Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in <i>Bacillus subtilis</i>

Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a “brute-force” approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for <i>Bacillus subtilis</i>. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong <i>veg</i> expression module. Analyses of the synthetic modules indicated that sequences at the 5′-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the <i>gfp</i> coding region was replaced by the coding region of the <i>xynA</i> gene, encoding the industrially relevant <i>B. subtilis</i> xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the <i>gfp</i> and <i>xynA</i> constructs were due to differences in 5′-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins

Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in <i>Bacillus subtilis</i>

Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a “brute-force” approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for <i>Bacillus subtilis</i>. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong <i>veg</i> expression module. Analyses of the synthetic modules indicated that sequences at the 5′-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the <i>gfp</i> coding region was replaced by the coding region of the <i>xynA</i> gene, encoding the industrially relevant <i>B. subtilis</i> xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the <i>gfp</i> and <i>xynA</i> constructs were due to differences in 5′-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins

ChECs antagonizing plant cell death and supporting multiplication of plant pathogenic bacteria.

<p>(A) Infiltration scheme for the transient co-expression assay. Agrobacteria containing constructs for ChEC or YFP expression were mixed with those for cell death-inducer (CDI) expression. Mixtures were infiltrated into opposite sides of <i>N. benthamiana</i> leaves to allow pair-wise comparisons and to take account of leaf-to-leaf variation in necrosis manifestation. Thus, an infiltrated site expressing YFP/ChNLP1 was included as an internal control in every infiltrated leaf, to which the site expressing ChEC/ChNLP1 was compared. (B, C) Examples of infiltration site pairs 8 dpi. ChEC3 abolishes ChNLP1-induced necrosis (B, dotted circle), but a fungal secreted chitinase does not (C). (D) Quantification of cell death-suppressing activity of four wave 2 effectors (ChEC3, 3a, 6, 36), three wave 3 effectors (ChEC89, 34, 13) and an <i>in vitro</i>-expressed effector (ChEC5). Histograms show the proportion of sites expressing ChEC/CDI that displayed reduced necrosis compared to control sites expressing YFP/CDI. *, ** and *** indicate significant difference from the respective chitinase control with and without signal peptide at P<0,02, <0.005 and <0.0002, respectively (Student's t-test). <i>P. infestans</i> effector Avr3a^KI was used as positive control for suppression of INF1-induced cell death. Data represent means of at least three independent experiments, with at least 15 leaves/experiment/co-expression combination (± standard error). (E) Bacterial titers in <i>Arabidopsis</i> Col-0 leaves infected with <i>Pseudomonas syringae</i> pv <i>tomato</i> expressing ChECs as fusions with a bacterial effector mediating delivery into plant cells <i>via</i> type III secretion. <i>Hyaloperonospora arabidopsidis</i> ATR13^Emco5<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643-Sohn1" target="_blank">[27]</a> and YFP were included as positive and negative controls, respectively. Colony forming units were determined 0 and 3 days after spray inoculation. * and ** indicate significant difference from the YFP control at P<0.03 and P<0.0005, respectively. Data represent means of 4 replicates (± standard error).</p

Expression profiling of selected biotrophy-associated ChEC and putative toxin genes by qRT-PCR.

<p>Expression levels are shown relative to the mean expression of actin and α-tubulin. Genes with highest expression <i>in planta</i> are highlighted with colours, indicating distinct waves of effector gene expression. <i>In vitro</i> cell types are: dormant spores (SP), saprotrophic mycelium (MY) and mature appressoria (VA). <i>In planta</i> stages are: unpenetrated appressoria (UA), penetrated appressoria with nascent biotrophic hyphae beneath (PA), biotrophy to necrotrophy switch (SW) and late necrotrophy (LN).</p

Appressorial pores as an interface for focal ChEC delivery.

<p>Transformant appressoria expressing the wave 2 effectors ChEC36:mRFP (A–O) and ChEC6:mRFP (P, Q). Appressoria or penetration sites after removal of appressoria were examined by confocal laser scanning microscopy viewed from above (A, B, E–I, O–Q) or from the side (C, D), and with transmission electron microscopy (J, K) and scanning electron microscopy (L–N). (A–D) Bright field and maximum fluorescence intensity overlay images of appressoria. Black arrows indicate the anticlinal plant cell wall and white arrows the penetration pore. (E) Fluorescence overlay image of an appressorium showing weak peripheral labelling of intracellular structures. (F, G) Fluorescence recorded with identical settings at the base (F) and the center (G) of the appressorium shown in (E). Arrow indicates a fluorescent ring surrounding the brightly fluorescing pore. (H, I) Fluorescence overlays recorded with identical settings focused on appressorial pores (H) or biotrophic hyphae (BH) formed beneath a penetrated appressorium (arrow). (J) Median section through an appressorium viewed with transmission electron microscopy (fixed with glutaraldehyde-osmium tetroxide and embedded in epoxy resin). A penetration hypha evaginates from the pore (P). An additional layer of the appressorial wall (asterisk) forms a thickened ring (arrowheads) around the pore, continuous with the penetration hypha wall. PW, plant cell wall. (K) Immunogold labelling of an appressorial pore (arrow) using antibodies recognizing mRFP (cells fixed in formaldehyde-glutaraldehyde and embedded in acrylic resin). PW, plant cell wall. WD, host cell wall deposits. (L) Scanning electron microscope image showing attached turgid appressorium (A) and collapsed conidium (C) on a leaf surface. (M) Plant-exposed underside of detached appressoria with penetration pores (black arrows) and remnants of extracellular matrix and/or plant cuticle (white arrow). (N, O) Penetration sites from which appressoria were detached completely. (N) The lobed outline of a former appressorium is still visible (arrowheads) with a mark representing the penetration point (arrow). (O) Micrograph series representing different focal planes as fluorescence overlay (top panels) and corresponding black on white conversion of the fluorescence channel (bottom panels), focusing from the penetration point (left) downwards into the plant cell wall (right). Arrow: inserted penetration hypha. (P, Q) Fluorescence overlays focused on the appressorial pore (P) and the underlying plant cell wall (Q). Arrow, anticlinal plant cell wall. Images were recorded at 24 hours post inoculation (hpi) (A–G, K, P, Q), 32 hpi (J, L–O), 40 hpi (H, I). Scale bars: 5 µm (A, H, L, N, O, P) and 2 µm (C, E, M), 1 µm (J), 500 nm (K). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s004" target="_blank">Figure S4</a>.</p

ChEC delivery to the biotrophic interface and host apoplast.

<p>Transformant biotrophic hyphae expressing CHEC89:mRFP (wave 3 effector) viewed with confocal laser scanning microscopy. (A) Maximum fluorescence intensity overlay projection of appressoria (arrows) and underlying biotrophic hyphae showing fluorescent foci (arrowheads) on the hyphal surface. (B) Single optical section from (A) showing labelling of the plant cell wall (arrows). (C, D) Mature biotrophic hypha, viewed as in (A and B), showing fluorescence accumulation in hyphal concavities (arrowheads). Arrow: appressorium. (E) Epidermal cell infected by a biotrophic hypha (arrows) showing fluorescence in the apoplastic space (*) enlarged by plasmolysis. Arrowheads demarcate the host plasma membrane. V, vacuole of the host protoplast. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s007" target="_blank">Figure S7</a> for the corresponding bright field image. (F) Unlabelled necrotrophic hypha (arrow) emerging from a biotrophic hypha. Images were recorded at 43 hpi (A–E) and 55 hpi (F). Scale bars: 10 µm (E) and 5 µm (A, C, F). BH, biotrophic hypha. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s006" target="_blank">Figure S6</a>.</p

ChECs accumulate in interfacial bodies and diffuse into the host cell wall.

<p>Transformant biotrophic hyphae expressing ChEC34:mRFP (wave 3 effector). (A, B) Bright field micrograph and corresponding maximum fluorescence intensity projection. Arrows: fluorescent foci. (C, D) Biotrophic hypha expressing CHEC34:mRFP viewed with confocal laser scanning microscopy settings optimized to show fluorescence in the penetrated epidermal cell wall (arrows). Arrowheads: unpenetrated wall of the same cell. (E, F) Transmission electron microscopy of a wild-type appressorium that produced a biotrophic hypha underneath with interfacial bodies (arrowheads). Arrows indicate the penetration site of the host cell wall. Cells were fixed with glutaraldehyde-osmium tetroxide and embedded in epoxy resin. (F) Close-up of an interfacial body (white asterisk) located between the plant plasma membrane (black arrowheads) and the fungal cell wall (black asterisk). (G, H) Immunogold cytochemistry using an antibody recognizing mRFP labels (G) interfacial bodies (arrows) or (H) the plant-fungal interface. Cells were fixed in formaldehyde-glutaraldehyde and embedded in acrylic resin. A, appressorium. FC, fungal cytoplasm. PC, plant cytoplasm. V, plant vacuole. BH, biotrophic hypha. (*) Fungal cell wall. Images were recorded at 40 hpi (A, B) and 43 hpi (C–H). Scale bars: 5 µm (A, C), 2.5 µm (E), 500 nm (F, G) and 250 nm (H).</p