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EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles.

ABSTRACT: The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (?10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.

SUBMITTER: Fan Y

PROVIDER: S-EPMC4570534 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles.

Fan Yao Y Tan Kemin K Chhor Gekleng G Butler Emily K EK Jedrzejczak Robert P RP Missiakas Dominique D Joachimiak Andrzej A

Protein science : a publication of the Protein Society 20150703 9

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality ...[more]

PMID: 26032645

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Project description:Purpose: Bacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed ‘PCVRs’ (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. We used RNA-Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Methods: Cultures were incubated in toxin-inducing conditions, and expression of each PCVR in the atxAacpAacpB-null background strain was induced with an IPTG concentration that yielded native protein levels for each PCVR. RNA was extracted when cultures reached late-exponential phase using saturated acid phenol extraction, followed by RNA precipitation from the aqueous phase. Creation of libraries for NGS analysis used total RNA (1.0 μg). Samples were treated with Ribo-Zero (Epicentre) to remove ribosomal RNA prior to fragmentation using divalent cations and heat. Libraries were created using an Illumina TruSeq sample preparation kit following the protocol as recommended by the manufacturer. NGS sequencing was performed as a paired-end 50 base sequence using an Illumina HiSeq 1500 following the protocol recommended by the manufacturer. Results: Plasmid and chromosome-borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥4-fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. Of the 203 transcripts associated with pXO1, 15 were altered at least fourfold: 13 by AtxA and 2 by AcpA. No pXO1-derived transcripts were affected fourfold or greater by AcpB. Read maps revealed that many of the regulated transcripts from pXO1 were associated with the 35 kb region that lies within a larger 44.8 kb pathogenicity island (PAI) (Thorne, 1993). We detected transcripts for 110 genes on pXO2, of which 21 were regulated fourfold or greater by at least one of the three PCVRs. The highly regulated genes clustered within a 35.5 kb region of the plasmid, which includes the capsule biosynthetic operon capBCADE followed by the weakly co-transcribed acpB gene. A significantly smaller proportion of PCVR-regulated genes mapped to the chromosome. Of the 5593 transcripts, 198 were altered fourfold or greater by one or more PCVRs. Clustering of PCVR-regulated chromosome genes was not apparent. Among the most highly regulated chromosomal genes were many genes associated with branched chain amino acid (BCAA) synthesis and uptake. Of the 17 genes implicated in BCAA biosynthesis, expression of 13 of these genes is repressed by AtxA and AcpB. In this work, we have shown that the three PRD-containing virulence regulators of B. anthracis exhibit overlapping and divergent target gene specificities. Conclusions: In this work, we show the relative activities of the PCVRs for specific and co-regulated genes. The results reveal the vast effects of the PCVRs on B. anthracis gene expression and indicate a high degree of functional similarity among the regulators. Our investigations provide insight into control of B. anthracis gene expression and expand our knowledge of structure and function of this emerging class of virulence gene regulators.

Dataset Information

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles.

Publications

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles.

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