32 research outputs found

    Formation and Characterization of Non-Growth States in Clostridium Thermocellum: Spores and L-Forms

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    Clostridium thermocellum is an anaerobic thermophilic bacterium that exhibits high levels of cellulose solublization and produces ethanol as an end product of its metabolism. Using cellulosic biomass as a feedstock for fuel production is an attractive prospect, however, growth arrest can negatively impact ethanol production by fermentative microorganisms such as C. thermocellum. Understanding conditions that lead to non-growth states in C. thermocellum can positively influence process design and culturing conditions in order to optimize ethanol production in an industrial setting

    Formation and characterization of non-growth states in <it>Clostridium thermocellum</it>: spores and L-forms

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    Abstract Background Clostridium thermocellum is an anaerobic thermophilic bacterium that exhibits high levels of cellulose solublization and produces ethanol as an end product of its metabolism. Using cellulosic biomass as a feedstock for fuel production is an attractive prospect, however, growth arrest can negatively impact ethanol production by fermentative microorganisms such as C. thermocellum. Understanding conditions that lead to non-growth states in C. thermocellum can positively influence process design and culturing conditions in order to optimize ethanol production in an industrial setting. Results We report here that Clostridium thermocellum ATCC 27405 enters non-growth states in response to specific growth conditions. Non-growth states include the formation of spores and a L-form-like state in which the cells cease to grow or produce the normal end products of metabolism. Unlike other sporulating organisms, we did not observe sporulation of C. thermocellum in low carbon or nitrogen environments. However, sporulation did occur in response to transfers between soluble and insoluble substrates, resulting in approximately 7% mature spores. Exposure to oxygen caused a similar sporulation response. Starvation conditions during continuous culture did not result in spore formation, but caused the majority of cells to transition to a L-form state. Both spores and L-forms were determined to be viable. Spores exhibited enhanced survival in response to high temperature and prolonged storage compared to L-forms and vegetative cells. However, L-forms exhibited faster recovery compared to both spores and stationary phase cells when cultured in rich media. Conclusions Both spores and L-forms cease to produce ethanol, but provide other advantages for C. thermocellum including enhanced survival for spores and faster recovery for L-forms. Understanding the conditions that give rise to these two different non-growth states, and the implications that each has for enabling or enhancing C. thermocellum survival may promote the efficient cultivation of this organism and aid in its development as an industrial microorganism.</p

    Transcription and translation of the sigG gene is tuned for proper execution of the switch from early to late gene expression in the developing Bacillus subtilis spore.

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    A cascade of alternative sigma factors directs developmental gene expression during spore formation by the bacterium Bacillus subtilis. As the spore develops, a tightly regulated switch occurs in which the early-acting sigma factor σF is replaced by the late-acting sigma factor σG. The gene encoding σG (sigG) is transcribed by σF and by σG itself in an autoregulatory loop; yet σG activity is not detected until σF-dependent gene expression is complete. This separation in σF and σG activities has been suggested to be due at least in part to a poorly understood intercellular checkpoint pathway that delays sigG expression by σF. Here we report the results of a careful examination of sigG expression during sporulation. Unexpectedly, our findings argue against the existence of a regulatory mechanism to delay sigG transcription by σF and instead support a model in which sigG is transcribed by σF with normal timing, but at levels that are very low. This low-level expression of sigG is the consequence of several intrinsic features of the sigG regulatory and coding sequence-promoter spacing, secondary structure potential of the mRNA, and start codon identity-that dampen its transcription and translation. Especially notable is the presence of a conserved hairpin in the 5' leader sequence of the sigG mRNA that occludes the ribosome-binding site, reducing translation by up to 4-fold. Finally, we demonstrate that misexpression of sigG from regulatory and coding sequences lacking these features triggers premature σG activity in the forespore during sporulation, as well as inappropriate σG activity during vegetative growth. Altogether, these data indicate that transcription and translation of the sigG gene is tuned to prevent vegetative expression of σG and to ensure the precise timing of the switch from σF to σG in the developing spore

    Late σ<sup>F</sup>-dependent expression of both P<sub><i>sigG</i></sub> and P<sub><i>spoIIQ</i></sub> requires SpoIIQ, σ<sup>E</sup>, and SpoIIIAA-AH.

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    <p>The activity of P<sub><i>sigG</i></sub><i>-lacZ</i> <b>(A, C, D)</b> or P<sub><i>spoIIQ</i></sub><i>-lacZ</i> <b>(B, E)</b> was monitored during sporulation of strains with the following genotypes: wild type (<i>WT</i>; open circles), <i>ΔsigG</i> (closed circles), <i>ΔsigG ΔsigF</i> (open squares), <i>ΔsigG ΔspoIIQ</i> (open diamonds), <i>ΔsigG ΔsigE</i> (open triangles), and <i>ΔsigG ΔspoIIIAA-AH</i> (closed triangles). (P<sub><i>sigG</i></sub><i>-lacZ</i> strains were JJB31, JJB73, JJB75, JJB79, JJB85, and JJB77, respectively. P<sub><i>spoIIQ</i></sub><i>-lacZ</i> strains were AHB881, AHB882, AHB915, AHB916, AHB917, and AHB1017, respectively.) For clarity, only data from a subset of these strains are presented in each graph (as labeled) and, also for clarity, the data for the <i>ΔsigG</i> strain of each (closed circles) is presented in all graphs. Note that (D) provides a zoomed view of the data from the boxed area of (C). The timing of the σ<sup>F</sup>-to-σ<sup>G</sup> switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all panels, error bars indicate ± standard deviations based on three independent experiments.</p

    Misexpression of <i>sigG</i> causes ectopic σ<sup>G</sup> activity in a subset of vegetative cells.

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    <p><b>(A, B)</b> GFP production from a σ<sup>G</sup>-dependent P<sub><i>sspB</i></sub><i>-gfp</i> reporter was monitored by fluorescence microscopy of vegetatively growing cells in which <i>sigG</i> was expressed from its wild type regulatory sequences (<sup>WT</sup>P<sub><i>sigG</i></sub>) or from regulatory sequences modified to remove or repair the four features identified in this study to dampen <i>sigG</i> expression (<sup><i>quad</i></sup>P<sub><i>sigG</i></sub>). Additionally, cells either harbored wild type <i>csfB</i>, or were deleted for the gene (<i>ΔcsfB</i>). (Strains EBM192 [<sup>WT</sup>P<sub><i>sigG</i></sub>], EBM276 [<sup><i>quad</i></sup>P<sub><i>sigG</i></sub>], EBM282 [<sup>WT</sup>P<sub><i>sigG</i></sub> <i>ΔcsfB</i>], and EBM287 [<sup><i>quad</i></sup>P<sub><i>sigG</i></sub>-<i>sigG ΔcsfB</i>].) In <b>(A)</b>, representative microscopy images are shown with GFP fluorescence (false-colored green) merged with membrane fluorescence from the dye FM 4–64 (false-colored red). Yellow arrowheads indicate vegetative cells with visible GFP fluorescence. Scale bar 5 μm. In <b>(B)</b>, GFP fluorescence intensity (with background subtracted) for more than 500 cells of each strain, including a “no GFP” control strain (PY79) lacking the P<sub><i>sspB</i></sub><i>-gfp</i> reporter, is shown in column scatter graph format, with each cell represented by a black dot. Cells exhibiting fluorescence intensity above the cut off value (three standard deviations above mean auto-fluorescence of the “no GFP” strain; gray dashed line) were determined to have detectable σ<sup>G</sup> activity. The percentage of cells with this activity is indicated above the respective strain. Note that ~60000 fluorescence units is the upper limit of detection under our microscopy settings. <b>(C)</b> Simultaneous deletion of <i>csfB</i> and misexpression of <i>sigG</i> from <sup><i>quad</i></sup>P<sub><i>sigG</i></sub> causes synthetic toxicity to vegetatively growing <i>B</i>. <i>subtilis</i>. The same strains described in (A) were struck onto LB agar and were photographed after ~18 hours of growth at 37°C.</p

    P<sub><i>sigG</i></sub> activity is detected both before and after the switch from σ<sup>F</sup> to σ<sup>G</sup>.

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    <p><b>(A, B)</b> The activities of P<sub><i>sigG</i></sub> reporters harboring the native GTG start codon, P<sub><i>sigG</i></sub><i>-sigG</i><sup><i>1-28</i></sup><i>-lacZ</i> (P<sub><i>sigG</i></sub><i>-GTG</i>; open circles) or engineered to harbor an ATG start codon, P<sub><i>sigG</i></sub><i>-ATG-sigG</i><sup><i>2-28</i></sup><i>-lacZ</i> (P<sub><i>sigG</i></sub><i>-ATG</i>; closed circles), were measured during a time course of sporulation. (Strains EBM177 and EBM175, respectively.) Background β-galactosidase activity was measured in a strain without a <i>lacZ</i> reporter, PY79 (no <i>lacZ</i>; asterisks). Note that (B) provides a zoomed view and statistical significance of early sporulation data points from the boxed area of (A). <b>(C, D)</b> Strains harboring P<sub><i>sigG</i></sub> reporters with altered <i>sigG</i> 5’ coding sequence, one engineered to reduce secondary structure (RSS) in <i>sigG</i> codons 2–28, P<sub><i>sigG</i></sub><i>-ATG-</i><sup><i>RSS</i></sup><i>sigG</i><sup><i>2-28</i></sup><i>-lacZ</i> (P<sub><i>sigG</i></sub><i>-ATG-RSS</i>; open triangles), and another harboring <i>comGA</i> codons 2–8 in place of <i>sigG</i> codons 2–28, P<sub><i>sigG</i></sub><i>-ATG-comGA</i><sup><i>2-8</i></sup><i>-lacZ</i> (P<sub><i>sigG</i></sub><i>-ATG-comGA</i>; open diamonds), were monitored for β-galactosidase production during sporulation. (Strains EBM237 and JJB31, respectively.) Data for the P<sub><i>sigG</i></sub><i>-ATG-sigG</i><sup><i>2-28</i></sup><i>-lacZ</i> (P<sub><i>sigG</i></sub><i>-ATG</i>; closed circles) strain (EBM175) and PY79 (no <i>lacZ</i>, asterisks), the same as shown in (A) and (B), are also included for reference. Note that (D) provides a zoomed view and statistical significance of early sporulation data points from the boxed area of (C). The timing of the σ<sup>F</sup>-to-σ<sup>G</sup> switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all panels, error bars indicate ± standard deviations based on three independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01, NS not significant, Student’s <i>t</i>-test.</p

    Misexpression of <i>sigG</i> causes premature σ<sup>G</sup> activity in the forespore during sporulation.

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    <p>GFP production from a σ<sup>G</sup>-dependent P<sub><i>sspB</i></sub><i>-gfp</i> reporter was monitored by fluorescence microscopy of sporulating cells (hour 3.5) in which <i>sigG</i> was expressed from its wild type regulatory sequences (<sup>WT</sup>P<sub><i>sigG</i></sub>) or from regulatory sequences modified to remove or repair the four features identified in this study to dampen <i>sigG</i> expression (<sup><i>quad</i></sup>P<sub><i>sigG</i></sub>). Additionally, cells either harbored wild type <i>csfB</i>, or were deleted for the gene (<i>ΔcsfB</i>). (Strains EBM192 [<sup>WT</sup>P<sub><i>sigG</i></sub>], EBM276 [<sup><i>quad</i></sup>P<sub><i>sigG</i></sub>] and EBM282 [<sup>WT</sup>P<sub><i>sigG</i></sub> <i>ΔcsfB</i>].) <b>(A)</b> Representative microscopy images for each strain. GFP fluorescence is depicted in grayscale (P<sub><i>sspB</i></sub>-GFP) or false-colored green (Merge). Membrane fluorescence from the dye FM 4–64 is shown in grayscale (Membranes) or false-colored red (Merge). Following asymmetric division and during engulfment, the FM 4–64 dye labels all membranes including the double membranes separating the mother cell and forespore; after the completion of engulfment, the membranes surrounding the forespore are no longer accessible to the dye and therefore remain unlabeled. Yellow arrowheads indicate late engulfment forespores, identifiable by their FM 4-64-labeled engulfing membranes, with visible GFP fluorescence. Scale bar 5 μm. <b>(B)</b> GFP fluorescence intensity (with background subtracted) for more than 200 late-engulfment forespores or <b>(C)</b> their corresponding mother cells for the three strains described in (A) as well as a “no GFP” control strain (PY79) lacking the P<sub><i>sspB</i></sub><i>-gfp</i> reporter are shown in column scatter graph format, with each cell represented by a black dot. Cells exhibiting fluorescence intensity above the cut off value (three standard deviations above mean auto-fluorescence of the “no GFP” strain; gray dashed line) were determined to have detectable σ<sup>G</sup> activity. The percentage of cells with this activity is noted above the respective strain.</p

    The identified transcriptional and translational regulation of <i>sigG</i> diminishes <i>sigG</i> expression by 4-6-fold and is required to prevent aberrant activity of σ<sup>G</sup>.

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    <p><b>(A, B)</b> Expression of a P<sub><i>sigG</i></sub> reporter is increased by 4-6-fold in the absence of the four regulatory features identified in this study. β-Galactosidase production was monitored during sporulation of strains harboring P<sub><i>sigG</i></sub><i>-sigG</i><sup><i>1-28</i></sup><i>-lacZ</i> (<sup>WT</sup>P<sub><i>sigG</i></sub><i>-lacZ</i>; open circles) or a variant in which all four features of <i>sigG</i> that dampen expression were simultaneously removed or repaired, <sup><i>15nt</i></sup>P<sub><i>sigG</i></sub><sup><i>mut7</i></sup><i>-ATG-</i><sup><i>RSS</i></sup><i>sigG</i><sup><i>2-28</i></sup><i>-lacZ</i> (<sup><i>quad</i></sup>P<sub><i>sigG</i></sub>-<i>lacZ</i>; closed circles) (strains EBM177 and EBM262, respectively.) Note that (B) provides a zoomed view of the data from the boxed area of (A). <b>(C, D)</b> Expression of <i>sigG</i> from regulatory sequences lacking the four regulatory features identified in this study causes aberrant σ<sup>G</sup> activity during a time course of sporulation. β-Galactosidase production from (C) the σ<sup>F</sup>-dependent P<sub><i>spoIIQ</i></sub><i>-lacZ</i> reporter or (D) the σ<sup>G</sup>-dependent P<sub><i>sspB</i></sub><i>-lacZ</i> reporter was monitored during sporulation of strains in which <i>sigG</i> was expressed from its wild type regulatory sequences (<sup>WT</sup>P<sub><i>sigG</i></sub><i>-sigG</i>; open squares) or from regulatory sequences modified to remove or repair the four features identified in this study to dampen <i>sigG</i> expression (<sup><i>quad</i></sup>P<sub><i>sigG</i></sub><i>-sigG</i>; closed squares). (P<sub><i>spoIIQ</i></sub><i>-lacZ</i> strains were CFB429 and CFB431, respectively. P<sub><i>sspB</i></sub><i>-lacZ</i> strains were CFB435 and CFB437, respectively.) The black arrow in (D) indicates aberrant σ<sup>G</sup> activity at early times of sporulation. The timing of the σ<sup>F</sup>-to-σ<sup>G</sup> switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all panels, error bars indicate ± standard deviations based on three independent experiments.</p

    Suboptimal spacing of the P<sub><i>sigG</i></sub> -10 and -35 elements diminishes <i>sigG</i> expression.

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    <p><b>(A)</b> Alignment of ten <i>B</i>. <i>subtilis</i> promoters activated by σ<sup>F</sup>, including P<sub><i>sigG</i></sub>. Nucleotides comprising the -10 and -35 elements of each promoter are in bold and shaded gray. Transcription start sites, if known, are underlined [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007350#pgen.1007350.ref041" target="_blank">41</a>]. The consensus promoter sequence for σ<sup>F</sup> is shown above [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007350#pgen.1007350.ref038" target="_blank">38</a>]. Red arrowheads indicate two notable features of P<sub><i>sigG</i></sub> that differ from the other σ<sup>F</sup>-target promoters: shorter spacing of the -10 and -35 promoter elements (14 nt as opposed to the more common 15 nt; left arrowhead), and a T at position -7 (more typically an A or G; right arrowhead). <b>(B, C)</b> P<sub><i>sigG</i></sub> activation is significantly stimulated by increasing the spacing between the -10 and -35 elements to 15 nt. β-Galactosidase production was monitored during sporulation of strains harboring P<sub><i>sigG</i></sub><i>-lacZ</i> (<i>WT</i> [14 nt]; open circles) and <sup><i>15nt</i></sup>P<sub><i>sigG</i></sub><i>-lacZ</i> (<i>15 nt</i>; closed circles), a variant in which a single nucleotide was inserted between the P<sub><i>sigG</i></sub> -10 and -35 elements to increase their spacing to 15 nt. (Strains JJB31 and JJB51, respectively.) Note that (C) provides a zoomed view of the data from the boxed area of (B). <b>(D, E)</b> The T at position -7 at most only modestly influences P<sub><i>sigG</i></sub> activation. The activity of P<sub><i>sigG</i></sub><i>-lacZ</i> (<i>WT</i>; closed circles),<sup><i>T→A</i></sup>P<sub><i>sigG</i></sub><i>-lacZ</i> (<i>T→A</i>; closed diamonds), and <sup><i>T→G</i></sup>P<sub><i>sigG</i></sub><i>-lacZ</i> (<i>T→G</i>; open diamonds) was measured during sporulation. (Strains JJB31, JJB87, and JJB89, respectively.) The <i>T→A</i> and <i>T→G</i> variants were engineered to harbor an A or G, respectively, at position -7 in place of T. Note that (E) provides a zoomed view of the data from the boxed area of (D). The timing of the σ<sup>F</sup>-to-σ<sup>G</sup> switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all relevant panels, error bars indicate ± standard deviations based on three independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01 Student’s <i>t</i>-test.</p
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