1,721,024 research outputs found
The molecular function of SwrA: an auxiliary factor modulating DegU transcriptional activity
No full textWe recently demonstrated that the main fla/che promoter PA(fla/che) can be bound by the phosphorylated form of DegU (DegU~P) with two opposite outcomes: complete repression if DegU~P alone is bound to PA(fla/che) DNA; transcriptional stimulation if DNA is bound by DegU~P complexed with SwrA.
Thus SwrA, which is necessary for swarming motility, constitutes an auxiliary factor that modulates the transcriptional activity of the response regulator DegU turning it from a repressor into an activator of PA(fla/che). Evidences indicate that SwrA might modulate other DegU~P-regulated promoters.
Also, we demonstrated that DegU32(Hy) is a mutant protein unable to functionally interact with SwrA at the fla/che promoter; the phenotype of degU32(Hy) strains differs from that of the wild type DegU~P and we suggest the use of degS200(Hy) mutant strains for studies aimed at analyzing the effect of the level of DegU phosphorylation.
SwrA is coded by a gene containing a slippery poly-adenine tract that allows phase variations between a functional and a non-functional allelic state.
In swrA+ cells (typically in undomesticated strains) fla/che transcription oscillates from the basal/medium level to the activated state that is required for swarming. When swrA is in the non-functional form (e.g. in the 168 laboratory strain) fla/che transcription can oscillate between a repressed state in which no flagella are made and a basal/medium level of transcription sufficient for a limited swimming motility. While in both swrA- and swrA+ strains oscillations depend on phosphorylation of DegU mediated by environmental stimuli, in swrA- cells the secondary fla/che promoter PD3(fla/che) plays an important role that might constitute the bistable switch acting on motility
Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical Review
No full textPoly-gamma glutamic acid (γ-PGA) is an anionic bacterial polymer constituted by glutamic acid residues only. It has the intrinsic ability to strongly interact with positively charged ions and flocculate them. For this reason, a large body of literature has accumulated on its application in bioremediation, particularly targeted to positively charged heavy metals. In this work, the most important characteristics of γ-PGA and of its production are summarized, highlighting the advantages, but also the limits, in its application in bioremediation
Bio-Driven Sustainable Extraction and AI-Optimized Recovery of Functional Compounds from Plant Waste: A Comprehensive Review
No full textThe agrifood industry produces copious amounts of waste, which represent an execrable wastage of natural resources and result in economic losses over the entire value chain. This review compares conventional and biological methods for the recovery of functional compounds from plant wastes to rescues part of the intrinsic valuable elements contained therein. Biological methods involve bioprocesses based on hydrolytic enzymes and engineered bacterial strains, to facilitate the release of valuable compounds. Then, this review explores the innovative and transformative role of artificial intelligence and machine learning techniques for real-time monitoring, optimizing, and digitizing the extraction procedures. By combining the potential of biological extraction and AI integration, this review provides insights on how these approaches can revolutionize the agrifood sector, increasing the efficiency and environmental sustainability of the plant waste valorization process
Poly-gamma-glutamate production in Bacillus subtilis
No full textPoly-gamma-glutamic acid (gamma-PGA) is an anionic polymer of increasing industrial interest, composed of thousands of glutamic acid residues linked by gamma-glutamyl bonds. Secretion of the polymer into the medium confers a mucoid colony morphology to the Bacillus producer strains grown on LB agar plates.
Although Bacillus subtilis possesses the functional biosynthetic pgs operon, containing four genes, laboratory strains do not have the ability to produce the polymer because pgs transcription is not active.
We dissected the genetic elements involved in the conversion of laboratory non-producer strains into gamma-PGA producers and established that the synergic action of two gene products is required. The co-presence of the wild-type swrAA allele, a gene involved in swarming motility, and the hyperphosphorylated form of the transcriptional factor degU, belonging to the two-component system degS/degU, is sufficient to drive pgs transcription and gamma-PGA production
Transcriptional autoregulation of swrAA, a gene involved in Bacillus subtilis motility
No full textThe swrAA gene, required for swarming migration in B. subtilis, is involved in transcriptional regulation of the fla/che operon which contains the genes necessary for flagellum biosynthesis and chemotaxis and the gene coding for the alternative sigma factor SigD. Nevertheless SwrAA does not bear resemblance to DNA binding proteins nor does it show any particular feature by in silico analysis.
In order to gain insight into its biological role we studied its expression profile in relationship with swarming and swimming motility.
We demonstrate that transcription of swrAA is driven by two promoters: a sigD-dependent promoter, active during growth in liquid LB medium, and a putative sigA-dependent promoter, triggered by the phosphorylated form of the two-component response regulator DegU and active during swarming migration.
Our data indicate that DegU is necessary not only for swarming (2, 3), but also for swimming and this requirement is independent from swrAA transcription, since SwrAA over-expression does not restore motility in strains carrying a degU deletion, suggesting that DegU concurs with SwrAA to achieve complete motility in B. subtilis
γ-Glutamyltranspeptidase-catalyzed enzymatic synthesis of flavour enhancers from Allium sp.
No full textThe use of flavour enhancers in the food industry could be beneficial for several reasons: they ensure homogeneity of the
final products, reduce costs for condiments and favor consumer's acceptance. On the other hand, the consumer's attention for
convenient, minimally-processed, nutritious, healthy, yet tasty food prompts the food industry to an accurate choice of the
ingredients. In this scenario, naturally occurring kokumi substances could play an important role. Kokumi is a japanese term that refers to mouthfulness, thickness and long-lasting savory sensations. Kokumi substances are represented mainly by gamma-glutamyl derivatives of amino acids. They are nearly tasteless for themselves, but they elicit a strong taste sensation,
expecially in conjunction with protein-rich food [Dunkel 2007]. In vegetables of the genus Allium, kokumi substances were
identified in gamma-glutamyl derivatives of S-alkyl and S-alkenyl cysteines and their S-oxides [Ueda 1990].There is a number of
difficulties connected with the supplying of these materials. Isolation from natural sources is laborious, and their content in
vegetables varies with cultivation and storage. In addition, upon crushing the plant, they are enzymatically degraded. The
chemical synthesis is not economical, due to the need of protection/deprotection steps. We exploited recently the enzymatic synthesis at the laboratory scale of the gamma -glutamyl derivatives of S-allyl cysteine, S-methyl cysteine and methionine, catalyzed by a commercially available mammalian GGT [Speranza 2012]. In this communication we report that such flavour enhancers can be obtained as well by using a purified, home-made bacterial GGT from Bacillus subtilis, a GRAS (Generally Recognized As Safe) organism, suited to food processing
SwrAA activates poly-gamma-glutamate synthesis inaddition to swarming in Bacillus subtilis
No full textPoly-gamma-glutamic acid (γ-PGA) is an extracellular polymer produced by various strains of Bacillus. Ιt was first described as the component of the capsule in B. anthracis, where it plays a relevant role in virulence. γ-PGA is also a distinctive component of “natto”, a Japanese traditional food consisting of soybean fermented by B. subtilis (natto). Domesticated B. subtilis strains do not synthesize γ-PGA although they possess the functional biosynthetic pgs operon. In the present work we explore the correlation between the genetic determinants, swrAA and degU, which allow a derivative of the domestic strain JH642 to display a mucoid colony morphology on LB agar plates due to the production of γ-PGA. Full activation of the pgs operon requires the co-presence of SwrAA and the phosphorylated form of DegU (DegU~P). The presence of either DegU~P or SwrAA alone has only marginal effects on pgs operon transcription and γ-PGA production.
Although SwrAA was identified as necessary for swarming and full swimming motility together with DegU, we show that motility is not involved in γ-PGA production. Activation of γ-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU~P display a cooperative effect
Cost-competitive γ-PGA production from low costs feedstock using an engineered Bacillus subtilis lab strain
Full text linkPoly-γ-glutamic acid (γ-PGA) represents one of the most promising biomaterial naturally secreted by microorganisms mainly belonging to the Bacillales order. This uncommon homo-polyamide is formed by D-/L- glutamic acid units polymerized by γ-amide linkages. The polymer, resistant to common proteases, is endowed with characteristics that are suitable for many biotechnological applications, e.g. as metal flocculant, drug carrier, food additive and many more.
However it is crucial to improve the economic viability of its production for γ-PGA industrial exploitation. Nowadays valorization of agro-food wastes is also a crucial issue.
This work aims at improving the cellulolytic capabilities of a B. subtilis strain to obtain γ-PGA exploiting the abundant and low-cost organic fraction present in rice straw as feedstock. Unfortunately rice straw is rich in silica which inhibits bacterial growth. However, an alkali treatment was established in which hemicelluloses are collected in the liquid fraction and silica can be extracted from the solid lignocellulosic part, concurrently enhancing cellulose bioavailability. The liquid hemicellulose-rich fraction and the remaining solid cellulose-rich part can then be used for bacterial fermentation. Preliminary results demonstrate that B. subtilis can grow on such a substrate as sole carbon source. Conveniently, the strain carrying the degU32(Hy) mutation, necessary for hyper-production of γ-PGA, shows more efficient hydrolysis of cellobiose and xylan than the wild type.
To further improve the strain cellulolytic potential genome and transcriptome data analyses were performed to select endogenous genes coding for enzymes important for saccharification of lignocellulose matrixes that could be modified. Genes bglC and xynA (encoding an endo-1,3,(4)-beta-glucanase and an endo-1,4-beta-xylanase, respectively) were chosen as targets, since their expression is poor and limited to the transition phase. An allelic-exchange approach was set up to increase xynA and bglC expression levels by optimizing the endogenous promoters and translation signals as well as by inserting a promoter UP element. The cellulolytic properties and γ-PGA yield of the engineered strains will be presented and discussed
Swarming and poly-gamma-glutamate synthesis depend on the synergic action of SwrAA and DegU in Bacillus subtilis
No full textIsogenic populations of Bacillus subtilis are multicellular communities consisting of distinct differentiated cell types and the two component system DegS-DegU has a central role in the control of single cell fates. The level of DegU phosphorylation attained inside each cell is one of the main parameters used to select specific cell fates but is not the only parameter that triggers a particular genetic programme. In laboratory strains, cooperativity between DegU and SwrAA has been observed in motility: a wild-type copy of the gene (swrAA+) confers a full swimming capacity and the ability to swarm on semi-solid surfaces when DegU is present but it does not show any motility advantage if the two component system DegS/U is deleted (1).
We will show that the synergic action of DegU and SwrAA is a conserved “modus operandi” since it is also observed in the production of poly-gamma-glutamic acid (gamma-PGA), an extracellular anionic polymer composed of thousands of glutamic acid residues linked by gamma-glutamyl bonds, produced and secreted by Bacilli. Domesticated B. subtilis strains synthesize gamma-PGA if they carry a degQH (2) or a degU32(Hy) / degS200(Hy) mutation and the wild type swrAA allele. These data indicate that the activation of the biosynthetic pgs operon is dependent on the co-presence of a high level of DegU~P and SwrAA. The presence of either SwrAA or DegU~P alone has only a marginal effect on gamma-PGA production and pgs operon transcription. The effect of SwrAA and DegU~P is cooperative rather than additive. Motility is not involved in gamma-PGA production since a sigD null mutation or a large deletion in the main flagellar operon (fla/che) do not affect gamma-PGA synthesis in degU32(Hy) swrAA+ strains. Moreover, a fla/che promoter up-mutation, that allows swarming and full swimming motilities in a degU32(Hy) swrAA- strain, does not confer the ability to produce gamma-PGA.
Activation of gamma-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU~P display a cooperative effect.
1) Calvio et al., 2008. J Bacteriol 190:5720-28.
2) Stanley NR and Lazazzera BA, 2005. Mol Microbiol 57:1143-
An Auto-Regulatory Loop Governing Motility in Bacillus subtilis
No full textWe will show that transcription of swrAA, the gene required for swarming migration in B. subtilis, is driven by two promoters: a sigD-dependent promoter, active during liquid growth, and a putative sigA-regulated promoter, active in the presence of the phosphorylated form of the response regulator DegU, and in swarming. Since sigD transcription is enhanced by SwrAA (Kearns and Losick, 2005), the finding that swrAA is, in turn, sigD-dependent, delineates the existence of a positive feed-back loop, one possible mechanism to set up bistability.
We will also demonstrate that the positive action played by SwrAA is prevented in strains carrying a deletion of the two component system degS-degU, and this effect is independent from swrAA transcription. As seen by other authors (Verhamme et al., 2007; Kobayashi, 2007), degU is necessary for swarming; however, we will show that also the positive effect of SwrAA on swimming motility is lost in delta-degU strains.
The epistatic effect of degU on swrAA points to a cooperation of the two gene products in the pathway leading to Bacillus motility. This effect might not be unique, as also the activation of the pgs operon, driving the synthesis of gamma-poly-glutamic acid, depends on the concerted action of DegU-P and SwrAA
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