMed Scopus (252) Google Scholar) indicated that the yrbE genes of all mce loci encode transmembrane proteins with similarity to permease components of ATP-binding cassette (ABC) 2The abbreviations used are:ABCATP-binding cassetteRT-PCRreverse transcription-PCRDCCDdicyclohexylcarbodiimideBCGBacille de Calmette et Guèrin transporters. The hallmark of ABC transporters is an ATPase with highly conserved features. Some mce loci include genes encoding such ATPases, which belong to the Mkl family, a subset of ABC transporter ATPases (15Casali N. Riley L.W. BMC Genomics. 2007; 8: 60Crossref PubMed Scopus (169) Google Scholar). Further, all actinobacteria containing mce loci were found to also contain genes encoding Mkl ATPases, usually not proximal to the mce loci. Based on genetic interaction mapping and mutagenesis, Joshi et al. (9Joshi S.M. Pandey A.K. Capite N. Fortune S.M. Rubin E.J. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11760-11765Crossref PubMed Scopus (133) Google Scholar) provided evidence that the Mce1 and Mce4 proteins in M. tuberculosis are both functionally linked to a single Mkl ATPase (MceG), encoded by a gene not linked to either the mce1 or the mce4 loci. These observations strongly suggest that mce loci encode a novel type of ABC transporter. However, direct evidence for this transport function has not been demonstrated. ATP-binding cassette reverse transcription-PCR dicyclohexylcarbodiimide Bacille de Calmette et Guèrin Many mce loci also include two or four genes downstream of the yrbE-mce genes (14Shimono N. Morici L. Casali N. Cantrell S. Sidders B. Ehrt S. Riley L.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15918-15923Crossref PubMed Scopus (175) Google Scholar). The latter genes have been referred to as mce-associated (mas) genes (15Casali N. Riley L.W. BMC Genomics. 2007; 8: 60Crossref PubMed Scopus (169) Google Scholar); however, there is already precedent for using Mas to denote mycocerosic acid synthase of M. tuberculosis (19Mathur M. Kolattukudy P.E. J. Biol. Chem. 1992; 267: 19388-19395Abstract Full Text PDF PubMed Google Scholar), so we will simply refer to the latter genes in mce loci as additional mce genes (i.e. mce4HI). We previously found that the mce4 locus of Rhodococcus jostii RHA1 was among a large cluster of genes up-regulated during growth on cholesterol (20van der Geize R. Heuser T. Hara H. Wilbrink M.H. Yam K. Anderton M.C. Sim E. Davies J.E. Dijkhuizen L. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (417) Google Scholar). Moreover, we found that RHA1 mutants with deletions of either the supAB genes (homologous to yrbE4AB genes; Δsup) or the mce4ABCDEF genes (Δmce4AF) lost the ability to grow on cholesterol. These findings led us to hypothesize that the Mce4 system of RHA1 functions in cholesterol uptake. Because the mce4 locus of M. tuberculosis is also clustered with cholesterol metabolism genes homologous to those in RHA1, we hypothesized the same function for the Mce4 systems in both organisms. Consistent with this, Pandey and Sassetti (10Pandey A.K. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4376-4380Crossref PubMed Scopus (722) Google Scholar) recently showed that an M. tuberculosis yrbE4A disruption mutant is impaired in mineralization of C-4 of cholesterol, assimilation of C-26 of cholesterol, and growth with cholesterol as a substrate. In the present study, we used mutational analysis and a direct assay of cholesterol uptake to conclusively determine the role of the Mce4 system of RHA1 in steroid uptake. We also used bioinformatic analysis to assess the role of Mce4 systems in other actinobacteria. Bacterial Growth—R. jostii RHA1 was grown at 30 °C on a shaker on defined Goodies medium (21Bauchop T. Elsden R. J. Gen. Microbiol. 1960; 23: 457-469Crossref PubMed Scopus (578) Google Scholar) plus either 10 mm pyruvate or 2 mm of a steroid substrate. Cholesterol was from Sigma-Aldrich, and other steroids were from Steraloids (Newport, RI). Biomass was estimated on the basis of total protein measured by disrupting cells with hot alkaline lysis and using the BCA protein assay (Pierce) with bovine serum albumin as the standard. PCR—Nucleic acids were extracted, and RNA was purified as described previously (20van der Geize R. Heuser T. Hara H. Wilbrink M.H. Yam K. Anderton M.C. Sim E. Davies J.E. Dijkhuizen L. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (417) Google Scholar). cDNA was synthesized with the SuperScript™ III reverse-transcriptase (Invitrogen) and random hexamers (Invitrogen). Quantitative PCR was done using TaqMan probes (Applied Biosystems), and gene expression differences were calculated as described previously (20van der Geize R. Heuser T. Hara H. Wilbrink M.H. Yam K. Anderton M.C. Sim E. Davies J.E. Dijkhuizen L. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (417) Google Scholar). Primer and probe sequences are in supplemental Table S1. PCR conditions for reverse-transcription PCR (RT-PCR) and RT-quantitative-PCR assays were: 95 °C for 5 min and then 35 cycles of 95 °C for 40 s, 66 °C (PCR 1) or 59 °C (PCR 2–10) for 40 s, 72 °C for 1 min. A final extension at 72 °C was for 7 min before holding at 4 °C. Gene Deletion and Complementation—The mce4H-mce4I-ro04706 genes of R. jostii RHA1 were deleted (Δmce4HI) using the sacB counter selection system essentially as described (22van der Geize R. Hessels G.I. van Gerwen R. van der Meijden P. Dijkhuizen L. FEMS Microbiol. Lett. 2001; 205: 197-202Crossref PubMed Scopus (136) Google Scholar). Sequences of all oligonucleotides used are in supplemental Table S1. Oligonucleotides ro04706-F and ro04706-R were used to amplify and clone a 1.8-kb region downstream of the three genes of RHA1, comprised of the last 13 codons and the stop codon of ro04706, into SmaI-digested pK18mobsacB, yielding pK18-Ro04706. Oligonucleotides ro04704-F and ro04704-R were used to amplify the upstream region of the three genes. The obtained 1.8-kb PCR product, composed of the first 20 codons including the start codon of ro04704, was cloned into pGEM-T. A double digestion with NcoI (blunt-ended with Klenow) and SpeI was performed on the resulting plasmid, and the liberated fragment was cloned into BamHI (blunt-ended with Klenow) and SpeI double-digested pK18-Ro04706, resulting in pK18-Ro04704-06 used for mce4H-mce4I-ro04706 gene deletion. The deletion was verified by PCR using oligonucleotides ro04704-06-F with ro04704-06-R and ro04704contr-F with ro04706contr-R, matching sequences flanking the targeted three genes. The supA-supB gene deletion (Δsup) and the mceH-mceI gene deletion (Δmce4HI) were complemented, resulting in the strains Δsup-C and Δmce4HI-C, respectively, as follows. The supA-supB genes were amplified using primers ro04696-F and ro04797-R. The resulting 1636-bp amplicon was digested with NdeI and HindIII and cloned in pTip-QC2 (23Nakashima N. Tamura T. Biotechnol. Bioeng. 2004; 86: 136-148Crossref PubMed Scopus (68) Google Scholar), yielding pTip-supAB. Similarly, the mce4H and mce4I genes were amplified using primers ro04704ex-F and ro04705ex-R. The resulting 1267-bp amplicon was subcloned, digested with BspHI and HindIII, and ligated into NcoI/HindIII-digested pTip-QC1 (23Nakashima N. Tamura T. Biotechnol. Bioeng. 2004; 86: 136-148Crossref PubMed Scopus (68) Google Scholar), yielding pTip-mce4HI. The pTip-supAB and pTip-mceHI plasmids were introduced into the Δsup strain and the Δmce4HI strain, respectively, by electroporation. A “vector control” strain without the supA-supB genes (ΔsupV) was also produced by electroporating Δsup with pTip-QC2. Cholesterol Uptake Assay—Cholesterol uptake was measured in resting cell suspensions. Cells were grown to mid-log phase on pyruvate, washed twice, and suspended at a cell density of 900 mg of protein/liter (A600 = 13.3) in Goodies buffer. Aliquots of 0.05 ml Goodies medium were placed in 4-ml vials, and [4-14C]cholesterol (53 mCi/mmol, PerkinElmer Life Sciences) was added from an ethanol stock solution. Aliquots of 0.05 ml of cell suspensions were added to the vials for a final cell density of 450 mg of protein/liter. The vials were incubated with shaking for 5 min at room temperature. Cholesterol uptake was stopped by diluting the suspensions with 1.0 ml of ice-cold buffer, collecting the cells on a 0.22-μm Millipore nitrocellulose filter (Fisher Scientific, Mississauga, Ontario), washing the cells with 10 ml of 5! Tween 20 (Fisher) and then with 10 ml of 50! ethanol, and finally, with 10 ml of 5! Tween 20. The filters were placed in Beckman Ready-Safe scintillation mixture (Beckman Coulter) and counted in a Beckman LS-600IC scintillation counter to determine the amount of cholesterol taken up by the cells. The rate of uptake was found to be constant for greater than 5 min, and there was a linear relationship between uptake rate and cell biomass over a cell density range of 69–1375 mg of protein/liter. Where indicated, 60 mm sodium azide, 2.0 mm dicyclohexylcarbodiimide (DCCD), or 10 mm sodium orthovanadate was added to cell suspensions 10 min prior to the addition of the labeled cholesterol. Steady-state kinetic parameters were analyzed using the least squares and dynamic weighting options of LEONORA (24Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, New York1995Google Scholar). The mce4 Locus—The RHA1 mce4 locus has the typical gene arrangement of other mce loci and appears to be an operon (Fig. 1). As reported previously (15Casali N. Riley L.W. BMC Genomics. 2007; 8: 60Crossref PubMed Scopus (169) Google Scholar), orthologous mce4 loci occur in Nocardia farcinica (IFM 10152), Mycobacterium bovis (AF2122/97), Mycobacterium avium subsp. paratuberculosis (k-10), Mycobacterium smegmatis (MC2 155), and M. tuberculosis (H37Rv and other strains). We also found mce4 loci in M. avium (104), M. bovis BCG (Bacille de Calmette et Guèrin), Mycobacterium marinum (M), Mycobacterium ulcerans (Agy99), and Mycobacterium vanbaalenii (PYR-1). The mce4 gene products of RHA1 have the greatest similarity to their respective orthologs in N. farcinica. The sup genes of the RHA1 mce4 locus are orthologs of yrbE4 genes in other mce4 loci. The RHA1 mce4 locus has the conserved configuration of two yrbE (sup) plus eight mce genes found in all known mce4 loci. However, the mce4 locus of RHA1 is unique in that mce4I is followed by two additional genes. The first of these genes, ro04706, is only two nucleotides downstream of mce4I and encodes a hypothetical protein with an N-terminal region similar to the conserved domain of the substrate-binding site of ketosteroid isomerases (Pfam family PF02136). The second of these genes, ro04707, encodes a protein with similarity to the conserved domain of the 3-β-hydroxysteroid dehydrogenase/isomerase family (Pfam family PF01073). Both of these genes appear to have orthologs in all of the above strains, but none of those orthologs is closely linked to mce4 loci. The orthologs in H37Rv are Rv2042c and Rv0139, respectively, which are not closely linked to the large steroid metabolism gene cluster of H37Rv. However, Rv0139 is part of a large regulon that also includes the steroid metabolism genes and mce4 genes of H37Rv (25Kendall S.L. Withers M. Soffair C.N. Moreland N.J. Gurcha S. Sidders B. Frita R. ten Bokum A. Besra G.S. Lott J.S. Stoker N.G. Mol. Microbiol. 2007; 65: 684-699Crossref PubMed Scopus (169) Google Scholar). The four genes upstream of supA (yrbE4A) are conserved in all of the above organisms and putatively encode a 3-ketoacyl-(acyl-carrier-protein) reductase, a ferredoxin and two acyl-CoA dehydrogenases. The fifth gene upstream of supA (yrbE4A) is conserved in all the above organisms, except N. farcinica, and putatively encodes an acyl-CoA synthetase. In RHA1, the 11 genes from supA through ro04706 are closely spaced (Fig. 1), with maximum intergenic regions of 20 bases and with six of the predicted genes overlapping the following ones by four nucleotides. Putative transcriptional promoters were found upstream of hsd4A, supA, and ro04707, but none were found within the 11-gene mce4 cluster, suggesting co-transcription of that cluster. This conclusion was further supported by RT-PCR analysis, which detected transcripts including intergenic regions within this cluster. Seven of the 10 intergenic regions were assayed, and all were detected. Transcripts including the intergenic regions on either side of the 11-gene cluster were also detected, suggesting some level of transcription through those regions. We used expression of the supA gene as a proxy for expression of mce4 locus genes in an RT-quantitative-PCR assay. The supA gene was up-regulated 4.0-fold during growth on cholesterol, relative to during growth on pyruvate. Together, the above results strongly suggest that the 11-gene mce4 cluster is co-transcribed and regulated as an operon. The results do not preclude the existence of additional smaller transcripts within the region nor larger transcripts including additional flanking genes. Like other mce4 loci, that of RHA1 is not proximal to a gene encoding an Mlk family ATPase. However, the RHA1 genome includes two genes, ro01974 and ro02744, encoding ATPases orthologous to MceG, the Mlk ATPase gene of M. tuberculosis. Both of these RHA1 genes are associated with other mce loci of RHA1. One or both of these ATPases may function in the RHA1 Mce4 system. Growth Phenotypes of Mutants—We made an unmarked, inframe deletion mutant of the mce4HI-ro04706 genes (Δmce4HI) in RHA1 (Fig. 1). Like the previous Δsup and Δmce4AF deletion mutants (20van der Geize R. Heuser T. Hara H. Wilbrink M.H. Yam K. Anderton M.C. Sim E. Davies J.E. Dijkhuizen L. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (417) Google Scholar), the Δmce4HI mutant completely lost the ability to grow on cholesterol (Fig. 2A). We constructed two complementation strains to confirm that deleted genes were the cause of the mutant phenotypes. The Δsup-C complementation strain consists of the Δsup mutant with plasmid pTip (23Nakashima N. Tamura T. Biotechnol. Bioeng. 2004; 86: 136-148Crossref PubMed Scopus (68) Google Scholar) containing supAB. The Δsup-C strain partially regained the ability to grow on cholesterol. An “empty vector” control strain (Δsup-V), consisting of the Δsup mutant with plasmid pTip, could not grow on cholesterol. Given this complementation and the complete inability of the Δmce4AF and Δmce4HI deletion mutants to grow on cholesterol, it is clear that the Δsup deletion did not have a polar effect preventing expression of the downstream genes. The unmarked, in-frame Δmce4AF and Δmce4HI deletion mutants are also very unlikely to have polar effects. The Δmce4HI-C complementation strain consists of the Δmce4HI mutant with plasmid pTip (23Nakashima N. Tamura T. Biotechnol. Bioeng. 2004; 86: 136-148Crossref PubMed Scopus (68) Google Scholar) containing mce4HI, but not ro04706. The Δmce4HI-C strain also partially regained the ability to grow on cholesterol. This indicates that the last gene of the mce4 operon, ro04706, is not essential for growth on cholesterol. Overall, the above results demonstrate that each group of genes, supAB, mce4ABCDEF, and mce4HI, encode one or more proteins essential for growth on cholesterol. Cholesterol Uptake—The role of the Mce4 system in cholesterol uptake was directly examined by measuring cholesterol uptake by wild-type RHA1 and the mutant strains. We assayed uptake of 14C-labeled cholesterol by resting cell suspensions. Attempts to use cholesterol-induced cells were unsuccessful as cholesterol used for induction could not be washed from the cells, making it impossible to control the concentration and specific activity of cholesterol in the assay. However, uninduced RHA1 cells grown on pyruvate had measurable cholesterol uptake activity. Uptake followed Michaelis-Menten kinetics and exhibited a low Km (0.6 ± 0.1 μm) for cholesterol (Fig. 3A). Uptake activity was abolished by preincubation of cells with the respiratory inhibitor, sodium azide, or either of two ATPase inhibitors, DCCD and vanadate, indicating that uptake is energy-dependent, and more specifically, ATP-dependent (Fig. 3B). The intended effect of DCCD in RHA1 is supported by previous studies (26Agarwal N. Kalra V.K. Biochim. Biophys. Acta. 1983; 723: 150-159Crossref PubMed Scopus (6) Google Scholar, 27Parrish N.M. Ko C.G. Hughes M.A. Townsend C.A. Dick J.D. J. Antimicrob. Chemother. 2004; 54: 722-729Crossref PubMed Scopus (31) Google Scholar) that have shown that DCCD inhibits proton-driven ATPases of mycobacteria that are phylogenetically related, and physiologically similar, to RHA1. Further, the consistent effect of these two, mechanistically distinct ATPase inhibitors strongly indicates that they indeed inhibited the ATPase. The maximum specific uptake activity for the uninduced cells was 15 ± 2 μm/min/mg of protein, but we predict a higher rate for induced cells. The Δsup, Δmce4AF, and Δmce4HI strains had no measurable cholesterol uptake activity (Fig. 3B). These uptake phenotypes are entirely consistent with the growth phenotypes of the mutants. Moreover, the Δsup-C and Δmce4HI-C complementation strains had wild-type cholesterol uptake activity. These phenotypes are consistent with the growth phenotypes, but it is interesting that complementation is more complete in the uptake assay. The explanation for this difference in degree of complementation may be the use of uninduced cells for the uptake assay. Thus, expression of the supAB and mce4HI genes from pTip may be sufficient for uninduced uptake activity but may not be sufficient for maximal growth on cholesterol. These results clearly indicate that products of the mce4 genes are part of a cholesterol uptake system. Uptake of Other Steroids—Wild-type RHA1 and the three deletion mutants were additionally tested for growth on a range of steroids and related compounds. RHA1 was able to grow on 4-androstene-3,17-dione, 5-α-cholestanol, 5-α-cholestanone, cholic acid, progesterone, and β-sitosterol, but not on cholcalciferol, cholestane, ergocalciferol, ergosterol, estradiol, or hydrocortisone. The Δsup, Δmce4AF, and Δmce4HI mutants also lost the ability to grow on β-sitosterol (Fig. 4), 5-α-cholestanol, and 5-α-cholestanone (not shown). Cholesterol, 5-α-cholestanol, 5-α-cholestanone, and β-sitosterol have very similar structures. The deletion mutants were unaffected in growth on 4-androstene-3,17-dione, cholic acid, and progesterone, which differ notably from the above four compounds in having shorter, polar side chains. These results indicate that the Mce4 system takes up cholesterol, 5-α-cholestanol, 5-α-cholestanone, and β-sitosterol. One or more other systems likely transport 4-androstene-3,17-dione, cholic acid, and progesterone, but we cannot exclude the possibility that the Mce4 system also transports the latter three compounds. Association of mce4 Genes with Steroid Metabolism Genes—To obtain evidence that Mce4 systems are involved in steroid uptake in other bacteria, we searched available genome sequences with mce4 loci for genetically linked steroid metabolism genes. We searched for homologs of the RHA1 kshA gene, hsaADCB, and hsaEGF gene clusters. These genes encode most of the steps involved in degradation of rings A and B of the steroid nucleus to citric acid cycle intermediates. We previously found that, although the gene order varied in the genomes of RHA1 versus H37Rv, these genes were all in the region upstream of the mce4 loci of both genomes, with the hsaADCB and hsaFGE putative operons conserved in both organisms (20van der Geize R. Heuser T. Hara H. Wilbrink M.H. Yam K. Anderton M.C. Sim E. Davies J.E. Dijkhuizen L. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (417) Google Scholar). For the current analysis, we examined the genomes of M. avium (104), M. bovis BCG, M. bovis (A
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