Subsystem: ESAT-6 proteins secretion system in Firmicutes

This subsystem's description is:

For more information, please check out the description and the additional notes tabs, below

DiagramFunctional RolesSubsystem SpreadsheetAdditional Notes 

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Group Alias
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This Subsystem has been initiated by RossO (SS: "282458.1-266-4-271") based solely on conserved clustering of several hypothetical ORFs in Firmicutes (scroll down for his original notes). Turns out, M.J. Pallen (7) has made similar observation before and even traced the cluster to ESAT-6 proteins and cognate secretion system in Mycobacteria. Pity, we were late!

See also related SS: ESAT-6 proteins secretion system in Actinobacteria

Variant codes:

1 = "minimal cluster" of ESAT-6-like protein EsxA and FtsK/SpoIIIE family protein EssC
2 = "full length" cluster: EsxA and EssC, plus EssA, EssB, EsaA, and EsaB
3 = "full length" cluster with additional concerved components
-1 = genome does not contain [ESAT-6-like + FtsK/SpoIIIE family protein]-related clusters


ESAT-6, the 6 kDa early secreted protein from Mycobacterium tuberculosis, a potent T-cell antigen (9), is the prototype of a novel Esat-6/Esx family of small proteins of unknown function produced by several Gram(+) microorganisms, many of them pathogenic (4, 7, 10). The biochemical function of these proteins is still unclear, but their prominent role in virulence has been documented in Mycobacteria (reviewed in 1) and recently – in Staphylococcus aureus (3).
All ESAT-6 family proteins lack known secretion signals, and their export requires novel secretory apparatus, which is encoded by a cluster of genes co-localized with ESAT-6 family proteins. It was originally identified by bioinformatics analysis (4, 10) and later experimentally confirmed (reviewed in 2). The genome of M. tuberculosis H37Rv and other Mycobacterial species has five copies of this cluster known as the ESAT-6 loci, with the region 4 (Rv3444c-3450c) believed to be ancestral (5). Orthologs of region 4 are also found in genomes of Corynebacteria, Streptomyces, Brevibacterium, and other Actinobacteria (5).
Conservative gene clusters encoding proteins of the ESAT-6/Esx family and the components of the putative cognate secretion system have been identified in genomes of other Gram-positive pathogens as well as, including Staphylococcus aureus, Bacillus anthracis, and Listeria monocytogenes (7). Although the level of sequence similarity between mycobacterial ESAT-6 homologs and their analogs in Firmicutes (EssxAB/YukE proteins) is very low, their inclusion in a single ESAT-6 superfamily is based on (i) conservation of the WXG motif, (ii) the protein length of ~100 AA, and – most importantly -- (iii) a tendency of the corresponding genes to cluster with other ESAT-6 family members as well as with EssC/YukA proteins – membrane-bound ATPases possessing two or more FtsK/SpoIIIE domains (7). Single copies of this domain, containing ATP_binding motif, are found in the FtsK cell division protein from E. coli and in the SpoIIIE sporulation protein from B. subtilis, where they are thought to be involved in DNA translocation. However, this domain is a specific example of the more general AAA+ domain (6), common to many ATPases involved in macromolecular secretion, including those from Gram-negative type IV protein secretion systems (7).
The clustering of the ESAT-6 and the FtsK/SpoIIIE family proteins (the “minimal ESAT-6 cluster”), preserved almost invariably in all species (i) gives credibility to categorizing the distant ESAT-6 homologs as one family and (ii) identifies the FtsK/SpoIIIE family as the most conserved (and likely most important) component of the cognate secretion system (7). Indeed, the essential role of the FtsK/SpoIIIE family proteins in the secretion of ESAT-6-homologs (and in virulence) has been demonstrated experimentally in Mycobacteria (reviewed in 2) and in S. aureus (3). Notably, other genes within the ESAT-6 cluster differ between mycobacteria (see SS: “ESAT-6 proteins secretion system in Actinobacteria”) and Firmicutes (see SS: “ESAT-6 proteins secretion system in Firmicutes”), yet their involvement in ESAT-6 secretion has been documented in each system (2, 3). The differences between the components of this novel Gram(+) secretion system in diverse species may indicate adaptations to different effector molecules. Extending a parallel with Gram(-) secretion, where secreted proteins are often components rather then targets of the translocation system, M. J. Pallen (7) has speculated that ESAT-6 family proteins are indeed a part of secretory apparatus, while effector proteins (or other macromolecules) remain to be identified and might in fact differ greatly between species. Notably, mutations in ESAT-6 cluster in M. smegmatis not only disrupt secretion of ESAT-6 and CFP-10, but also result in increased conjugal DNA transfer (11). Furthermore, the YueB protein, a B. subtilis homolog of the “putative secretion accessory protein EsaA” has been identified as the bacteriophage SPP1 receptor (8), leading the authors to speculate that membrane products of the ESAT-6 operon (YueB, YukC, and YukA) “could form a complex required for channeling the ejected DNA through the membrane."

Given its role in pathogenesis, it is noteworthy that the ESAT-6 secretion system also functions in non-pathogenic Mycobacteria, Clostridia, and other species. While the function of this secretion system is likely to be different in various bacteria, conservation of the gene cluster suggests that the core mechanism of this novel Gram(+) secretion system is largely conserved.

Open problems, conjectures:

I. Several lines of evidence suggest that ESAT-6 protein family are not the actual effectors (or at least not the only effectors) of this secretion system (7). It is tempting to speculate that large hypothetical proteins with clear amino-terminal similarity to ESAT-6 identified in B. anthracis (7) can potentially be secreted via this system. We believe that this hypothesis can be supported be the following observations:

(i) One of B. anthracis large proteins with amino-terminal similarity to ESAT-6 is located immediately downstream of the ESAT-6 gene cluster in several strains (BA2198 homologs)
(ii) Distant homologs of BA2198 are co-localized with ESAT-6 cluster in genomes of Listeria monocytogenes and S. aureus as well (Fig. 1, green arrows). These proteins display distant similarity with binary ADP-ribosylating toxins of Bacillus and Clostridia (Ran Nir-Paz, personal communication), indicating their potential involvement in virulence (currently annotated in SEED as “Lmo0066 homolog within ESAT-6 gene cluster, similarity to ADP-ribosylating toxins”)
(iii) Although N-termini of these Listeria and Staphylococcal proteins do not posses explicit similarity with the ESAT-6 family, the do contain the characteristic WXG motif.

II. An observation: the presence of species-specific tandem arrays of homologous proteins in the vicinity of the ESAT-6 secretion cluster -- is a recurrent theme in genomes of Staphylococcus aureus, Bacillus anthracis, Listeria monocytogenes, and Clostridium acetobutilicum:
(i) Different strains of S. aureus contain from 6 to 11 copies of ~160 AA hypothetical ORF (DUF600)
(ii) Different strains of Listeria harbor from none to 3 copies of “Lmo0066 homolog within ESAT-6 gene cluster, similarity to ADP-ribosylating toxins”
(iii) In Clostridium acetobutilicum up to 3 copies of a TPR-repeat-containing protein (cd00189) are present in various strains near the minimum [ESAT-6-like protein -- FtsK/SpoIIIE family protein] cluster (tetratricopeptide (TPR) repeat domains are present in various chaperone, cell-cycle, transciption, and protein transport complexes and are believed to be involved in protein-protein interaction
These proteins might be a part of the secretion apparatus or it’s potential effectors.

1. Brodin, P., I.Rosenkrands, P.Andersen, S.T. Cole, Roland Brosch. 2004. ESAT-6 proteins: protective antigens and virulence factors? Trends. Microbiol, 12:500-508
2. Brodin P, Majlessi L, Marsollier L, de Jonge MI, Bottai D, Demangel C, Hinds J, Neyrolles O, Butcher PD, Leclerc C, Cole ST, Brosch R. Dissection of ESAT-6 System 1 of Mycobacterium tuberculosis and Impact on Immunogenicity and Virulence. Infect Immun. 2006 Jan;74(1):88-98.
3. Burts M.L., W.A. Williams, K.DeBord, and D.M. Missiakas. 2005. EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. PNAS, 102:1169 –1174
4. Cole, S.T. et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544
5. Gey van Pittius N. et al. 2001. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria. Genome Biology, 2(10): research0044.1–0044.18
6. Neuwald, A.F. et al. 1999. AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27–43
7. Pallen M.J. 2002. The ESAT-6/WXG100 superfamily – and a new Gram-positive secretion system? Trends Microbiol., 10(5):209-212
8. Sao-Jose ́C., C. Baptista, and M.A. Santos. 2004. Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1. J. Bact, 186(24):8337–8346
9. Sørensen, A.L. et al. 1995. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 63, 1710–1717
10. Tekaia, F. et al. 1999. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber. Lung Dis. 79, 329–342
11. Flint J. L., J.C. Kowalski, P.K. Karnati, and K.M. Derbyshire. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. PNAS, 101(34):12598 –12603.

Notes from original SS by RossO:

Original genomes were required to contain at least 1 of the signature columns

SIG1 = EsxA
SIG2 = EsaA
SIG3 = EssB
NSIG1 = EssC
CONS3 = EsaB
CONS4 = EssA




Clostridium acetobutylicum ATCC 824 has SIG2 where you would expect it:

Length = 3940880

Score = 189 bits (480), Expect = 3e-50
Identities = 96/96 (100%), Positives = 96/96 (100%)
Frame = -3





Bacillus subtilis:

YueB = SIG2 = EsaA in Staph. aureus
YucBA = NSIG1 = EssC in Staph. aureus
YucC = SIG3 = EssB in Staph. aureus

The authors in the following paper found that

1. At least these genes (and probably a few more) occur in an operon (in B.subtilis)

2. "yueB is the essential gene involved in the irreversible binding of SPP1 to its host."

They conjecture

3. "a putative protein complex formed by the yukE operon products could be involved in

double-stranded DNA uptake in B. subtilis.

4. "YukBA could play an auxiliary role in driving phage DNA movement across the cell membrane."

To sum up,

"We speculate that membrane products of the yukE operon (YueB, YukC, and YukBA) could
form a complex required both for proper phage binding (involving YueB) and for
channeling the ejected DNA through the membrane."

3189: Bacteriophage SPP1 adsorption protein yueB [UniProt]

J Bacteriol. 2004 December; 186(24): 8337–8346.
doi: 10.1128/JB.186.24.8337-8346.2004.

Bacillus subtilis Operon Encoding a Membrane Receptor for Bacteriophage SPP1†

Carlos São-José, Catarina Baptista, and Mário A. Santos

The results reported here have identified yueB as the essential gene involved in irreversible binding of bacteriophage SPP1 to Bacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of the yueB gene, could be complemented by a xylose-inducible copy of yueB inserted at amyE. Second, disruption of yueB by insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-β-d-thiogalactopyranoside). YueB homologues are widely distributed in gram-positive bacteria. The protein Pip, which also serves as a phage receptor in Lactococcus lactis, belongs to the same family. yueB encodes a membrane protein of ∼120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation of yueB and the surrounding genes indicated that yueB is part of an operon which includes at least the upstream genes yukE, yukD, yukC, and yukBA. Disruption of each of the genes in the operon allowed efficient irreversible adsorption, provided that yueB expression was retained. Under these conditions, however, smaller plaques were produced, a phenotype which was particularly noticeable in yukE mutant strains. Interestingly, such reduction in plaque size was not correlated with a decreased adsorption rate. Overall, these results provide the first demonstration of a membrane-bound protein acting as a phage receptor in B. subtilis and suggest an additional involvement of the yukE operon in a step subsequent to irreversible adsorption.



SIG1 = EsxA
SIG2 = EsaA
SIG3 = EssB
NSIG1 = EssC
CONS3 = EsaB
CONS4 = EssA

EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections.

Burts ML, Williams WA, DeBord K, Missiakas DM.

Department of Microbiology, University of Chicago, Chicago, IL 60637, USA.

Mycobacterium tuberculosis secretes ESAT-6, a virulence factor that triggers cell-mediated immune responses and IFN-gamma production during tuberculosis. ESAT-6 is transported across the bacterial envelope by a specialized secretion system with a FSD (FtsK-SpoIIIE domain) membrane protein. Although the presence of ESAT-6-like genes has been identified in the genomes of other microbes, the possibility that they may encode general virulence functions has hitherto not been addressed. Herein we show that the human pathogen Staphylococcus aureus secretes EsxA and EsxB, ESAT-6-like proteins, across the bacterial envelope. Staphylococcal esxA and esxB are clustered with six other genes and some of these are required for synthesis or secretion of EsxA and EsxB. Mutants that failed to secrete EsxA and EsxB displayed defects in the pathogenesis of S. aureus murine abscesses, suggesting that this specialized secretion system may be a general strategy of human bacterial pathogenesis.