Poly-gamma-glutamate production in Bacillus subtilis

Poly-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

The 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

Autoregulation of swrAA and Motility in Bacillus subtilis▿

We demonstrate that transcription of the gene swrAA, required for swarming migration in Bacillus subtilis, is driven by two promoters: a sigD-dependent promoter and a putative sigA-dependent promoter, which is inactive during growth in liquid Luria-Bertani medium and becomes active in the presence of the phosphorylated form of the response regulator DegU or on semisolid surfaces. Since sigD transcription is enhanced by SwrAA, this finding reveals that swrA expression is controlled by a positive feedback loop. We also demonstrate that the positive action of SwrAA in swimming and swarming motility is prevented in strains carrying a deletion of the two-component system degS-degU and that this effect is independent of swrAA transcription. Therefore, both DegU and SwrAA must be present to achieve full motility in B. subtilis

Transcriptional regulation of swrAA, a gene involved in motility and in the synthesis of gamma-poly-glutamic acid in Bacillus subtilis

We demonstrate that transcription of the gene swrAA, required for swarming migration in B. subtilis, is driven by two promoters: a sigD-dependent promoter, active during growth in liquid LB medium, and a putative sigA-dependent promoter, active in the presence of the phosphorylated form of the response regulator DegU and in swarming conditions. The existence of a positive feed-back loop, with SwrAA enhancing sigD expression and being in turn SigD-dependent, is confirmed by the level of the swrA operon transcription in swrAA+ and swrAA- strains. Furthermore, our data indicate that DegU is necessary for swimming as well as for swarming and this requirement is independent from swrAA transcription, since SwrAA over-expression does not restore motility in strains carrying a deletion of the two component system degSdegU. Besides motility, we show that SwrAA and DegU, the latter in the constitutively active form degU32(Hy), are both necessary for the activation of the pgs operon, driving the synthesis of gamma-poly-glutamic acid (PGA)

Swarming and poly-gamma-glutamate synthesis depend on the synergic action of SwrAA and DegU in Bacillus subtilis

Isogenic 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-

SwrAA activates poly-gamma-glutamate synthesis inaddition to swarming in Bacillus subtilis

Poly-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

An Auto-Regulatory Loop Governing Motility in Bacillus subtilis

We 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

The swrAA gene, regulating swarming behaviour in Bacillus subtilis, is sigD-dependent but displays additional regulative features

In Bacillus subtilis, a wild-type copy of swrAA gene is necessary for swarming and stimulates swimming motility. The biological function of its product is presently unknown. SwrAA shows no homology to any entry in Protein databases, and no particular feature can be found by in silico analysis. We therefore concentrated our efforts on the expression profile of this gene, in order to gain some insight on the role it plays in the activation of the swarming behavior. Accurate inspection of swrAA upstream sequence, suggested the presence of sequences (TTGCCT-N17-TACAAT and TAAA-N12-CCCGATAT) for the binding of Sigma A and Sigma D respectively, the latter being almost identical to the consensus. In in vitro transcription assay, we confirmed the promoter dependence on the presence of SigD but no transcription could be detected with E(sigA). In vivo, site-directed mutagenesis of the SigD consensus sequence completely abolishes transcription of a PswrAA-lacZ transcriptional fusion. swrAA expression is extremely low in conditions where other sigD-dependent genes are normally well expressed but displays additional regulative features which do not affect other sigD-dependent promoters. We explored the expression profile of PswrAA in the presence of mutations which are known to impair motility. In swimming assays, the positive role of SwrAA is lost in the absence of the DegS/DegU two component system. Taken together our results (see also the work presented by Amati et al.) point to a central role of SwrAA in the decision process B. subtilis cells face at the end of the exponential growth, possibly finely tuning the responses regulated by DegS/DegU

Pentraxins and Alzheimer’s disease: At the interface between biomarkers and pharmacological targets

Alzheimer’s disease (AD) is a neurodegenerative disorder involving deposition of misfolded proteins in vulnerable brain regions leading to inexorable and progressive neuronal loss and deterioration of cognitive functions. The AD brain displays features typical of chronic inflammation as defined by the presence of activated microglia and by an excessive amount of neuroinflammatory components such as cytokines and acute-phase proteins. This review aims to shed light on the role of the immune processes involved in AD, focusing on a family of inflammatory modulators belonging to the acute-phase proteins and crucial components of the humoral arm of innate immunity: pentraxins. In particular we analyze function of the pentraxins in AD, their upregulation in the brain and their contribution to neurodegeneration. Additionally, we highlight the role of pentraxins as putative AD biomarkers and as pharmacological therapeutic targets