Biosurfactant-Producing Bacillus velezensis PW192 as an Anti-Fungal Biocontrol Agent against Colletotrichum gloeosporioides and Colletotrichum musae

In this study, plant-root-associated Bacillus species were evaluated as antifungal biocontrol agents by analyzing the production of surface bioactive molecules known as lipopeptide biosurfactants. This study aimed to isolate and characterize antifungal biosurfactant-producing Bacillus bacterium. Bacillusvelezensis PW192 was isolated from the rhizosphere of Lagerstroemia macrocarpa var macrocarpa and identified based on phylogenetic analysis of the 16S rRNA gene. The biosurfactant was excreted to cultured supernatant and exhibited emulsification power up to 60% and a decrease in surface tension from 72 in distilled water to 21 mN/m. The surface tension properties were stable in a broad range of pH from 6 to 10, in high temperatures up to 100 °C, and in salinities with a NaCl concentration up to 12% (w/v). Starting from 0.5 mg of acid, precipitated crude biosurfactant exhibited antifungal activity toward Anthracnose, caused by the phytopathogens Colletotrichum gloeosporioides and C. musae. The chemical structures of the biosurfactant were structurally characterized as lipopeptides fengycin A and fengycin B. The stability of the biosurfactant, as well as the antifungal properties of B. velezensis PW192, can potentially make them useful as agricultural biocontrol agents, as well as in other biotechnological applications

The CodY-dependent <i>clhAB2</i> operon is involved in cell shape, chaining and autolysis in <i>Bacillus cereus</i> ATCC 14579

<div><p>The Gram-positive pathogen <i>Bacillus cereus</i> is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of <i>B</i>. <i>cereus</i> ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the <i>clhAB</i>_<i>2</i> operon is required for this chain lengthening phenotype. The <i>clhAB</i>_<i>2</i> operon is specific to the <i>B</i>. <i>cereus</i> group (i.e., <i>B</i>. <i>thuringiensis</i>, <i>B</i>. <i>anthracis</i> and <i>B</i>. <i>cereus</i>) and encodes two membrane proteins of unknown function, which are homologous to the <i>Staphylococcus aureus</i> CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (Δ<i>clhAB</i>_<i>2</i>) was constructed and our quantitative image analyses show that Δ<i>clhAB</i>_<i>2</i> cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of Δ<i>clhAB</i>_<i>2</i> were significantly wider than wild-type cells (1.47 μm ±CI_95% 0.04 vs 1.19 μm ±CI_95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, Δ<i>clhAB</i>_<i>2</i> cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the <i>B</i>. <i>cereus clhAB</i>_<i>2</i> operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of <i>clhAB</i>_<i>2</i> using a <i>lacZ</i> transcriptional reporter in wild-type, <i>ccpA</i> and <i>codY</i> deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of <i>clhAB</i>_<i>2</i> expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of <i>clhAB</i>_<i>2</i> in glucose-grown cells.</p></div

Expression of <i>clhAB</i>_<i>2</i> in the presence or presence of glucose.

<p>(A) Genetic organization of <i>clhAB</i>_<i>2</i> locus in the <i>Bc</i> genome and schematic representation of the <i>clhAB</i>_<i>2</i>’Z transcriptional fusion construct. (B) <i>Bc</i> strain <i>clhAB</i>_<i>2</i>’Z was grown in LB in the presence (open circles) or absence (closed circles) of 0.35% glucose. Optical densities (OD₆₀₀, in blue circles) of cell cultures and β-galactosidase specific activities (U/mg protein, in black losanges) in the presence (open losanges) or absence (closed losanges) of 0.35% glucose are shown. The levels of <i>lacZ</i> expression of pHT304-18’Z (background level) were around 15 U/mg protein. (C) Filtered supernatants were measured for glucose concentration (Glucose Assay Kit, Sigma, in blue squares) and for pH determination (black triangles). The start of the transition growth phase is indicated as <i>t</i>₀ for time zero. The glucose concentration of LB medium (closed squares) was below 0.01% and pH (closed triangles) was 7 ± 0.2. The data presented are representative of four independent experiments.</p

Phenotypic analyses of Δ<i>clhAB</i>_<i>2</i> isogenic mutant.

<p>(A) Box plot of growth rates in the absence (LB) or presence of 0.35% glucose (LBG). Growth rates were determined using output files of OD₆₁₀ values from the microplate reader (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials & Methods</a>). NS—no statistical significance. (B, C) Stationary phase survival of <i>Bc</i> and Δ<i>clhAB</i>_<i>2</i> isogenic mutant in LB and in LBG. Cell viability tests were performed at 0, 4, 24, 48, and 72h. Significance is based on Mann & Whitney test with a <i>P</i> <0.05*.</p

Inter-constriction cell arrangements and cell width measures in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB2ΩclhAB</i>_<i>2</i> populations in the presence or absence of glucose.

<p>(A) Close view of <i>Bc</i>, Δ<i>clhAB2</i>, and Δ<i>clhAB2</i>,<i>ΩclhAB2</i> chains at <i>t</i>₀ using fluorescence microscopy. Cells were grown in LB and LB with 0.35% glucose (LBG). Division septa and cytoplasmic membranes were imaged using the FM4-64 lipophilic dye. Chains exhibited constrictions that occurred at septa spaced 4 cells apart in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i><i>ΩclhAB</i>_<i>2</i> in LB medium and in Δ<i>clhAB</i>_<i>2</i> in LBG medium. They also exhibited constrictions that occurred at septa spaced 8 cells apart in <i>Bc</i> and Δ<i>clhAB</i>_<i>2</i>,Ω<i>clhAB</i>_<i>2</i> in LBG medium. (B) Distributions of “short” (≤4) and “long” (>4) inter-constriction cell types in the <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and complemented mutant populations (N>250 cell arrangements). Two inter-constriction arrangement types in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> populations were defined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>). The first type, containing cell arrangements with two to four cells ("short") and the second type, including cell arrangements with six to eight cells ("long"). The significant effects of glucose (dashed line) and <i>clhAB</i>_<i>2</i> mutation (solid line) are based on a Binomial analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) with <i>P</i> <0.01** and <0.05*. (C) Cell width measures in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i> and Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> populations (N>250 bacilli) in LB and LBG media. The significant effect of <i>clhAB</i>_<i>2</i> mutation in LBG medium is based on a Student’s t test and ANOVA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) with <i>P</i> <0.001***. A <i>P</i> value close to the cutoff 0.05 was considered as non significant (NS). Mean ± CI _95% is depicted.</p

CodY-dependent regulation of the <i>clhAB</i>_<i>2</i> operon in the presence or absence of glucose.

<p>Effect of <i>codY</i> mutation on the expression of <i>clhAB</i>_<i>2</i>. Cells of <i>Bc</i> and isogenic mutant strains (Δ<i>codY</i>, <i>codY</i>-complemented mutant), which all harbored the transcriptional P_{<i>clhAB2</i>}’-<i>lacZ</i> fusion construct, were grown in LB (closed symbols) or in LBG (open symbols) media. See legend of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a> for additional informations.</p

Two models for <i>B</i>. <i>cereus clhAB</i>_<i>2</i> regulation.

<p>Left: regulation of <i>clhAB</i>_<i>2</i> expression in an amino acid- rich medium. In a nutrient-rich medium (such as LB medium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref013" target="_blank">13</a>]), ILV uptake is sufficient to maintain the endogenous pool of ILV. The CodY global regulatory protein displays enhanced affinity for its DNA target when bound to ILV[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref027" target="_blank">27</a>]. ILV-bound CodY binds to the CodY binding sequence upstream of <i>clhAB</i>_<i>2</i> and assists RNA polymerase with transcribing the <i>clhAB</i>_<i>2</i> operon (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g004" target="_blank">4</a>). The expression level of <i>clhAB</i>_<i>2</i> is constant and moderate (i.e. basal level) during the late exponential and transition growth phases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g001" target="_blank">Fig 1</a>). Right: regulation of <i>clhAB</i>_<i>2</i> expression in an amino acid- glucose- rich medium. In LB medium with 0.35% glucose, ILV-CodY binds to the CodY motif and activates <i>clhAB</i>_<i>2</i> expression, but the expression profile is different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g001" target="_blank">Fig 1</a>). CcpA plays a positive role by indirectly regulating the transcription of <i>clhAB</i>_<i>2</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a>) and this regulatory pathway remains to be characterized. The role of known or hypothetical effector molecules is depicted with a dashed arrow. In the left part, the ILV effector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref027" target="_blank">27</a>] is depicted with a green dashed arrow; in right part, the Fru-6-P [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref025" target="_blank">25</a>] and unknown glycolytic intermediate effectors are depicted with blue dashed arrows. Moderate and constant <i>clhAB</i>_<i>2</i> expression is depicted by three identical gray arrows. High and gradual <i>clhAB</i>_<i>2</i> expression is depicted by three non-identical gray arrows. The intracellular ILV pool is depicted as a green box. The CodY motif sequence is depicted as a red box. The unknown DNA-binding motif is depicted as a blue box.?, unknown CcpA-dependent signaling pathway; ?, unknown transcriptional regulator. Fru-6-P, fructose-6-phosphate, Glc-6-P, glucose-6-phosphate. ILV, isoleucine, leucine, valine. CodY, CodY transcriptional regulator. CcpA, CcpA global carbon regulator; PTS, phosphotransferase system. T, unknown transporter of ILV.</p

The glucose-activated expression of <i>clhAB</i>_<i>2</i> is abolished by the deletion of <i>ccpA</i>.

<p>Effect of <i>ccpA</i> mutation on the activation of <i>clhAB</i>_<i>2</i>. Cells of <i>Bc</i> and isogenic mutant strains (Δ<i>ccpA</i>, <i>ccpA</i>-complemented mutant), which all harbored the transcriptional P_{<i>clhAB2</i>}’-<i>lacZ</i> fusion construct, were grown in LB (closed symbols) or in LBG (open symbols) media. Samples were harvested at the indicated times and were assayed for β-galactosidase specific activity. Glucose 0.35% was added, when appropriate, at the onset of the culture. <i>t</i>_n is the number of hours before (-) or after <i>t</i>₀. SD bars are shown.</p

Chain lengths in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i><i>ΩclhAB</i>_<i>2</i> populations in the presence or absence of glucose.

<p>(A) Phase-contrast images of cell-chains at <i>t</i>₀ of wild-type (<i>Bc</i>), Δ<i>clhAB</i>_<i>2</i> (Δ), complemented (ΔΩ) and pHT315 (Δp) mutant strains grown in LB medium and LB medium supplemented with glucose (LBG). Images of chains revealed strong constrictions (deeper invaginations) corresponding to cells undergoing separation. Scale bar (5μm) is shown for each image. (B) Fluorescent micrographs of <i>Bc</i>, Δ, and ΔΩ cell-chains at <i>t</i>₀ in LB and LBG. Division septa and cytoplasmic membranes were imaged using the FM4-64 lipophilic dye. Scale bar (5μm) is shown for each image. (C) Box plots of chain length (number of cells per chain) at <i>t</i>₀ in <i>Bc</i> (blue), Δ<i>clhAB</i>_<i>2</i> (red), Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> (green) populations. Between 90 and 130 chains from at least three independent cultures were analysed. No fewer than 1,000 cells were quantitated for each strain represented in the graph using fluorescence micrographs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>). Median (strong line in the box), interquartile range (IQR; box), whiskers (1.5 x IQR) and outliers (dot) are presented. Significance is based on two tests, Mann-Whitney and Two-Sample Fisher-Pitman Permutation, with a <i>P</i> of <0.01**. Non significative (NS), <i>P</i>> 0.05.</p