DataSheet1_Frequency dependent growth of bacteria in living materials.pdf

The fusion of living bacteria and man-made materials represents a new frontier in medical and biosynthetic technology. However, the principles of bacterial signal processing inside synthetic materials with three-dimensional and fluctuating environments remain elusive. Here, we study bacterial growth in a three-dimensional hydrogel. We find that bacteria expressing an antibiotic resistance module can take advantage of ambient kinetic disturbances to improve growth while encapsulated. We show that these changes in bacterial growth are specific to disturbance frequency and hydrogel density. This remarkable specificity demonstrates that periodic disturbance frequency is a new input that engineers may leverage to control bacterial growth in synthetic materials. This research provides a systematic framework for understanding and controlling bacterial information processing in three-dimensional living materials.</p

Msb1 Interacts with Cdc42, Boi1, and Boi2 and May Coordinate Cdc42 and Rho1 Functions during Early Stage of Bud Development in Budding Yeast

<div><p>Msb1 is not essential for growth in the budding yeast <i>Saccharomyces cerevisiae</i> since <i>msb1</i>Δ cells do not display obvious phenotypes. Genetic studies suggest that Msb1 positively regulates Cdc42 function during bud development, since high-copy <i>MSB1</i> suppressed the growth defect of temperature-sensitive <i>cdc24</i> and <i>cdc42</i> mutants at restrictive temperature, while deletion of <i>MSB1</i> showed synthetic lethality with <i>cdc24</i>, <i>bem1</i>, and <i>bem2</i> mutations. However, the mechanism of how Msb1 regulates Cdc42 function remains poorly understood. Here, we show that Msb1 localizes to sites of polarized growth during bud development and interacts with Cdc42 in the cells. In addition, Msb1 interacts with Boi1 and Boi2, two scaffold proteins that also interact with Cdc42 and Bem1. These findings suggest that Msb1 may positively regulate Cdc42 function by interacting with Cdc42, Boi1, and Boi2, which may promote the efficient assembly of Cdc42, Cdc24, and other proteins into a functional complex. We also show that Msb1 interacts with Rho1 in the cells and Msb1 overproduction inhibits the growth of <i>rho1-104</i> and <i>rho1-3</i> but not <i>rho1-2</i> cells. The growth inhibition appears to result from the down-regulation of Rho1 function in glucan synthesis, specifically during early stage of bud development. These results suggest that Msb1 may coordinate Cdc42 and Rho1 functions during early stage of bud development by promoting Cdc42 function and inhibiting Rho1 function. Msb1 overproduction also affects cell morphology, septin organization, and causes increased, aberrant deposition of 1,3-β-glucan and chitin at the mother-bud neck. However, the stimulation of glucan synthesis mainly occurs during late, but not early, stage of bud development.</p></div

<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

Msb1 overproduction inhibits the growth and glucan synthesis in <i>rho1-104</i> cells.

<p>(<b>A</b>) Overexpression of <i>MSB1</i> inhibits the growth of <i>rho1-104</i> cells. Cells of yeast strain NY1537 (WT) and NY1538 (<i>rho1-104</i>) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing dextrose (Dex) or galactose and raffinose (Gal) at 30°C. Pictures were taken after 3 d. (<b>B</b>) Overexpression of <i>MSB1</i> in <i>rho1-104</i> cells causes the accumulation of small-budded cells. The percentage of small buds in the population of budding cells overexpressing <i>MSB1</i> as in panel A was quantitated. More than 200 buds were scored. (<b>C</b>) Glucan distribution in <i>rho1-104</i> cells overexpressing <i>MSB1</i>. Cells as in panel A were stained for 1,3-β-glucan. Bar, 5 µm.</p

Table_2_Bacterial growth stage determines the yields, protein composition, and periodontal pathogenicity of Porphyromonas gingivalis outer membrane vesicles.xls

IntroductionP. gingivalis (W83), as the keystone pathogen in chronic periodontitis, has been found to be tightly bound to systemic diseases. Outer membrane vesicles (OMVs) produced by P. gingivalis (W83) are thought to serve key functions in bacterial virulence and pathogenicity. This study aims to comprehend the biological functions of P. gingivalis OMVs isolated from different growth stages by comparing their physicochemical properties and pathogenicity.MethodsProtein composition was analyzed via isotope-labeled relative and absolute quantification (iTRAQ). Macrophage polarization and the expression of IL-6 and IL-1β were detected. The proliferation, migration, osteogenic differentiation, and IL-1b/NLRP3 expression of periodontal ligament stem cells (PDLSCs) were evaluated. P. gingivalis/P. gingivalis OMVs-induced periodontal models were also constructed in Sprague Dawley rats.ResultsThe protein composition of P. gingivalis OMVs isolated from different growth stages demonstrated obvious differences ranging from 25 KDa to 75 KDa. In the results of flow cytometry, we found that in vitro experiments the M1 subtype of macrophages was more abundant in the late-log OMVs and stationary OMVs groups which boosted the production of inflammatory cytokines more than pre-log OMVs. Compared to pre-log OMVs, late-log OMVs and stationary OMVs had more pronounced inhibitory effects on proliferation, migration, and early osteogenesis of PDLSCs. The NLRP3 inflammasome was activated to a larger extent in the stationary OMVs group. Micro-computed tomography (Micro CT), hematoxylin-eosin staining (HE), and tartrate acid phosphatase (TRAP) results showed that the periodontal damage in the stationary OMVs group was worse than that in the pre-log OMVs and late-log OMVs group, but almost equal to that in the positive control group (P. gingivalis).DiscussionIn general, both in vivo and in vitro experiments showed that late-log OMVs and stationary OMVs have more significant pathogenicity in periodontal disease.</p

Msb1 inhibits Rho1 function and interacts with Rho1 <i>in vivo</i>.

<p>(<b>A</b>) Overexpression of <i>MSB1</i> inhibits the growth of <i>rho1-3</i> cells. Cells of yeast strain NY2284 (WT), NY2285 (<i>rho1-2</i>), and NY2286 (<i>rho1-3</i>) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing dextrose (Dex) or galactose and raffinose (Gal) at 30°C. Pictures were taken after 3 d. (<b>B</b>) Overexpression of <i>MSB1</i> does not inhibit the growth of <i>cdc42</i>-Ts cells. Cells of yeast strain YEF473A (WT), YEF2258 (<i>cdc42-201</i>), and JPC241 (<i>cdc42^G60D</i>) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing dextrose (Dex) or galactose and raffinose (Gal) at 30°C. Pictures were taken after 4 d. (<b>C</b>) Msb1 interacts with Rho1 by GST pull-down assay. Cells of yeast strain YEF1395 (<i>msb1</i>Δ) carrying YEp181-3HA-MSB1 along with pEGKT (GST), pEGKT-CDC42 (Cdc42), or pEGKT-RHO1 (Rho1) were used in the assay. Molecular weight: GST (27 kDa), GST-Cdc42 (46 kDa), GST-Rho1 (48 kDa).</p

Msb1 localizes to sites of polarized growth and interacts with Cdc42.

<p>(<b>A</b>) GFP-Msb1 localization during bud development. Cells of yeast strain YEF1395 (<i>msb1</i>Δ) carrying plasmid pRS426-GFP-MSB1 were grown in SC-Ura medium and examined for GFP fluorescence. Bar, 5 µm. (<b>B</b>) Msb1 interacts with Cdc42 by GST pull-down assay. Cells of yeast strain YEF473A carrying YEp181-3HA-MSB1 along with pEGKT (GST), pEGKT-CDC42 (GST-Cdc42, WT), pEGKT-CDC42^Q61L (GST-Cdc42, Q61L), or pEGKT-CDC42^T17N (GST-Cdc42, T17N) were grown in SC-Leu-Ura medium containing 2% raffinose at 30°C. Galactose was added to a final concentration of 2%, and the cultures were grown for 4 h to induce the expression of GST-fusion proteins. GST or GST-tagged proteins were pulled down by glutathione-Sepharose beads from equal amounts of Triton X-100-solubilized cell lysates. Molecular weight: GST (27 kDa), GST-Cdc42 (46 kDa), HA-Msb1 (130 kDa).</p

Functional interaction between Msb1 and Boi1/Boi2.

<p>(<b>A</b>) Morphology of <i>boi1</i>Δ, <i>boi2</i>Δ, and <i>boi1</i>Δ <i>boi2</i>Δ cells overexpressing <i>MSB1</i>. Cells of yeast strains JGY2425 (<i>boi1</i>Δ), JGY2349 (<i>boi2</i>Δ), and JGY2821 (<i>boi1</i>Δ <i>boi2</i>Δ) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing galactose and raffinose for 12 h. (<b>B</b>) Morphology of cells with elevated expression of <i>MSB1</i> and <i>BOI2</i>. Cells of yeast strain YEF473A carrying plasmids YEp13-MSB1/pUG36 (MSB1↑), YEp13/pUG36-BOI2 (BOI2↑), or YEp13-MSB1/pUG36-BOI2 (MSB1↑ BOI2↑) were grown on SC-Leu-Ura plate containing dextrose at 30°C for 16 h. Bars, 5 µm.</p

Detection of Msb1 interaction with Bem1, Cdc24, Boi1, and Boi2.

<p>(<b>A</b>) Msb1 interacts with Boi2 and Boi1 by GST pull-down assay. Cells of yeast strain YEF1395 (<i>msb1</i>Δ) carrying YEp181-3HA-MSB1 along with pEGKT (GST), pEGKT-CDC42 (Cdc42), pEGKT-BEM1 (Bem1), pEGKT-CDC24 (Cdc24), pEGKT-BOI1 (Boi1), or pEGKT-BOI2 (Boi2), as well as cells of yeast strain JGY2425 (<i>boi1</i>Δ) or JGY2349 (<i>boi2</i>Δ) carrying YEp181-3HA-MSB1/pEGKT or YEp181-3HA-MSB1/pEGKT-BOI1 were used in the assay. (<b>B</b>) Msb1 interacts with the C-terminal region of Boi1 and Boi2 lacking the proline-rich motif. Left panel, the schematic diagram of domain structure in Boi1 and Boi2. P, proline-rich motif. Right panel, GST pull-down assay with GST-tagged Boi2, Boi2-C, and Boi1-C in yeast strains YEF473A (WT), JGY2425 (<i>boi1</i>Δ), and JGY2349 (<i>boi2</i>Δ). (<b>C</b>) Msb1 interacts with Cdc42 in <i>boi1</i>Δ <i>boi2</i>Δ cells. GST pull-down assay was performed in cells of yeast strain YEF473A (WT) and JGY2821 (<i>boi1</i>Δ <i>boi2</i>Δ) carrying YEp181-3HA-MSB1/pEGKT or YEp181-3HA-MSB1/pEGKT-CDC42. Molecular weight of GST-tagged proteins: Cdc42 (46 kDa), Bem1 (86 kDa), Cdc24 (120 kDa), Boi1 (133 kDa), Boi2 (140 kDa), Boi1-C (88 kDa), and Boi2-C (90 kDa).</p

Phenotypes of cells overexpressing <i>MSB1</i>.

<p>(<b>A</b>) Morphology and septin organization (shown by GFP-Cdc3) in cells overexpressing <i>MSB1</i>. Cells of yeast strain JGY881 (<i>GFP-CDC3</i>) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing galactose and raffinose at 30°C for 16 h. DIC and GFP fluorescence images were taken. (<b>B</b>, <b>C</b>) Chitin deposition (<b>B</b>) as well as 1,3-β-glucan and mannan distribution (<b>C</b>) were visualized in cells of strain YEF473A carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) grown in SC-Ura medium containing galactose and raffinose at 30°C. The cells were stained for chitin, 1,3-β-glucan, and mannan. (<b>D</b>) Msb1 localizes to sites of aberrant glucan deposition. Cells of strain JGY139A (<i>GAL1-GFP-MSB1</i>) was grown in SC-Ura medium containing galactose and raffinose at 30°C. Cells were stained for 1,3-β-glucan with aniline blue. Bars, 5 µm.</p