Exoprotease Production Research Articles

Attenuation of biofilm and quorum sensing regulated virulence factors of an opportunistic pathogen Pseudomonas aeruginosa by phytofabricated silver nanoparticles

Green-synthesized nanoparticles provide an effective strategy for inhibiting microbial pathogenesis by affecting biofilm formation, quorum sensing (QS), and other surface properties of microorganisms. QS is a density-dependent communication signaling cascade that regulates biofilm formation and other pathogenic factors of Pseudomonas aeruginosa. In this context, the effect of phytofabricated silver nanoparticles (CC-AgNPs) synthesized using Cuphea carthagenensis extract on biofilm, QS, and QS-dependent virulence factors of P. aeruginosa were evaluated in this study. CC-AgNPs demonstrated significant attenuation of biofilm, QS, and QS-dependent virulence factors at sub-MICs. A significant inhibition of 88.39 ± 4.32 %, 79.64 ± 3.31 %, 73.07 ± 3.0 %, and 61.67 ± 1.5 % of biofilm formation, quorum sensing, pyocyanin, and LasB elastase, respectively was reported in the study at 20 μg/mL. The study also demonstrated a significant reduction of LasA Staphylolytic activity and 91.37 ± 1.05 % exoprotease production in comparison to untreated control. The lower concentrations of CC-AgNPs also demonstrated significant attenuation of biofilm and other virulence factors suggesting the strong potency of NPs against P. aeruginosa. XTT analysis reported the effect of CC-AgNPs on sessile cells of P. aeruginosa without impacting growth of planktonic cells at sub-MICs. Cell-proliferation study in human cell lines (HEK 293 and Caco-2 cells) demonstrated the safe nature of CC-AgNPs at tested concentrations. This study is novel in a way that environmentally friendly CC-AgNPs were used to inhibit QS at sub-MICs without killing the tested strains, therefore, could be developed as an anti-virulent drug to overcome biofilm and antimicrobial resistance problems.

Inhibition of growth, biofilm formation, virulence, and surface attachment of Agrobacterium tumefaciens by cinnamaldehyde derivatives.

Agrobacterium tumefaciens, a soil-borne, saprophytic plant pathogen that colonizes plant surfaces and induces tumors in a wide range of dicotyledonous plants by transferring and expressing its T-DNA genes. The limited availabilities and efficacies of current treatments necessitate the exploration of new anti-Agrobacterium agents. We examined the effects of trans-cinnamaldehyde (t-CNMA) and its derivatives on the cell surface hydrophobicity, exopolysaccharide and exo-protease production, swimming motility on agar, and biofilm forming ability of A. tumefaciens. Based on initial biofilm inhibition results and minimum inhibitory concentration (MIC) data, 4-nitro, 4-chloro, and 4-fluoro CNMAs were further tested. 4-Nitro, 4-chloro, and 4-fluoro CNMA at ≥150 μg/ml significantly inhibited biofilm formation by 94–99%. Similarly, biofilm formation on polystyrene or nylon was substantially reduced by 4-nitro and 4-chloro CNMAs as determined by optical microscopy and scanning electron microscopy (SEM) and 3-D spectrum plots. 4-Nitro and 4-chloro CNMAs induced cell shortening and concentration- and time-dependently reduced cell growth. Virulence factors were significantly and dose-dependently suppressed by 4-nitro and 4-chloro CNMAs (P ≤ 0.05). Gene expressional changes were greater after 4-nitro CNMA than t-CNMA treatment, as determined by qRT-PCR. Furthermore, some genes essential for biofilm formation, motility, and virulence genes significantly downregulated by 4-nitro CNMA. Seed germination of Raphanus sativus was not hindered by 4-nitro or 4-fluoro CNMA at concentrations ≤200 μg/ml, but root surface biofilm formation was severely inhibited. This study is the first to report the anti-Agrobacterium biofilm and anti-virulence effects of 4-nitro, 4-chloro, and 4-fluoro CNMAs and t-CNMA and indicates that they should be considered starting points for the development of anti-Agrobacterium agents.

PLANT GROWTH-PROMOTING TRAITS OF ANTARCTIC ENDOPHYTIC BACTERIA

Successful colonization of Antarctic lands by vascular plants Deschampsia antarctica and Colobanthus quitensis and their adaptation to stressful environments is associated not only with climate change but also with the functioning of microbial groups of phylo- and endosphere of these plants. The aim of our study was to screen plant growth-promoting traits in endophytic bacteria of antarctic vascular plants. Materials and methods. We have studied 8 bacterial cultures isolated from D. antarctica collected during the 25th Ukrainian Antarctic Expedition (January-April 2020) along the Western part of the Antarctic Peninsula. Overnight liquid cultures were obtained on Nutrient Broth medium (HiMedia, Ltd.) in a shaking incubator (26 ℃, 160 rpm). Bacterial isolates were grown on Ashby's combined-nitrogen-free medium with sucrose. Drop collapse assay for cyclic lipopeptide production (CLP), motility assay, exoprotease production and phosphate solubilizing ability were performed using generally accepted methods. Results. All studied isolates have shown plant growth-promoting traits. The most abundant were nitrogen-fixing activity and motility. Both these play important role in plant colonization and promoting the growth of plants in harsh environments. The evidences of CLP were shown by two strains only. There was no notice of phosphate solubilizing ability and exoprotease production. Conclusions. Endophytic bacteria of antarctic vascular plants could support the growth and nutrition needs of the plants.

Effect of the Monothiol Glutaredoxin GrxD on 2,4-Diacetylphloroglucinol Biosynthesis and Biocontrol Activity of Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a plant root-associated bacterium that suppresses several soilborne plant diseases due to its production of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The biosynthesis of 2,4-DAPG is controlled by many regulatory elements, including the global regulator of the Gac/Rsm regulon and the pathway-specific repressor PhlF. In this work, a novel genetic element grxD, which encodes the monothiol glutaredoxin GrxD, was identified and characterized in the production of 2,4-DAPG in P. fluorescens 2P24. Our data showed that the mutation of grxD remarkably decreased 2,4-DAPG production. GrxD lost its ability to alter the production of 2,4-DAPG when the active-site CGFS motif of GrxD was mutated by site-directed mutagenesis. Further studies showed that the RsmA and RsmE proteins were essential for the GrxD-mediated regulation of 2,4-DAPG and exoprotease production. In addition, our data revealed that the deletion of grxD increased the expression of phlF, which negatively regulated the production of 2,4-DAPG. In addition, the grxD mutant was severely impaired in the biocontrol effect against the bacterial wilt of tomato. Overall, our results indicated that the monothiol glutaredoxin GrxD is involved in the production of 2,4-DAPG of P. fluorescens by influencing the Gac/Rsm global signaling pathway and transcriptional regulator PhlF and is essential for the biocontrol properties.

Effect of halogenated indoles on biofilm formation, virulence, and root surface colonization by Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease in several plant species by transferring its T-DNA to the host genome. Its chemotactic response to a range of chemical compounds released by hosts facilitates its colonization to host surfaces, and thus, novel anti-agrobacterium compounds are needed to prevent its biofilm formation. Here, we investigated 83 indole derivatives against A. tumefaciens, and based on the screening, 4-chloroindole, 6-iodoindole, and 5-chloro-2-methyl indole were selected as candidates that at 50 μg mL−1 significantly inhibited the adherence and biofilm formation of A. tumefaciens to abiotic (nitrocellulose and polystyrene) and biotic (roots of Brassica juncea) surfaces. Furthermore, they reduced bacterial growth in a time and concentration-dependent manner and significantly reduced log CFU mL−1 and survival (%). Changes in biofilm morphologies and biomasses, thicknesses, and substratum coverages were determined, and 2-D and 3-D analyses were performed using a crystal violet assay and bright field, CLSM, and SEM microscopies. Virulence factors such as swimming motility, exopolysaccharide, and exo-protease production, and cell surface hydrophobicity were markedly inhibited by the three compounds. Transcriptional analysis showed multi-fold downregulation of biofilm, virulence, motility, and stress-related genes; however, the degrees of these downregulations were variably affected. B. juncea seed germination was only severely affected by 4-chloroindole. This study demonstrates the promising antibiofilm and antivirulence activities of the three indole derivatives tested and their potentials for targeting and curbing A. tumefaciens infections.

Iron limitation by transferrin promotes simultaneous cheating of pyoverdine and exoprotease in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13-33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.

Rhamnolipids stabilize quorum sensing mediated cooperation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main models to study social behaviors in bacteria since it synthesizes several exoproducts, including exoproteases and siderophores and release them to the environment. Exoproteases and siderophores are public goods that can be utilized by the individuals that produce them but also by non-producers, that are considered social cheaters. Molecularly exoprotease cheaters are mutants in regulatory genes such as lasR, and are commonly isolated from chronic infections and selected in the laboratory upon serial cultivation in media with protein as a sole carbon source. Despite that the production of exoproteases is exploitable, cooperators have also ways to restrict the growth and selection of social cheaters, for instance by producing toxic metabolites like pyocyanin. In this work, using bacterial competitions, serial cultivation and growth assays, we demonstrated that rhamnolipids which production is regulated by quorum sensing, selectively affect the growth of lasR mutants and are able to restrict social cheating, hence contributing to the maintenance of cooperation in Pseudomonas aeruginosa populations.

Seeding Public Goods Is Essential for Maintaining Cooperation in Pseudomonas aeruginosa.

Quorum sensing in Pseudomonas aeruginosa controls the production of costly public goods such as exoproteases. This cooperative behavior is susceptible to social cheating by mutants that do not invest in the exoprotease production but assimilate the amino acids and peptides derived by the hydrolysis of proteins in the extracellular media. In sequential cultures with protein as the sole carbon source, these social cheaters are readily selected and often reach equilibrium with the exoprotease producers. Nevertheless, an excess of cheaters causes the collapse of population growth. In this work, using the reference strain PA14 and a clinical isolate from a burn patient, we demonstrate that the initial amount of public goods (exoprotease) that comes with the inoculum in each sequential culture is essential for maintaining population growth and that eliminating the exoprotease in the inoculum leads to rapid population collapse. Therefore, our results suggest that sequential washes should be combined with public good inhibitors to more effectively combat P. aeruginosa infections.

Repression of VvpM Protease Expression by Quorum Sensing and the cAMP-cAMP Receptor Protein Complex in Vibrio vulnificus.

Septicemia-causing Vibrio vulnificus produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is induced in the stationary phase by multiple transcription factors, including sigma factor S, SmcR, and the cAMP-cAMP receptor protein (cAMP-CRP) complex. Distinct roles of VvpM, such as induction of apoptosis, lead us to hypothesize VvpM expression is different from that of the other exoproteases. Its transcription, which was found to be independent of sigma S, is induced at the early exponential phase and then becomes negligible upon entry into the stationary phase. SmcR and CRP were studied regarding the control of vvpM expression. Transcription of vvpM was repressed by SmcR and cAMP-CRP complex individually, which specifically bound to the regions -2 to +20 and +6 to +27, respectively, relative to the vvpM transcription initiation site. Derepression of vvpM gene expression was 10- to 40-fold greater in an smcR crp double mutant than in single-gene mutants. Therefore, these results show that the expression of V. vulnificus exoproteases is differentially regulated, and in this way, distinct proteases can engage in specific interactions with a host.IMPORTANCE An opportunistic human pathogen, Vibrio vulnificus produces multiple extracellular proteases that are involved in diverse interactions with a host. The total exoproteolytic activity is detected mainly in the supernatants of the high-cell-density cultures. However, some proteolytic activity derived from a metalloprotease, VvpM, was present in the supernatants of the low-cell-density cultures sampled at the early growth period. In this study, we present the regulatory mechanism for VvpM expression via repression by at least two transcription factors. This type of transcriptional regulation is the exact opposite of those for expression of the other V. vulnificus exoproteases. Differential regulation of each exoprotease's production then facilitates the pathogen's participation in the distinct interactions with a host.

ComX-Induced Exoproteases Degrade ComX in Bacillus subtilis PS-216.

Gram-positive bacteria use peptides as auto-inducing (AI) signals to regulate the production of extracellular enzymes (e.g., proteases). ComX is an AI peptide, mostly known for its role in the regulation of bacterial competence and surfactant production in Bacillus subtilis. These two traits are regulated accordingly to the bacterial population size, thus classifying ComX as a quorum sensing signal. ComX also indirectly regulates exoprotease production through the intermediate transcriptional regulator DegQ. We here use this peptide-based AI system (the ComQXPA system) as a model to address exoprotease regulation by ComX in biofilms. We also investigate the potential of ComX regulated proteases to degrade the ComX AI peptide. Results indicate that ComX indeed induces the expression of aprE, the gene for the major serine protease subtilisin, and stimulates overall exoprotease production in biofilms of B. subtilis PS-216 and several other B. subtilis soil isolates. We also provide evidence that these exoproteases can degrade ComX. The ComX biological activity decay is reduced in the spent media of floating biofilms with low proteolytic activity found in the comP and degQ mutants. ComX biological activity decay can be restored by the addition of subtilisin to such media. In contrast, inhibition of metalloproteases by EDTA reduces ComX biological activity decay. This suggests that both serine and metalloproteases, which are induced by ComX, are ultimately capable of degrading this signaling peptide. This work brings novel information on regulation of exoproteases in B. subtilis floating biofilms and reveals that these proteolytic enzymes degrade the AI signaling peptide ComX, which is also a major determinant of their expression in biofilms.

Functionality of Root-Associated Bacteria along a Salt Marsh Primary Succession.

Plant-associated bacteria are known for their high functional trait diversity, from which many are likely to play a role in primary and secondary succession, facilitating plant establishment in suboptimal soils conditions. Here we used an undisturbed salt marsh chronosequence that represents over 100 years of soil development to assess how the functional traits of plant associated bacteria respond to soil type, plant species and plant compartment. We isolated and characterized 808 bacterial colonies from the rhizosphere soil and root endosphere of two salt marsh plants, Limonium vulgare and Artemisia maritima, along the chronosequence. From these, a set of 59 strains (with unique BOX-PCR patterns, 16S rRNA sequence and unique to one of the treatments) were further screened for their plant growth promoting traits (siderophore production, IAA production, exoprotease production and biofilm formation), traits associated with bacterial fitness (antibiotic and abiotic stress resistance – pH, osmotic and oxidative stress, and salinity) and metabolic potential. An overall view of functional diversity (multivariate analysis) indicated that the distributional pattern of bacterial functional traits was driven by soil type. Samples from the late succession (Stage 105 year) showed the most restricted distribution, harboring strains with relatively low functionalities, whereas the isolates from intermediate stage (35 year) showed a broad functional profiles. However, strains with high trait performance were largely from stage 65 year. Grouping the traits according to category revealed that the functionality of plant endophytes did not vary along the succession, thus being driven by plant rather than soil type. In opposition, the functionality of rhizosphere isolates responded strongly to variations in soil type as observed for antibiotic resistance (P = 0.014). Specifically, certain Pseudomonas sp. and Serratia sp. strains revealed high resistance against abiotic stress and antibiotics and produce more siderophores, confirming the high plant-growth promoting activity of these two genera. Overall, this study contributes to a better understanding of the functional diversity and adaptation of the microbiome at typical salt marsh plant species across soil types. Specifically, soil type was influential only in the rhizosphere but not on the endosphere, indicating a strong plant-driven effect on the functionality of endophytes.

Biofilm formation by Vibrio parahaemolyticus on food and food contact surfaces increases with rise in temperature

Vibrio parahaemolyticus is recognized as a human foodborne pathogen that is mostly associated with seafood. This pathogen can form a mature biofilm on food and food contact surfaces during food processing. The present study investigated V. parahaemolyticus biofilm formation at various temperatures on shrimp, crab, and stainless steel coupons, using the biofilm formation index (BFI) method on microtiter plates. The results were also confirmed by field emission scanning electron microscopy. Both the BFI values and cultural counts revealed that V. parahaemolyticus biofilm formation was stronger at higher temperatures than at lower temperatures. Biofilm formation differed according to the growth surface type and growth temperature. It was found that higher temperatures (15–37 °C) induced stronger biofilm formation whereas 4 and 10 °C resulted in attachment of the bacterial cells as monolayers. It could be concluded that temperatures of 25–37 °C result in significantly stronger biofilm formation as well as exoprotease and AI-2 production on food and food contact surfaces, indicating that these temperatures might be threatening conditions for food safety.

Quantification of the potential biocontrol and direct plant growth promotion abilities based on multiple biological traits distinguish different groups of Pseudomonas spp. isolates

Members of the Pseudomonas genus colonize the rhizosphere of different plant species and display plant-probiotic traits. Therefore, they are interesting candidates for biological agricultural inputs to stimulate plant development and/or promote crop health. We have generated a collection of 19 isolates of pseudomonads obtained from either bulk soil or the rhizosphere of healthy individuals of major extensive crops from different plots under no-till management located in Argentina. Isolates were selected for their ability to antagonize several fungal pathogens recovered from infected soybean and maize plants. Partial sequencing of 16S rDNA, oprF and rpoB genes positioned isolates within all major pseudomonads groups. Most isolates colonized the rhizosphere of soybean and maize seedlings without affecting germination or development. Members of the Pseudomonas chlororaphis subgroup, and one isolate of the Pseudomonas putida subgroup, displayed the widest antifungal spectrum and strongest antagonistic potential. A principal component analysis based on the outcome of quantitative and qualitative tests related to biocontrol of fungal pathogens, direct plant growth promotion, and other root colonization-related traits, showed that isolates with the highest antagonistic potential and mostly of soil origin were grouped together, whereas rhizospheric isolates and those with strong flagellar-dependent motility, exoprotease production and biofilm development, were clustered in a separate group. Interestingly, quantitative comparison of newly developed biocontrol and direct growth promotion indices revealed that these two plant probiotic traits are rather associated to different set of isolates, and rarely present in the same pseudomonad strains. These indices could be employed to screen and categorize isolates by their potential to act as a biocontrol or a biofertilizer agent.

Integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models reveals that pathogenicity is host specific.

Pseudomonas aeruginosa is a multi-host opportunistic pathogen causing a wide range of diseases because of the armoury of virulence factors it produces, and it is difficult to eradicate because of its intrinsic resistance to antibiotics. Using an integrated whole-genome approach, we searched for P. aeruginosa virulence genes with multi-host relevance. We constructed a random library of 57 360 Tn5 mutants in P. aeruginosa PAO1-L and screened it in vitro for those showing pleiotropic effects in virulence phenotypes (reduced swarming, exo-protease and pyocyanin production). A set of these pleiotropic mutants were assayed for reduced toxicity in Drosophila melanogaster, Caenorhabditis elegans, human cell lines and mice. Surprisingly, the screening revealed that the virulence of the majority of P. aeruginosa mutants varied between disease models, suggesting that virulence is dependent on the disease model used and hence the host environment. Genomic analysis revealed that these virulence-related genes encoded proteins from almost all functional classes, which were conserved among P. aeruginosa strains. Thus, we provide strong evidence that although P. aeruginosa is capable of infecting a wide range of hosts, many of its virulence determinants are host specific. These findings have important implication when searching for novel anti-virulence targets to develop new treatments against P. aeruginosa.

The phosphorylation flow of the Vibrio harveyi quorum-sensing cascade determines levels of phenotypic heterogeneity in the population.

Quorum sensing (QS) is a communication process that enables a bacterial population to coordinate and synchronize specific behaviors. The bioluminescent marine bacterium Vibrio harveyi integrates three autoinducer (AI) signals into one quorum-sensing cascade comprising a phosphorelay involving three hybrid sensor kinases: LuxU; LuxO, an Hfq/small RNA (sRNA) switch; and the transcriptional regulator LuxR. Using a new set of V. harveyi mutants lacking genes for the AI synthases and/or sensors, we assayed the activity of the quorum-sensing cascade at the population and single-cell levels, with a specific focus on signal integration and noise levels. We found that the ratios of kinase activities to phosphatase activities of the three sensors and, hence, the extent of phosphorylation of LuxU/LuxO are important not only for the signaling output but also for the degree of noise in the system. The pools of phosphorylated LuxU/LuxO per cell directly determine the amounts of sRNAs produced and, consequently, the copy number of LuxR, generating heterogeneous quorum-sensing activation at the single-cell level. We conclude that the ability to drive the heterogeneous expression of QS-regulated genes in V. harveyi is an inherent feature of the architecture of the QS cascade. V. harveyi possesses one of the most complex quorum-sensing (QS) cascades known, using three different autoinducers (AIs) to control the induction of, e.g., bioluminescence, virulence factors, and biofilm and exoprotease production. We constructed various V. harveyi mutants to study the impact of each component and subsystem of the QS signaling cascade on QS activation at the population and single-cell levels. We found that the output was homogeneous only in the presence of all AIs. In the absence of any one AI, QS activation varied from cell to cell, resulting in phenotypic heterogeneity. This study elucidates a molecular design principle which enables a tightly integrated signaling cascade to control the expression of diverse phenotypes within a genetically homogeneous population.

Effect of salinity and incubation time of planktonic cells on biofilm formation, motility, exoprotease production, and quorum sensing of Aeromonas hydrophila

The aim of this study was to determine the effect of salinity and age of cultures on quorum sensing, exoprotease production, and biofilm formation by Aeromonas hydrophila on stainless steel (SS) and crab shell as substrates. Biofilm formation was assessed at various salinities, from fresh (0%) to saline water (3.0%). For young and old cultures, planktonic cells were grown at 30 °C for 24 h and 96 h, respectively. Biofilm formation was assessed on SS, glass, and crab shell; viable counts were determined in R2A agar for SS and glass, but Aeromonas-selective media was used for crab shell samples to eliminate bacterial contamination. Exoprotease activity was assessed using a Fluoro™ protease assay kit. Quantification of acyl-homoserine lactone (AHL) was performed using the bioreporter strain Chromobacterium violaceum CV026 and the concentration was confirmed using high-performance liquid chromatography (HPLC). The concentration of autoinducer-2 (AI-2) was determined with Vibrio harveyi BB170. The biofilm structure at various salinities (0–3 %) was assessed using field emission electron microscopy (FESEM). Young cultures of A. hydrophila grown at 0–0.25% salinity showed gradual increasing of biofilm formation on SS, glass and crab shell; swarming and swimming motility; exoproteases production, AHL and AI-2 quorum sensing; while all these phenotypic characters reduced from 0.5 to 3.0% salinity. The FESEM images also showed that from 0 to 0.25% salinity stimulated formation of three-dimensional biofilm structures that also broke through the surface by utilizing the chitin surfaces of crab, while 3% salinity stimulated attachment only for young cultures. However, in marked contrast, salinity (0.1–3%) had no effect on the stimulation of biofilm formation or on phenotypic characters for old cultures. However, all concentrations reduced biofilm formation, motility, protease production and quorum sensing for old culture. Overall, 0–0.25% salinity enhanced biofilm formation and expression of quorum sensing regulatory genes in young cultures, whereas these responses were reduced when salinity was >0.25%. In old cultures, salinity at any concentrations (0.1–3%) induced stress in A. hydrophila. The present study provides insight into the ecology of A. hydrophila growing on fish and crustaceans such as shrimp and crabs in estuarine and seawater.

Phosphorylation of DegU is essential for activation of amyE expression in Bacillus subtilis.

Alpha-amylase (amyE) is one of the major exo-enzymes secreted by Bacillus subtilis during the post-exponential phase. The DegS-DegU two-component system regulates expression of majority of post-exponentially expressed genes in B. subtilis. It has been demonstrated that varying levels of the phosphorylated form of DegU (DegU-P) control different cellular processes. Exo-protease production is observed when effective concentration of DegU-P rises in the cell, whereas swarming motility is favoured at very low amounts of DegU-P. In this study we show that like other exo-proteases, expression of amyE is positively regulated by increase in DegU-P levels in the cell. We also demonstrate that residues at the DNA-binding helix-turn-helix (HTH) motif of DegU are necessary for the amyE expression. This observation is further reinforced by demonstrating the direct interaction of DegU on amyE promoter.

Phosphorylation of Bacillus subtilis gene regulator AbrB modulates its DNA‐binding properties

AbrB is a global gene regulator involved in transition phase phenomena in Bacillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B. subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86D and S86A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator.

Regulation of the Response Regulator Gene degU through the Binding of SinR/SlrR and Exclusion of SinR/SlrR by DegU in Bacillus subtilis

Bacillus subtilis DegU is a response regulator of the DegS-DegU two-component regulatory system. Phosphorylated DegU (DegU-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the degU gene, by binding to target gene promoters. Nonphosphorylated DegU and low levels of DegU-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed degU. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in degU expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the degU promoter and that SlrR formed a complex with SinR on the degU promoter. In EMSA, DegU-P excluded the SinR/SlrR complex but not SinR from the degU promoter in the presence of RNA polymerase. These findings suggest that DegU-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased degU expression. This newly identified regulatory mechanism for degU is considered to be sequential transcription factor replacement.

The prevalence and origin of exoprotease-producing cells in the Bacillus subtilis biofilm

Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.