The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria.

BACKGROUND: The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.
RESULTS: Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor.
CONCLUSIONS: Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.

Background

Mycobacterium tuberculosis remains a serious threat to human health and in spite of significant investment in research on this organism, the mechanisms of its pathogenicity are still not clearly understood. One strategy used to determine these mechanisms is to compare the presence and absence of genes in different species (for example, virulent and avirulent) and extrapolate these differences to variation in phenotype. The genomes of M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis and the attenuated M. bovis BCG have been compared in different combinations using a variety of methods (subtractive genomic hybridization [1], bacterial artificial chromosome (BAC) restriction profile analysis [2,3,4,5], BAC arrays [6], DNA microarrays [7] and Southern blotting [8]). This has identified a number of regions of difference (RD) between the various organisms.

One of these regions, designated the RD1 (region of difference 1) deletion region [1], is a 9,505 bp region absent in all M. bovis BCG strains. RD1 is commonly thought to be the primary deletion that occurred during the serial passage of M. bovis by Calmette and Guérin between 1908 and 1921, and is thus thought possibly to be responsible for the primary attenuation of M. bovis to M. bovis BCG [5,7]. Consequently, the genes contained in RD1 have been the object of a number of studies focusing on diagnosis of M. tuberculosis infection, the search for efficient vaccine candidates and virulence [9,10,11,12]. RD1 encompasses the genes Rv3871 to Rv3879c (annotation according to [13]), which include the genes for the 6 kDa early-secreted antigenic target ESAT-6 (esx or esat-6) and L45 homologous protein CFP-10 (lhp) [14,15]. The esat-6 and lhp genes are situated immediately adjacent to each other and encode potent T-cell antigens that are secreted but lack detectable secretion signals [16,17].

During the genome sequencing of M. tuberculosis H37Rv, Cole et al. [13] identified at least 11 additional genes encoding small proteins of approximately 100 amino acids that share sequence similarities with ESAT-6, and grouped them into the esat-6 gene family. In addition, they found several small genes with similarity to lhp (which encodes the protein CFP-10) that are also situated directly adjacent to the esat-6 family genes. Sequence analyses indicated that the lhp family members belong to and extend this esat-6 gene family. It was also found that the lhp gene is co-transcribed and thus forms part of an operon with esat-6 [15].

The genes encoding the originally annotated CFP-10 and ESAT-6 proteins within the RD1 deletion region lie in a cluster of 12 other genes (encompassing the deletion region), which seems to have been duplicated five times in the genome of M. tuberculosis. The duplicated gene clusters have been previously described as the ESAT-6 loci in an analysis of the proteome of M. tuberculosis [18]. An examination of the sets of genes in the clusters reveals that each of the clusters also contains (in addition to a copy of esat-6 and lhp), genes encoding putative ABC transporters (integral inner-membrane proteins), ATP-binding proteins, subtilisin-like membrane-anchored cell-wall-associated serine proteases (the mycosins [19]), and other amino-terminal membrane-associated proteins [18].

We have compared sequences to establish the relationship between the multiple copies of the ESAT-6 gene cluster. Our results show that the ESAT-6 gene cluster is of ancient origin, is present in, and restricted to, the genomes of other members of the high G+C Gram-positive bacteria such as Corynebacterium diphtheriae and Streptomyces coelicolor, and is duplicated multiple times in M. tuberculosis and other mycobacteria. We discuss the conservation of this gene cluster in the context of its possible functional importance and its use in diagnosis of mycobacterial infection.

Results

Individual gene families and genomic organization in M. tuberculosis

The five ESAT-6 gene clusters present in Mycobacterium tuberculosis H37Rv were named region 1 (Rv3866-Rv3883c), 2 (Rv3884c-Rv3895c), 3 (Rv0282-Rv0292), 4 (Rv3444c-Rv3450c) and 5 (Rv1782-Rv1798), consistent with the arbitrary numbering system used previously to classify the five mycosin (subtilisin-like serine protease) genes identified from these regions [19]. Orthologs of the ESAT-6 gene clusters of M. tuberculosis H37Rv could be identified in the genomes of eight other strains and species belonging to the genus Mycobacterium, as well as in two species belonging to other genera (Table 1). Up to 12 different genes representing different gene families were identified in the five gene cluster regions and were designated families A to L according to their position in region 1 (Table 2).

Table 1

Bacterial genome sequencing projects of species and strains containing ESAT-6 gene clusters

	Organism	Strain	Status	Last access date	Last update	Sequencing center(s)	References
1	Mycobacterium tuberculosis	H37Rv	Completed	5-Mar-2001	11-Jun-1998	Sanger Centre/ Pasteur Institute	[13,20,47]
2	M. tuberculosis	CDC1551 (Oshkosh strain or CSU#93)	Completed	5-Mar-2001	28-Jan-1999	TIGR	[48] R.D. Fleischmann et al., unpublished data
3	M. tuberculosis	210	Partial sequencing project completed, no additional sequencing anticipated.	21-May-2001	4-May-2001	TIGR	[49]
4	M. bovis	AF2122/97 (spoligotype 9)	Shotgun in progress	5-Mar-2001	29-Aug-2000	Sanger Centre/ Pasteur Institute	[50]
5	M. bovis BCG	Pasteur 1173P2	Unfinished	-	-	Pasteur Institute	[51]
6	M. leprae	TN	Completed	7-Mar-2001	21-Feb-2001	Sanger Centre/ Pasteur Institute	[25,52,53]
7	M. avium	104	Gap closure finished	6-Mar-2001	22-Feb-2001	TIGR	[49]
8	M. paratuberculosis	K10	Unfinished (6.9 × coverage)	6-Mar-2001	25-Feb-2001	University of Minnesota	[29]
9	M. smegmatis	MC2 155	Shotgun completed, assembly	6-Mar-2001	22-Feb-2001	TIGR	[49]
10	Corynebacterium diphtheriae	NCTC13129	Finishing/gap closure	5-Mar-2001	26-Feb-2001	Sanger Centre	[54]
11	Streptomyces coelicolor	A3(2)	Cosmid sequencing	5-Mar-2001	1-Mar-2001	Sanger Centre	[55]

Table 2

Presence of genes in gene clusters of all available finished and unfinished genome sequences

				Presence and names of genes in each species
Gene family	Description	Protein size (in M. tb)	ESAT-6 cluster region	M. tuberculosis H37Rv	M. tuberculosis CDC1551 (CSU#93)	M. tuberculosis* 210	M. bovis* AF2122/97 (spoligotype 9)	M. bovis* BCG Pasteur 1173P2
A	ABC transporter family signature, 19-27% homology	283	1	Rv3866	MT3980	ND	MB851A	No sequence data
		276	2	Rv3889c	MT4004	MTB12A	MB727.3A (partly deleted #)	No sequence data
		295	3	Rv0289	MT0302	MTB203A	MB548A	No sequence data
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication
		300	5	Rv1794	MT1843	MTB196A	MB557A	No sequence data
B	AAA+ class ATPases, CBXX/CFQX family, SpoVK, 1× ATP/GTP-binding site, 29-39% homology	573	1	Rv3868	MT3981	MTB44B	MB851B	No sequence data
		619	2	Rv3884c	MT3999	MTB12B	MB727.1B	No sequence data
		631	3	Rv0282	MT0295	MTB23B	MB672B	No sequence data
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication
		610	5	Rv1798	MT1847	MTB196B	MB542B	No sequence data
C	Amino-terminal transmembrane protein, possible ATP/GTP-binding motif, 31-41% homology	480	1	Rv3869	MT3982	MTB44C	MB851C	No sequence data
		495	2	Rv3895c	MT4011	MTB136C	MB780.1C	No sequence data
		538	3	Rv0283	MT0296	MTB23C	MB672C	No sequence data
		470	4	Rv3450c	MT3556	MTB45C	MB493.1C	No sequence data
		506	5	Rv1782	MT1832	MTB46C	MB771.1C	No sequence data
D	DNA segregation ATPase, ftsK chromosome partitioning protein, SpoIIIE, yukA, 3× ATP/GTP-binding sites, 2× amino-terminal transmembrane protein, 28-39% homology	747 + 591	1	Rv3870+71	MT3983+85	MTB44Da+Db	MB851D	MB851D (partly deleted)
		1396	2	Rv3894c	MT4010	MTB3D	MB780.1D	No sequence data
		1330	3	Rv0284	MT0297	MTB23D	MB672D	No sequence data
		1236	4	Rv3447c	MT3553	MTB45D	MB585.1D	No sequence data
		435 + 932	5	Rv1783+84	MT1833	MTB46Da+Db	MB771.1D	No sequence data
E	PE, 18-90% homology	99	1	Rv3872	MT3986	MTB44E	MB851E	Deleted
		77	2	Rv3893c	MT4008	MTB3E	MB780.1E	No sequence data
		102	3	Rv0285	MT0298	MTB23E	MB389E	No sequence data
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication
		99 & 99	5	Rv1788 & 91	MT1837 & 40	MTB196Ea & Eb	MB771.0E & MB557E	No sequence data
F	PPE, 19-88% homology	368	1	Rv3873	MT3987	MTB44F	MB851F	Deleted
		399	2	Rv3892c	MT4007	MTB3F	MB780.1F	No sequence data
		513	3	Rv0286	MT0299	MTB472F	MB528F	No sequence data
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication
		365, 393 & 350		5	Rv1787 & 89 & 90	MT1836 & 38 & 39	MTB196Fa & Fb & Fc	MB771.0Fa & Fb & MB557F	No sequence data
G	lhp or CFP-10, also MTSA-10, grouped into ESAT-6 family, potent secreted T-cell antigens, 9-32% homology	100	1	Rv3874	MT3988	MTB44G	MB851G	Deleted
		107	2	Rv3891c	MT4006	MTB12G	MB727.3G	No sequence data
		97	3	Rv0287	MT0300	MTB472G	MB548G	No sequence data
		125	4	Rv3445c	MT3550	MTB45G	MB585.0G	No sequence data
		98	5	Rv1792 (Stop)	MT1841 (Stop)	MTB196G (Stop)	MB557G	No sequence data
H	ESAT-6 family, cfp7, L45 or l-esat, also Mtb9.9 family, potent secreted T-cell antigens, 15-27% homology	95	1	Rv3875	MT3989	MTB44H	MB851H †	Deleted
		95	2	Rv3890c	MT4005	MTB12H	MB727.3H	No sequence data
		96	3	Rv0288	MT0301	MTB203H	MB548H	No sequence data
		100	4	Rv3444c	MT3549	MTB45H	MB585.0H	No sequence data
		94	5	Rv1793	MT1842	MTB196H	MB557H	No sequence data
I	ATPases involved in chromosome partitioning, 1× ATP/GTP-binding motif, -33% homology-	666	1	Rv3876	MT3990	MTB60I	MB477I	Deleted
		341	2	Rv3888c	MT4003	MTB12I	Deleted #	No sequence data
		-	3	No duplication	No duplication	No duplication	No duplication	No duplication
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication
		-	5	No duplication	No duplication	No duplication	No duplication	No duplication
J	Integral inner membrane protein, binding-protein-dependent transport systems inner membrane component signature, putative transporter protein, 19-27% homology	511	1	Rv3877	MT3991	MTB369J	MB477J	Deleted
		509	2	Rv3887c	MT4002	MTB12J	MB727.3J (partly deleted #)	No sequence data
		472	3	Rv0290	MT0303	MTB203J	MB548J	No sequence data
		467	4	Rv3448	MT3554	MTB45J	MB585.1J	No sequence data
		503	5	Rv1795	MT1844	MTB196J	MB506J	No sequence data
K	Mycosins, subtilisin-like cell-wall associated serine proteases, 43-49% homology	446	1	Rv3883c	MT3998	MTB12Ka	MB727.0K	No sequence data
		550	2	Rv3886c	MT4001(Frame)	MTB12Kb	MB727.2K	No sequence data
		461	3	Rv0291	MT0304	MTB203K	MB548K	No sequence data
		455	4	Rv3449	MT3555	MTB45K	MB585.1K	No sequence data
		585	5	Rv1796	MT1845	MTB196K	MB506K	No sequence data
L	2× amino-terminal transmembrane protein, 16-27% homology	462	1	Rv3882c	MT3997	MTB12La	MB727.0L	No sequence data
		537	2	Rv3885c	MT4000 (Frame)	MTB12Lb	MB727.2L	No sequence data
		331	3	Rv0292	MT0305	MTB203L	MB694.0L	No sequence data
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication
		406	5	Rv1797	MT1846	MTB196L	MB542L	No sequence data
				Presence and names of genes in each species
Gene family	Description	Protein size (in M. tb)	ESAT-6 cluster region	M. leprae TN	M. avium* 104	M. paratuberculosis K 10	M. smegmatis* MC2 155	C. diphtheriae* NCTC13129	S. coelicolor A3 (2)
A	ABC transporter family signature, 19-27% homology	283	1	ML0057(pseudo)	ND	ND	MS29A	ND	ND
		276	2	MLabc (pseudo)‡	MA138A	MP3889c	ND	ND	ND
		295	3	ML2530	MA141A	MP0289	MS32A	ND	ND
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
		300	5	ML1540	MA310A	MP1794	ND	ND	ND
B	AAA+ class ATPases, CBXX/CFQX family, SpoVK, 1x ATP/GTP binding site, 29-39% homology	573	1	ML0055	ND	ND	MS29B	ND	ND
		619	2	ML0039(pseudo)	MA177B	MP3884c	ND	ND	ND
		631	3	ML2537	MA78B	MP0282	MS32B	ND	ND
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
		610	5	ML1536	MA310B	MP1798	ND	ND	ND
C	Amino-terminal transmembrane protein, possible ATP/GTP- binding motif, 31-41% homology	480	1	ML0054	ND	ND	MS29C	ND	ND
		495	2	Deleted	MA144C	MP3895c	ND	ND	ND
		538	3	ML2536	MA78C	MP0283	MS32C	ND	ND
		470	4	Deleted	MA94C	MP3450c	MS8C	CORDmem	SC3C3.07
		506	5	ML1544	MA221C	MP1782	ND	ND	ND
D	DNA segregation ATPase, ftsK chromosome partitioning protein, SpoIIIE, yukA, 3× ATP/GTP-binding sites 2 × amino-terminal transmembrane protein, 28-39% homology	747+591	1	ML0053+52	ND	ND	MS29D (Stop$)	ND	ND
		1396	2	Deleted	MA144D	MP3894c	ND	ND	ND
		1330	3	ML2535	MA78D	MP0284	MS32D	ND	ND
		1236	4	Deleted	MA504D	MP3447c	MS8D	CORDyuk	SC3C3.20c
		435+932	5	ML1543	MA221D	MP1783	ND	ND	ND
E	PE, 18-90% homology	99	1	Deleted	ND	ND	MS29E	ND	ND
		77	2	Deleted	MA138E	MP3893c	ND	ND	ND
		102	3	ML2534	MA78E	MP0285	MS32E	ND	ND
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication	No
		99 & 99	5	Deleted	MA310Ea & Eb	MP1788 & 91	ND	ND	ND
F	PPE, 19-88% homology	368	1	ML0051	ND	ND	MS29F	ND	ND
		399	2	Deleted	MA138F	MP3892c	ND	ND	ND
		513	3	ML2533 (pseudo)	MA78F	MP0286	MS32F	ND	ND
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
		365, 393 & 350		5	Deleted	MA310Fa & Fb & Fc	MP1787 & 89 & 90	ND	ND	ND
G	lhp or CFP-10, also MTSA-10, grouped ESAT-6 family, potent secreted T-cell antigens, 9-32% homology	100	1	ML0050	ND	ND	MS29G	ND	SC3C3.10 and SC3C3.11(c)
		107	2	Deleted	MA138G	MP3891c §	ND	ND	ND
		97	3	ML2532	MA141G	MP0287	MS32G	ND	ND
		125	4	Deleted	MA319G	MP3445c	MS8G	CORDcfp10	ND
		98	5	MLcfp (pseudo)‡	MA310G	MP1792	ND	ND	ND
H	ESAT-6 family, cfp7, L45 or l-esat, also Mtb9.9 family, potent secreted T-cell antigens, 15-27% homology	95	1	ML0049	ND	ND	MS29H	ND	SC3C3.10 and SC3C3.11¶
		95	2	ML0034 (pseudo)	MA138H	MP3890c §	ND	ND	ND
		96	3	ML2531	MA141H	MP0288	MS32H	ND	ND
		100	4	ML0363	MA319H	MP3444c	MS8H	CORDesat6	ND
		94	5	MLesat (pseudo)‡	MA310H	MP1793	ND	ND	ND
I	ATPases involved in chromosome partitioning, 1x ATP/GTP-binding motif, 33% homology	666	1	ML0048	ND	ND	MS29I	ND	SC3C3.03c
		341	2	ML0035 (pseudo)	MA138I	MP3888c	ND	ND	ND
		-	3	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
		-	5	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
J	Integral inner membrane protein, binding-protein-dependent transport systems inner membrane component signature, putative transporter protein, 19-27% homology	511	1	ML0047	ND	ND	MS29J	ND	ND
		509	2	ML0036 (pseudo)	MA138J	MP3887c	ND	ND	ND
		472	3	ML2529	MA141J	MP0290	MS32J	ND	ND
		467	4	Deleted	MA504J	MP3448	MS8J	CORDtransporter	SC3C3.21
		503	5	ML1539	MA310J	MP1795	ND	ND	ND
K	Mycosins, subtilisin-like cell-wall associated serine proteases, 43-49% homology	446	1	ML0041	ND	ND	MS65K	ND	ND
		550	2	ML0037 (pseudo)	MA177K	MP3886c	ND	ND	ND
		461	3	ML2528	MA141K	MP0291	MS32K	ND	ND
		455	4	Deleted	MA439K	MP3449	MS8K	CORDsub	SC3C3.17c and SC3C3.08
		585	5	ML1538	MA310K	MP1796	ND	ND	ND
L	2× amino-terminal transmembrane protein, 16-27% homology	462	1	ML0042	ND	ND	MS65L	ND	ND
		537	2	ML0038 (pseudo)	MA177L	MP3885c	ND	ND	ND
		331	3	ML2527	MA81L	MP0292	MS32L	ND	ND
		-	4	No duplication	No duplication	No duplication	No duplication	No duplication	No duplication
		406	5	ML1537	MA310L	MP1797	ND	ND	ND
Other region-specific genes of known functions (not assigned to a family)
Region 5 (not present in M. smegmatis, C. diphtheriae and S. coelicolor)			Rv1785c	Probable member of the cytochrome P450 family (pseudogene in M. leprae)
			Rv1786	Probable ferredoxin (pseudogene in M. leprae)
Other region-specific genes of unknown functions (not assigned to a family)
Region 1(deleted in M. avium and M. paratuberculosis, not present in C. diphtheriae and S. coelicolor)			Rv3867	Unknown, annotated as part of MT3980 (Rv3866) in M. tuberculosis CDC1551 sequence with a frameshift (functional in M. leprae)
			Rv3878	Unknown, some similarity to PPE family, deleted with RD1 deletion region in M. bovis BCG (pseudogene in M. leprae)
			Rv3879c	Unknown, repetitive, highly proline-rich N-terminus, deleted with RD1 deletion region in M. bovis BCG (pseudogene in M. leprae)
			Rv3880c	Unknown (functional in M. leprae)
			Rv3881c	Unknown (pseudogene in M. leprae)
Region 4 (not present in S. coelicolor)			Rv3446c	Unknown, may contain a possible ABC transporter signature (deleted in M. leprae)

*Names of genes of these organisms were given arbitrarily by the authors of this paper. †Gene not identified by BLAST, data obtained from [1], GenBank accession no. U34848 and AAC44033. ‡The gene is present in the sequence, but not annotated (name given arbitrarily by authors of this paper). §Genes identified by BLAST as well as data obtained from GenBank, accession no. AJ250015. ¶Orthologs in S. coelicolor are equally similar to family G and H. ND, Not detected - not necessarily absent from genome but possibly not detected because of unfinished sequencing process. No duplication, no duplication of this gene is present in this region. No sequence data, no sequence data is available for this organism, published deletion information is included ([1] and others). Deleted, deleted from the genome of this particular species or strain (# = deleted in only some strains of this species). Frame, frameshift. Stop, in-frame stop codon. Stop$, stop codon corresponds to stop codon in M. tuberculosis H37Rv, which splits gene into Rv3870 and Rv3871. Pseudo, confirmed pseudogene due to multiple frameshifts and stop codons.

Figure 1 shows a schematic representation of the genomic organization of the gene families present in each of the five ESAT-6 cluster regions of M. tuberculosis. Annotations and descriptions of single genes in these regions can be found at [20]. Regions 1 and 2 are situated directly adjacent to each other in the genome and are transcribed in opposite directions. In both regions 1 and 5 the large gene belonging to family D (encoding the ATPase protein) has been disrupted by an insertion (Figure 1). This insertion has resulted in an in-frame stop codon, giving rise to two smaller genes (containing all the motifs of the larger homolog) located directly adjacent to each other. The gene positions of members of families C, D, G and H are maintained in all five regions (see Figure 1), whereas most of the families that are not present in region 4 seem to be more flexible with regard to their position within the gene clusters (families A, B, I and L). There are also genes present within some of the ESAT-6 gene clusters that do not have any homologs in the other clusters, suggesting subsequent insertions or deletions from the ancestral region (indicated by black arrows in Figure 1, see also Table 2).

Figure 1

Schematic representation of the genomic organization of the genes present in the five ESAT-6 gene cluster regions of Mycobacterium tuberculosis H37Rv as well as the regions in C. diphtheriae and S. coelicolor. ORFs are represented as blocked arrows showing the direction of transcription, with the different colors reflecting the specific gene family and the length of the arrow reflecting the relative lengths of the genes. Annotations of M. tuberculosis H37Rv genes are according to Cole et al. [13]. Black arrows indicate unconserved genes present in these regions. Gaps between genes do not represent physical gaps between genes on the genome, but have been inserted to aid in indicating conservation among gene positions. Gene families were named arbitrarily according to their position in M. tuberculosis H37Rv region 1. The regions were named after the numbering system of Brown et al. [19] used arbitrarily for the five mycosin (subtilisin-like serine protease) genes identified from these regions (family K). M. tuberculosis regions are shown in order of suggested duplication events (see phylogenetic results) and not by numbering. The results of the analyses of the primary features of these genes and their corresponding proteins are included in a short summary at the bottom of the figure (see also Table 2).

The esat-6/lhp operon is not only present in the ESAT-6 gene clusters, but is distributed as six additional copies in the genome of M. tuberculosis (Figure 2). In four cases, the esat-6/lhp operon is flanked by ppe and pe genes (encoding proteins that have proline-proline-glutamic acid (PPE) and proline-glutamic acid (PE) motifs, respectively), indicating possible linked duplication between the esat-6/lhp operon and the pe/ppe gene pair.

Figure 2

Schematic representation of the six additional esat-6/lhp operon duplications and the regions that surround them in the genome of M. tuberculosis H37Rv. ORFs are represented by blocked arrows indicating direction of transcription, with the different colors reflecting the specific gene family and the length of the arrow reflecting the relative lengths of the genes as in Figure 1. The esat-6 and lhp genes deleted in M. bovis RD07 and RD09 deletion regions [7] are indicated.

ESAT-6 gene cluster identification in other mycobacteria

Table 2 presents the results of the similarity searches and all available data for the 12 identified gene families present in the different regions. All the mycobacteria currently being sequenced contain multiple copies of these regions in their genomes. As these different copies are also found in the same respective genomic locations (corresponding flanking genes) in all the mycobacteria, it indicates that the duplication events took place prior to the divergence of the different species.

M. tuberculosis CDC1551, M. tuberculosis 210 and M. bovis

The genomes of the M. tuberculosis CDC1551 and 210 clinical strains as well as the genome of M. bovis contain all five of the ESAT-6 gene cluster regions present in the genome of M. tuberculosis H37Rv (sharing between 99 and 100% similarity to M. tuberculosis H37Rv at protein level). It is interesting to note, however, that two of the genes present in region 2 in CDC1551 (MT4000 and MT4001) contain frameshifts in their sequences, indicating that they and the rest of the region may no longer be functional in CDC1551. Part of region 2 (a 2,405 bp fragment containing Rv3887c, Rv3888c and Rv3889c) is also deleted in certain strains of M. bovis only, including the strain AF2122/97 that is currently being sequenced [21]. An in-frame stop codon found in Rv1792 (family G) is also present in the orthologs in CDC1551 (MT1841) and strain 210 (MTB196G), indicating that the mutation may have taken place before divergence of the three strains. Two of the H37Rv genes as well as the strain 210 family D genes (in regions 1 and 5) have acquired in-frame stop codons, resulting in two genes lying adjacent to each other, whereas the family D Rv1783 and Rv1784 orthologs in CDC1551 are still one intact gene (MT1833). The orthologs of this gene in M. bovis (MB771.1D), M. leprae (ML1543), M. avium (MA221D), and M. paratuberculosis(MP1783) are also intact, implying that the mutation in the H37Rv and strain 210 orthologs must have occurred after divergence of the three M. tuberculosis strains.

M. leprae

Figure 3 shows a schematic representation of the genomic organization of the respective gene families present in each of the five ESAT-6 gene clusters of M. leprae. The genome sequence of M. leprae contains functional copies of two of the five ESAT-6 gene cluster regions (regions 1 and 3, which have between 50 and 70% similarity to M. tuberculosis H37Rv at protein level). Most of the genes from region 2 are deleted, and all the remaining genes in this region have become pseudogenes as a result of extensive point mutations. This is in contrast to the genes from region 1 (which lies directly adjacent to region 2), which contains no pseudogenes. It is thus conceivable that these clusters should function as a unit, and that genes could become non-functional when part of the unit is disrupted. Furthermore, all the genes immediately flanking the putative functional regions, as well as five of the eight genes only present in one of the regions as depicted in Table 2 (the Rv1785c, Rv1786, Rv3878, Rv3879c and Rv3881c orthologs ML1542, ML1541, ML0046, ML0045 and ML0043), are probable pseudogenes, indicating that the genes present in the functional clusters are being maintained as a unit.

Figure 3

Schematic representation of the genomic organization of the genes present in the five ESAT-6 gene cluster regions of Mycobacterium leprae. ORF's are represented as blocked arrows showing the direction of transcription, with the different colors reflecting the specific gene family and the length of the arrow reflecting the relative lengths of the genes as in Figure 1. Black arrows indicate unconserved genes present in these regions, while open arrows indicate pseudogenes. Annotations of M. leprae genes are according to Cole et al. [25].

M. avium and M. paratuberculosis

The genomes of the M. avium strain 104 and the closely related species M. paratuberculosis (or M. avium subsp. paratuberculosis) has revealed four of the five ESAT-6 gene cluster regions (sharing between 65 and 75% similarity to M. tuberculosis H37Rv at protein level), with region 1 being absent in both species (Figure 4). Closer inspection of the gene sequence surrounding region 1 in both these species has revealed a deletion of the region containing region 1 and some upstream flanking genes (from the Rv3861 gene ortholog up to and including the Rv3883c ortholog). This deletion coincided with the insertion of a ± 2,292 bp sequence containing the genes for a putative hydroxylase (± 818 bp) and the sigI sigma factor (± 824 bp). The presence of this sequence in both genomes (99% DNA sequence identity) indicates that the insertion/deletion may have occurred before the divergence of the two species. The genes from the remaining ESAT-6 gene cluster regions that are present in M. avium and M. paratuberculosis contain no stop codons or frameshifts and thus appear to be functional.

Figure 4

Schematic representation of the genomic organization of the genes present in the four ESAT-6 gene cluster regions of Mycobacterium avium and Mycobacterium paratuberculosis, as well as the flanking genes of the region 1 deletion. ORFs are represented as blocked arrows showing the direction of transcription, with the different colors reflecting the specific gene family and the length of the arrow reflecting the relative lengths of the genes as in Figure 1. Black arrows indicate unconserved genes present in these regions. M. avium and M. paratuberculosis genes were arbitrarily annotated by the authors of this paper.

M. smegmatis

The genome sequence of the avirulent, fast-growing mycobacterial species M. smegmatis contains three of the five ESAT-6 gene cluster regions, namely regions 1, 3 and 4 (sharing between 60 and 75% similarity to M. tuberculosis H37Rv at protein level), with regions 2 and 5 being absent (Figure 5). No deletions, frameshifts or stop codons were identified in any of the genes present in the regions 1, 3 and 4 and therefore it is concluded that these regions are functional.

Figure 5

Schematic representation of the genomic organization of the genes present in the three ESAT-6 gene cluster regions of Mycobacterium smegmatis. ORFs are represented as blocked arrows showing the direction of transcription, with the different colors reflecting the specific gene family and the length of the arrow reflecting the relative lengths of the genes as in Figure 1. Black arrows indicate unconserved genes present in these regions. M. smegmatis genes were arbitrarily annotated by the authors of this paper.

ESAT-6 gene cluster identification in bacteria other than the mycobacteria

Corynebacterium diphtheriae

The genome sequence of the closely related C. diphtheriae has revealed a copy of the region 4 ESAT-6 gene cluster (Figure 1, see Table 3 for percentage similarity between sequences), situated in the same genomic location as in the mycobacteria (indicated by the large stretch of flanking genes homologous to the genes flanking region 4 in M. tuberculosis H37Rv). All the genes present within this cluster appear to be fully functional, as no deletions, stop codons or frameshifts were identified. No duplications of the gene cluster could be detected in the genome of this organism.

Table 3

Similarity of M. tuberculosis H37Rv region 4-encoded proteins to proteins encoded by the C. diphtheriae and S. coelicolor regions

		Percentage similarity
M. tuberculosis region 4 proteins	Family
		C. diphtheriae	S. coelicolor
Rv3450c	C	47%	36%
Rv3447c	D	53%	57%
Rv3445c	G	47%	47 and 51%*
Rv3444c	H	58%	41 and 44%*
Rv3448	J	33%	45%
Rv3449	K	49%	45 and 47%

* Orthologs in S. coelicolor are equally similar to families G and H.

Streptomyces coelicolor

The S. coelicolor genome has revealed distinct orthologs for four of the six most conserved genes from the ESAT-6 gene cluster regions located in close proximity to each other (Figure 1). These genes show the highest similarity to the corresponding orthologs in region 4 of M. tuberculosis (see Table 3 for percentage similarity between sequences). There is also a very distinct ortholog (SC3C3.03c) of the region 1 family I gene (Rv3876) in the S. coelicolor region. There is no homolog for this gene in region 4 of M. tuberculosis. A sequence-similarity search using the sequences of the other two proteins encoded in region 4, namely ESAT-6 (Rv3444c) and CFP-10 (Rv3445c), has also revealed some similarity to two small genes situated within the same region in the genome of S. coelicolor (Table 3, Figure 1). These genes (SC3C3.10 and SC3C3.11) encode small proteins (124 and 103 amino acids) of unknown function, are very similar to each other, and lie adjacent to each other, similar to the observation for the esat-6/lhp operon. The sequences of both these proteins also contain the motif W-X-G, a feature present in most of the ESAT-6 and CFP-10 proteins. The higher degree of similarity between the genes from region 4 of the mycobacteria (and C. diphtheriae) and those present in the region in S. coelicolor suggests that region 4 may be the ancestral region in the mycobacteria, although a number of differences between these regions do exist.

Taxonomy

It is evident from the taxonomy (Figure 6) of the different species of bacteria in which copies of the ESAT-6 gene clusters could be found, that the presence of these clusters appears to be a specific characteristic of the high G+C Gram-positive Actinobacteria, and that multiple copies thereof are only found in the mycobacteria. No copies of the clusters could be found in the completed genome sequenceof Bacillus subtilus and that of other related species, which also form part of the Firmicutes (Gram-positive bacteria), but fall under the Bacillus/Clostridium group (low G+C Gram-positive bacteria). No copies of these clusters could be found in the genomes of any other bacteria or organism outside of the Firmicutes and thus the ESAT-6 gene clusters appear to be unique to the Actinobacteria.

Figure 6

Taxonomic position of the bacterial species that have the ESAT-6 gene clusters present in their genomes. This indicates that the ESAT-6 gene clusters seem to be a feature of only the high G+C Gram-positive bacteria (Actinobacteria) and that the presence of multiple copies of the gene clusters seems to be a characteristic only found in the mycobacteria. Phylogenetic relationships of members of the genus Mycobacterium indicated are based on 16S rRNA gene sequence information [56].

Phylogeny of the ESAT-6 gene cluster

To calculate the phylogenetic relationships between the five duplicated ESAT-6 gene cluster regions in M. tuberculosis and to identify the ancestral region, detailed phylogenetic analyses were performed on each of the six protein families present in all five of these regions (families C, D, G, H, J and K). Figure 7a shows a neighbor-joining tree of the protein sequences of the ATP/GTP-binding protein family (family D) from the ESAT-6 gene clusters of mycobacteria and C. diphtheriae, with the protein ortholog from S. coelicolor as the outgroup. This tree is representative of all six trees that were drawn using the six families (data for the other trees are not shown). To confirm the results obtained with the S. coelicolor orthologs as outgroups, the same analyses were done using the C. diphtheriae orthologs as outgroups, with comparable results (data not shown). This tree topology was not due to systematic error, as trees drawn using the FITCH algorithm gave the same results (data not shown). To confirm the basic structure of the trees and to verify that this structure is not influenced by the choice of outgroup, unrooted trees without any outgroup were constructed using the KITSCH algorithm, once again with comparable results (data not shown). To further verify the relationships among these clusters, the conserved sequences of all six proteins from M. tuberculosis were combined into one protein sequence and the same analysis performed (Figure 7b).

Figure 7

Phylogenetic trees showing the relationships between the five duplicated gene cluster regions. (a) Neighbor-joining phylogenetic tree of all available protein sequences of the ATP/GTP-binding protein family (family D in Table 2) with the protein ortholog of Streptomyces coelicolor as the outgroup. This tree is representative of all the trees drawn using the six most conserved proteins in these regions as well as using the protein ortholog of Corynebacterium diphtheriae as the outgroup. (b) Neighbor-joining phylogenetic tree of all six conserved proteins from the M. tuberculosis gene clusters combined into one protein per region. The combined protein of C. diphtheriae was used as the outgroup. (c) Neighbor-joining phylogenetic tree of the ESAT-6 and CFP-10 protein families combined (family G and H), using the combined protein of C. diphtheriae as the outgroup.

To investigate whether the non-conserved protein families (those that are not present in region 4 of the mycobacteria, C. diphtheriae or S. coelicolor) show the same basic phylogenetic relationships as the conserved families (present in all five regions), an analysis was done on the AAA+ class ATPase family (family B). This family does not have a homolog in region 4 and there is also no C. diphtheriae or S. coelicolor ortholog to use as outgroup. The tree constructed from the data from this family clearly showed once again that regions 2 and 5, and region 1 and 3, respectively, are phylogenetically closer to each other (data not shown).

Neighbor joining, FITCH, KITSCH and concatenated sequence comparison analyses all supported a single phylogeny that indicated that region 4 seems to be the most ancient of the mycobacterial ESAT-6 gene cluster regions. Region 4 is also the closest region to the S. coelicolor and C. diphtheriae regions. The order of duplication seems to extend from region 4, through 1 and 3 to regions 2 and 5. The phylogenetic relationships between corresponding clusters in the different mycobacteria are maintained throughout the different protein-family trees, and agree with the proposed phylogenetic order (or taxonomic position) of the mycobacterial species according to 16S rRNA data (see Figure 6).

As the genome of M. tuberculosis contains 11 copies of the esat-6/lhp gene pair that appears to be duplicated together, phylogenetic trees were constructed using the ESAT-6 or CFP-10 proteins separately (data not shown), or in combination as one ESAT-6/CFP-10 protein (Figure 7c). Using the combined C. diphtheriae ESAT-6/CFP-10 ortholog protein as outgroup, the same organization of duplication events was obtained with regions 1, 3, 2 and lastly 5 being duplicated from the ancient region 4. The other copies of the esat-6/lhp operon pair that are present in the M. tuberculosis genome sequence, but are not part of the ESAT-6 gene cluster regions, seem to have arisen from singular duplication events originating from different cluster regions. It is interesting to note that esat-6 and lhp from region 5 seem to be highly prone to duplication, as there are four additional copies of these two genes present in the genome, compared to just one additional copy originating from region 4 and region 3, respectively. These four gene duplicates of esat-6 and lhp from region 5 are also nearly identical (93-100% similarity at protein level), indicating their recent duplication.

Discussion

It was recently estimated in an in silico analysis of the genome sequence of M. tuberculosis H37Rv, that 52% of the proteome has been derived from gene duplication events [18]. One such involves the formation of multiple copies of the genes for the secreted T-cell antigens ESAT-6 and CFP-10 [14,16,17] together with a number of associated genes. A total of twelve gene families were identified in five regions (which were termed the ESAT-6 loci).

Phylogenetic analyses of the protein sequences of the six most conserved gene families, present within the five regions, predict that region 4 (Rv3444c to Rv3450c) is the ancestral region. Region 4 also contains the least number of proteins (only 6 compared to the 12 of region 1 (Rv3866-3883c) and region 2 (Rv3884c-3895c)), and does not contain the genes for PE and PPE, which may have been inserted into this region after the first duplication. Phylogenetic analyses using different methods and protein family data also suggests that subsequent duplications took place in the following order: region 1 (Rv3866-3883c) → 3 (Rv0282-0292) → 2 (Rv3884c-3895c) → 5 (Rv1782-1798). Furthermore, these analyses support the taxonomic order observed for the mycobacteria, with M. smegmatis being taxonomically the farthest removed from M. tuberculosis. The presence of a copy of region 4 and its flanking genes in C. diphtheriae strengthens the taxonomic data that implies that the corynebacteria and mycobacteria have a common ancestor. It appears that C. diphtheriae diverged from the mycobacteria before the multiple duplications of the ESAT-6 gene cluster, as only one copy of this cluster could be identified in the genome of this organism.

The loss of region 1 from the genomes of the species M. avium and M. paratuberculosis (belonging to the M. avium complex) is confirmed by clinical data showing that patients seronegative for the human immunodeficiency virus (HIV) and infected with mycobacteria belonging to the M. avium complex do not respond to ESAT-6 from region 1, but do recognize purified protein derivative (PPD) and M. avium sensitins [22]. The genes for ESAT-6 and CFP-10 (esat-6 and lhp) in region 1 are also not found in M. bovis BCG and have thus been the focus of recent research because of their application as diagnostic markers to differentiate between BCG vaccination and M. tuberculosis, M. bovis or M. avium infection (see for example [17,23]). In this study we have found several copies of the ESAT-6 and CFP-10 genes (with differing degrees of similarity) in the genomes of different mycobacteria (80% and 71% protein sequence similarity for ESAT-6 and CFP-10 respectively from region 1 in avirulent M. smegmatis), as well as orthologs in species outside the mycobacteria; care should therefore be taken when using these proteins for diagnostic purposes. It will be important to look at the protein sequence similarity between the copies of ESAT-6 and CFP-10 of different virulent and environmental mycobacterial species before a member of these immunodominant protein families can be chosen as a definite marker of M. tuberculosis infection. Studies to determine the production of interferon-γ in response to exposure to ESAT-6 and CFP-10 from environmental mycobacteria (for example M. smegmatis) by peripheral blood mononuclear cells from infected patients have not been done. Until these results are available, indicating that the T-cell responses against these proteins are not comparable to those against the M. tuberculosis proteins, care should be taken with claims regarding the potential diagnostic value of these antigens.

Most of the sequences of the genes belonging to the ESAT-6 gene cluster regions contain no stop codons or frameshifts and thus appear to be functional. This is significant when placed in the context of a bacterium such as M. leprae, as it is hypothesized that the genome of M. leprae may contain the minimal gene set required by a pathogenic mycobacterium [5,24,25] and that the activities of some functional genes once present in the genome of M. leprae have been silenced (they became pseudogenes through multiple stop codon mutations and frameshifts) because they are no longer needed for the bacterium's intracellular survival [13]. It appears that M. leprae contains at least two functional copies of the ESAT-6 gene cluster in its genome (regions 1 and 3). The M. leprae ESAT-6 copy from region 1 (the L45-antigen or L-ESAT antigen from clone L45) was shown to be strongly reactive to sera from leprosy patients [26], providing experimental evidence that at least one of the cluster regions is functional in M. leprae.

As most of the genes present within the ESAT-6 gene cluster regions encode proteins that are predicted to be associated with transport and energy-providing systems, we hypothesize that these proteins may be involved in the secretion of a substrate across the mycobacterial cell wall. It is well known that the T-cell antigens ESAT-6 and CFP-10 are found in short-term culture filtrates (ST-CF) of M. tuberculosis, although the mechanism of secretion is unknown, as these proteins do not possess any of the usual Sec-dependent secretion signals [14,15,16]. It is therefore possible that the genes in the ESAT-6 gene cluster regions act together to provide a system for the secretion of ESAT-6 and CFP-10. There is evidence for the processing of the TB10.4 protein (the ESAT-6 family member from region 3) to a lower molecular weight product [27], suggesting a possible role for the cell-wall-associated mycosin proteases [19] in the suggested transport system. Most of region 1 is situated in the RD1 deletion region of M. bovis BCG, possibly explaining the absence of expression of the mycosin-1 gene (Rv3883c) in BCG [19].

The hypothesis that an interdependent functional relationship exists between the proteins encoded in these regions is further supported by the M. leprae sequence data, which shows that deletions of parts of the ESAT-6 gene cluster region 2 apparently caused the remaining genes in the region to become pseudogenes. Furthermore, Wards and co-workers [12] produced an M. bovis knockout mutant of the ATPase gene Rv3871 (family D) in the ESAT-6 gene cluster region 1, resulting in a strain that did not sensitize guinea pigs to an ESAT-6 skin test. These results indicate a close relationship between the genes contained within these regions.

Wards et al. [12] showed that an esat-6/lfp knockout mutant of M. bovis was less virulent than its parent if gross pathology, histopathology and mycobacterial culture from tissues were taken into account. These results, combined with the fact that multiple copies of the ESAT-6 gene clusters are found in all the mycobacteria, clearly indicate that they form an important part of the mycobacterial genome. The presence of multiple duplications of the ESAT-6 gene cluster regions in the mycobacteria may be a significant difference between the members of this genus and other high G+C Gram-positives. Although the function of this cluster is presently unknown, there is sufficient evidence to indicate that it is of crucial importance to the mycobacteria and needs to be investigated further.

Materials and methods

Genome sequence data and analyses

Annotations and descriptions of individual genes as well as gene and protein sequences of individual organisms were obtained from the publicly available finished and unfinished genome sequence databases listed in Table 1. Preliminary sequence data for M. tuberculosis 210, M. avium 104 and M. smegmatis MC2 155 was obtained from The Institute for Genomic Research (TIGR) website [28]. Preliminary sequence data for M. paratuberculosis K10 was obtained from the University of Minnesota M. paratuberculosis website [29]. Preliminary sequence data for M. bovis AF2122/97(spoligotype 9), C. diphtheriae NCTC13129 and S. coelicolor A3 (2), was obtained from the Sanger Centre website [30]. All gene and protein sequences were subjected to analysis with the following programs to confirm annotation and to look for additional information: SignalP V2.0.b2 [31,32], ClustalW WWW server at the European Bioinformatics Institute [33,34], TMHMM v0.1 [35,36], MOTIF [37] and BLASTP [38,39]. No data, progress report or BLAST search function is available for the genome sequencing of M. bovis BCG Pasteur 1173P2 at the Pasteur Institute, but information concerning genome deletions was obtained from published data [1,2,3,5,6,7] and from the Pasteur Institute website [40].

Analyses of similar gene clusters

BLAST similarity searches [38], using the BLAST 2.0 program with tblastn and the BLOSUM-62 weight matrix, were used to identify stretches of DNA containing putative ORFs homologous to the genes found in the M. tuberculosis ESAT-6 gene cluster regions from finished and unfinished genome sequences available at the National Center for Biotechnology Information (NCBI) website [41]. A total of 98 finished and unfinished genome sequences (35 from Gram-positive species) were used in the analysis, as summarized in Table 4. Where applicable, BLAST servers in database search services of individual sequencing centers were also used for protein identification. The Sanger Centre and The Institute for Genomic Research (TIGR) use the program WU-BLAST version 2.0 [42], while the University of Minnesota uses BLASTN with supplied defaults [43]. Sequences were only admitted to analysis when found to be part of one of the five gene clusters. In other words, no single homologous genes in the mycobacteria or other organisms (for example B. subtilis) that did not form part of a similar gene cluster were considered for the analyses, to exclude any potential unassociated similarity that could lead to false positives.

Table 4

Publicly available finished and unfinished genome sequence databases used in this study

Acidithiobacillus ferrooxidans	Escherichia coli O157:H7 EDL933	Rhodobacter sphaeroides
Actinobacillus actinomycetemcomitans	*Enterococcus faecalis	Salmonella dublin
Aquifex aeolicus	Geobacter sulfurreducens	Salmonella enteritidis
*Bacillus anthracis	Haemophilus ducreyi 35000HP	Salmonella paratyphi
*Bacillus halodurans	Haemophilus influenzae Rd	Salmonella typhi
*Bacillus subtilis	Helicobacter pylori 26695	Salmonella typhimurium LT2
*Bacillus stearothermophilus	Helicobacter pylori J99	Shewanella putrefaciens
Bordetella bronchiseptica	Klebsiella pneumoniae	Sinorhizobium meliloti
Bordetella parapertussis	*Lactococcus lactis subsp. lactis	*Staphylococcus aureus COL
Bordetella pertussis	Legionella pneumophila	*Staphylococcus aureus MRSA
Borrelia burgdorferi	*Listeria monocytogenes	*Staphylococcus aureus MSSA
Brucella melitensis biovar Suis	Mesorhizobium loti	*Staphylococcus aureus Mu50
Buchnera sp. APS	Methylococcus capsulatus	*Staphylococcus aureus N315
Burkholderia mallei	*Mycobacterium avium	*Staphylococcus aureus NCTC 8325
Burkholderia pseudomallei	*Mycobacterium avium subsp. paratuberculosis	*Staphylococcus epidermidis
Campylobacter jejuni NCTC 11168	*Mycobacterium bovis	*Streptococcus equi
Carboxydothermus hydrogenoformans	*Mycobacterium leprae	*Streptococcus gordonii
Caulobacter crescentus	*Mycobacterium smegmatis	*Streptococcus mutans
Chlamydia muridarum	*Mycobacterium tuberculosis 210	*Streptococcus pneumoniae
Chlamydia pneumoniae	*Mycobacterium tuberculosis CDC1551	*Streptococcus pyogenes
Chlamydia trachomatis D/UW-3/CX	*Mycobacterium tuberculosis H37Rv
Chlamydophila pneumoniae AR39	*Mycoplasma genitalium G37	*Streptococcus pyogenes Manfredo
Chlamydophila psittaci	*Mycoplasma pneumoniae M129	*Streptomyces coelicolor A3(2)
Chlorobium tepidum	Neisseria gonorrhoeae	Synechocystis PCC6803
*Clostridium acetobutylicum	Neisseria meningitidis MC58	Thermotoga maritima
*Clostridium difficile	Neisseria meningitidis Z2491	Treponema denticola
*Corynebacterium diphtheriae	Pasteurella multocida PM70	Treponema pallidum
Coxiella burnetii	Porphyromonas gingivalis W83	*Ureaplasma urealyticum
Dehalococcoides ethenogenes	Pseudomonas aeruginosa	Vibrio cholerae
Desulfovibrio vulgaris	Pseudomonas putida KT2440	Wolbachia
Deinococcus radiodurans	Pseudomonas putida PRS1	Xylella fastidiosa
Escherichia coli K-12 MG1655	Pseudomonas syringae pv. tomato	Yersinia enterocolitica
Escherichia coli O157:H7	Rickettsia prowazekii	Yersinia pestis

Finished genome sequences are indicated in bold, * indicates Gram-positive species.

Contig sequences corresponding to the gene clusters were obtained from their respective genome databases and used in further analyses. The Genetics Computer Group (Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin) program FRAMESEARCH was used to obtain whole sequence ORFs from the contigs. These ORFs were translated to protein sequences with the program TRANSLATE (also from GCG). All multiple sequence alignments and phylogenetic analyses were conducted on the protein level with these translated protein sequences.

Multiple sequence alignments

Multiple sequence alignments were performed on separate gene families belonging to the different clusters using ClustalW 1.5 [33] with the default parameters. The alignments were manually checked for errors and refined where appropriate. Multiple sequence alignments were also manually edited in some analyses during which unaligned regions (inserts) were removed (resulting in so-called edited alignments).

Phylogenetic trees

Bootstrapping resampling of the data sets were performed on the edited alignments, which generated 100 randomly chosen subsets of the multiple sequence alignment. Pairwise distances were determined with PROTDIST using the Dayhoff PAM matrix and neighbor-joining phylogenetic trees were calculated using NEIGHBOR (PHYLIP 3.5, [44]). In the case of each family of proteins, the C. diphtheriae sequence was first used as the outgroup after which the S. coelicolor sequence was used. Further phylogenetic analyses were performed using the programs FITCH and KITSCH with and without the outgroups respectively. A majority rule and strict consensus tree of all bootstrapped sequences were obtained using CONSENSE. The same analyses as described above were performed on a combined protein consisting of the edited aligned sequences of all six conserved proteins in these gene clusters as well as a combined protein constructed from the edited aligned sequences of all available ESAT-6 and CFP-10 family members. Finally, to confirm the results obtained with the single proteins, an analysis was performed with whole, unedited aligned sequences of the six most conserved proteins, using the program Paup 4.0b4a [45], during which negative branches were collapsed and 1,000 subsets were generated for bootstrapping resampling of the data. The consensus trees of all the above were drawn using the program Treeview 1.5 [46].