Deciphering the genetic makeup of the Bacillus subtillis Esat-6-like secretion system

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2013The locus yukEDCByueBC of the Gram-positive model bacterium Bacillus subtilis was recently shown to encode a functional Esat-6-like secretion system (Ess), in natural isolates of this bacterium, with YukE being a typical secretion substrate of the WXG100 superfamily. This genetic locus has been renamed BsEss. Type VII/Ess secretion systems are widespread among bacteria of the phyla Actinobacteria and Firmicutes and in some species they play an important role in bacterial pathogenesis, as in Mycobacterium tuberculosis and Staphylococcus aureus. Thus, the BsEss is viewed as an attractive model to study molecular details of this important secretion pathway. Interestingly, the system is impaired in the classical B. subtilis lab strain 168, although carrying an intact BsEss locus. There are several reported mutations in the genome of strain 168, when compared to ancestral or undomesticated strains like NCIB 3610 and ATCC 6051, all of them affecting genes involved in Deg-regulated cellular processes. The BsEss is known to be activated by the phosphorylated state of DegU, so in this work we aimed to identify genetic mutations present in strain 168 that could account for the defective operation of the secretion system, and thus to contribute to the understanding of its functioning and regulation. Among the studied mutations, the ones that mostly impaired BsEss operation were those affecting the expression of genes sfp and degQ. Sfp is necessary for surfactin production, a requisite for B. subtilis swarming motility, functioning also as a signaling molecule in biofilm formation. DegQ was shown to enhance phosphotransfer from DegS~P to DegU, being also necessary to robust biofilm development. By complementing these two mutations in strain 168 we could restore YukE secretion to the levels observed in undomesticated strains. Other genes were included in this study, related to the DegS-DegU regulon. Among these we found swrB, a gene involved in the synthesis of flagella, as being important for YukE export. B. subtilis YeeF, a homologue of the S. aureus Ess-associated protein EsaD, seemed also to be important for BsEss functioning. Finally, in this work we have further explored previous results that suggested a negative role of BsEss expression in competence development, in an effort to uncover potential cellular functions for this secretion system.Foi recentemente demonstrado que o locus genómico yukEDCByueBC da bactéria Gram-positiva Bacillus subtilis codifica para um sistema de secreção do tipo Esat-6 (Ess). Este sistema é funcional em isolados naturais desta bactéria, sendo YukE um substrato de secreção típico da superfamília WXG100. Este locus foi recentemente renomeado BsEss. Os sistemas de secreção do Tipo VII/Ess são bastante prevalentes em bactérias dos filos Actinobacteria e Firmicutes, sendo que em algumas espécies desempenham um papel importante na patogénese bacteriana, nomeadamente em Mycobacterium tuberculosis e Staphylococcus aureus. Assim, o BsEss é visto como um modelo atractivo para estudar os detalhes moleculares deste importante sistema de secreção. Curiosamente, este sistema parece inibido na estirpe 168 de B. subtilis, a estirpe laboratorial mais utilizada em trabalhos de investigação, apesar de esta possuir o locus BsEss intacto. São conhecidas várias mutações na estirpe 168 quando comparada com estirpes ancestrais ou não domesticadas, como por exemplo os isolados NCIB 3610 e ATCC 6051. Essas mutações afectam genes envolvidos em processos celulares regulados pelo sistema de dois componentes DegS-DegU. Sabe-se que o BsEss é activado pela forma fosforilada de DegU, pelo que neste trabalho pretendeu-se identificar mutações presentes na estirpe 168 responsáveis pela inibição do BsEss, ao mesmo tempo contribuindo para uma melhor compreensão do seu funcionamento e regulação. Entre as mutações reportadas para a estirpe 168, verificámos que aquelas que mais influenciam negativamente a função do BsEss são as que afectam os genes sfp e degQ. Sfp é necessário para a produção de surfactina, um surfactante essencial para a motilidade de B. subtilis em superfícies sólidas (“swarming”), para além de funcionar também como molécula sinalizadora no desenvolvimento de biofilmes. DegQ aumenta a fosforilação de DegU através de DegS e está também envolvido na formação de biofilmes. Ao complementar ambas a mutações na estirpe 168, observou-se um restauro da secreção de YukE para níveis semelhantes aos das estirpes não domesticadas. Outros genes foram incluídos neste estudo, relacionados com o regulão DegS-DegU. De entre estes, descobrimos swrB, um gene envolvido na síntese dos flagelos, como sendo importante para a exportação de YukE. A proteína YeeF de B. subtilis, a qual é homóloga à proteína EsaD do Ess de S. aureus, revelou-se também importante para o funcionamento do BsEss. Finalmente, num esforço para desvendar possíveis funções celulares para este sistema de secreção, foi ainda explorado com mais detalhe resultados anteriores que sugeriam uma influência negativa da expressão de BsEss no desenvolvimento da competência de B. subtilis

High Levels of DegU-P Activate an Esat-6-Like Secretion System in <i>Bacillus subtilis</i>

<div><p>The recently discovered Type VII/Esat-6 secretion systems seem to be widespread among bacteria of the phyla <i>Actinobacteria</i> and <i>Firmicutes</i>. In some species they play an important role in pathogenic interactions with eukaryotic hosts. Several studies have predicted that the locus <i>yukEDCByueBC</i> of the non-pathogenic, Gram-positive bacterium <i>Bacillus subtilis</i> would encode an Esat-6-like secretion system (Ess). We provide here for the first time evidences for the functioning of this secretion pathway in an undomesticated <i>B. subtilis</i> strain. We show that YukE, a small protein with the typical features of the secretion substrates from the WXG100 superfamily is actively secreted to culture media. YukE secretion depends on intact y<i>ukDCByueBC</i> genes, whose products share sequence or structural homology with known components of the <i>S. aureus</i> Ess. Biochemical characterization of YukE indicates that it exists as a dimer both <i>in vitro</i> and <i>in vivo</i>. We also show that the <i>B. subtilis</i> Ess essentially operates in late stationary growth phase in absolute dependence of phosphorylated DegU, the response regulator of the two-component system DegS-DegU. We present possible reasons that eventually have precluded the study of this secretion system in the <i>B. subtilis</i> laboratory strain 168.</p></div

Multiple sequence alignment of representative WXG100 proteins from <i>Actinobacteria</i> and <i>Firmicutes</i>.

<p>The absolutely conserved W and G residues forming the WXG signature are in bold and underlined. WXG is part of the loop that in the tridimensional structures of the subunits EsxA, GBS1074 and ESAT-6 connects the two antiparallel α-helices α1 and α2 (formed by the gray-shaded residues) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Renshaw1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Sundaramoorthy1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Shukla1" target="_blank">[46]</a>. YukE segments predicted to form the α1 and α2 helices are also indicated by gray shading. The presence of a proline residue (boldface and enlarged font) in helix α2 was proposed to be a signature of WXG100 homodimer formation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Sundaramoorthy1" target="_blank">[45]</a>. The consensus sequence I-K/R-M/V/L-S/T-P-E-E-L, highlighted in the top 6 sequences, is conserved in a large subset of YukE closest homologues from <i>Firmicutes</i>, with residues I and P being absolutely conserved (not shown). Note that <i>B. subtilis</i> YfjA is a distant homologue of YukE. Asterisk, fully conserved residues; colon, conservation of residues with strongly similar properties; period, conservation of residues with weakly similar properties.</p

Oligomeric state of YukE.

<p>A. Purified YukE-His₆ from the affinity chromatography step was loaded into a size exclusion column (see methods) and the eluted material was continuously monitored by taking absorbance measurements at 280 nm (A_{280 nm}). The elution volume (ml) of the YukE-His₆ peak (solid curve) and the derived Stokes radius (<i>R_s</i>) and relative mass (<i>M_r</i>) are indicated. Note that the mass was estimated assuming that YukE-His₆ has the same hydrodynamic properties of the globular polypeptides composing the protein standard (dashed curve). The column void volume (<i>V₀</i>), the elution volume of standard proteins and their corresponding masses are also indicated. B. SDS-PAGE and Coomassie blue staining of YukE-His₆ from the peak fraction of the size exclusion chromatography. C. Modeling of the YukE subunit 3D structure by Phyre² (97% YukE sequence coverage, 99.8% confidence, template = GBS1074). D,E. <i>In vitro</i> cross-linking of YukE. Two concentration sets of purified YukE-His₆, from 84 to 20 µM (D) and from 10.3 to 0.4 µM (E) were incubated in the presence or absence of the cross-linking agent BS³. After SDS-PAGE separation (D, 2 µg protein per lane; E, 50 ng protein per lane), the cross-linking products were revealed by Coomassie blue staining (D) or by immunodetection with anti-YukE-His₆ antibodies (E). F. <i>In vivo</i> YukE cross-linking. YukE present in a concentrated culture supernatant of strain W654 (2.5 µg total protein, see methods for details) was cross-linked in presence of 1 or 5 mM BS³ and the cross-linked products detected with anti-YukE-His₆ as in panel E.</p

YukE production and secretion by <i>B. subtilis</i> strains ATCC 6051 (WT) and 168 and by their derivatives carrying mutations in <i>deg</i> genes.

<p>A. Growth curve of WT strain in LB at 37°C. A fitting curve for the exponential growth phase is presented. Growth curves of <i>deg</i> strains showed no significant differences (not shown). The arrows indicate the time points where culture samples were collected for production of protein extracts. T0, end of exponential growth phase; T2, 2 hours after entry in stationary growth phase. B. Immunodetection of YukE and TrxA (cytosolic control protein) in supernatant precipitates (SN) and cell extracts (C) prepared from T0 and/or T2 culture samples of strain ATCC 6051 (WT) and its derivatives W654 (<i>degU32</i>(Hy)), WTF28 (<i>degU</i>) and W648 (<i>degS</i>). Each lane was loaded with 20 µg total protein, except that of pure YukE-His₆ (100 ng)_. C. Immunodetection of YukE and TrxA in SN and C fractions prepared from T0 and/or T2 culture samples of strain 168 and its <i>degU32</i>(Hy)) derivative. The loaded protein amounts were as in panel B, except for the LB precipitate control that was with a volume equivalent to the maximum loaded SN fraction. The corresponding Coomassie blue stained gels of SN and C extracts are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840.s001" target="_blank">Figure S1</a>.</p

Schematic representation of the gene clusters encoding core components and substrates of T7S-like systems in <i>M. tuberculosis</i> (ESX-1), <i>S. aureus</i> (Ess) and <i>B. subtilis</i> (Ess).

<p>The nomenclature of ESX-1 genes is that proposed by Bitter et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Bitter1" target="_blank">[15]</a>. Note that, for simplicity, several ESX-1 secretion-associated protein (e<i>sp</i>) genes, located immediately or far upstream of <i>M. tuberculosis</i> e<i>ccA1</i>, are not represented. Genes consistently described as essential (or important) for secretion of cognate WXG100 proteins, or of other specific substrates, are marked with the letter “E” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Abdallah1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Burts1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Ohol1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Anderson1" target="_blank">[17]</a> (see text for <i>B. subtilis</i> Ess). Genes coding for products sharing conserved domains are depicted with the same color code, whereas those specific of each system are colored in white. Features of conserved gene products are indicated below (TMD stands for transmembrane domain). EssB and YukC harbor a pseudokinase domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Burroughs1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840-Zoltner1" target="_blank">[19]</a>.</p

YueB production and phage SPP1 plating in the undomesticated <i>B. subtilis</i> strain ATCC 6051 (WT) and its <i>deg</i> mutants.

<p>A. Western blot analysis of YueB polypeptides in cell extracts of the WT and derivative strains carrying the mutations <i>degU32</i>(Hy), <i>degU</i> and <i>degS</i>. The protein extracts were prepared from T0 and/or T2 samples as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone-0067840-g003" target="_blank">Figure 3</a>. Lane “<i>yueB</i>” is a control extract prepared from a <i>yueB</i> mutant strain to help identify the immune reactive species that are YueB specific. FL and TR indicate the position of the full length and truncated polypeptides of YueB, respectively (absent in “<i>yueB</i>” lane). Each lane was loaded with 20 µg total protein. B. Phage SPP1 efficiency of plating (EoP) in <i>deg</i> strains. The EoP value and the phage dilution (10⁻⁸ or 10⁻⁹) that produced each image are indicated in parenthesis. Note the reduced plaque size in <i>degU</i> and <i>degS</i> mutants.</p

YukE production and secretion by <i>BsEss</i> mutant strains.

<p>A. YukE and TrxA (cytosolic control protein) were immunodetected in supernatant precipitates (SN) and cell protein extracts (C) of strain W654 (<i>degU32</i>(Hy)) and its derivatives with individual knockouts of <i>BsEss</i> genes. Note that the YukE signal shown for cell extracts results from an overexposed film when compared to that of SN fractions. Immunodetection of YueB polypeptides is also shown for cell extracts. Each lane was loaded with 20 µg total protein, except that of the control that was loaded with 100 ng of pure YukE-His₆. B. Immunodetection of YukE and TrxA in SN and C fractions of the <i>yueB</i> complementation strain, in absence or presence of 0.5% xylose. The immunodetection of YueB polypeptides and the amount of loaded protein was as in panel B. The corresponding Coomassie blue stained gels of SN and C extracts are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067840#pone.0067840.s002" target="_blank">Figure S2</a>.</p

<i>B. subtilis</i> strains used in this work.

*<p>American Type Culture Collection.</p