Caracterização microbiológica e avaliação de uma cepa de Bacillus subtilis no desempenho de bezerros da raça Holandesa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inibição da divisão celular em Xanthomonas citri subsp. citri e Bacillus subtilis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of a Hexameric Exo-Acting GH51 alpha-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization and optimization of levan production by Bacillus subtilis NATTO

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Assessing Bacillus subtilis biosurfactant effects on the biodegradation of petroleum products

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bacillus subtilis na qualidade dos pintinhos ao nascer, desempenho e características intestinais dos frangos

Pós-graduação em Zootecnia - FCAV

A dual control mechanism synchronizes riboflavin and sulphur metabolism in Bacillus subtilis

Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for biosynthesis and/or transport of riboflavin (rib genes). Cytoplasmic riboflavin is rapidly and almost completely converted to FMN by flavokinases. When cytoplasmic levels of FMN are sufficient (high levels), FMN binding to FMN riboswitches leads to a reduction of rib gene expression. We report here that the protein RibR counteracts the FMN-induced turn-off activities of both FMN riboswitches in Bacillus subtilis, allowing rib gene expression even in the presence of high levels of FMN. The reason for this secondary metabolic control by RibR is to couple sulfur metabolism with riboflavin metabolism.

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.

DivIVA-Mediated Polar Localization of ComN, a Posttranscriptional Regulator of Bacillus subtilis

ComN (YrzD) is a small, 98-amino-acid protein recently shown to be involved in the posttranscriptional control of the late competence comE operon in Bacillus subtilis. We show here that ComN localizes to the division site and cell poles in a DivIVA-dependent fashion. Yeast two-hybrid and glutathione S-transferase pulldown experiments showed that ComN interacts directly with DivIVA. ComN is not essential for the polar assembly of the core competence DNA uptake machinery. Nevertheless, polar localization of ComN should play some role in competence acquisition because delocalization of ComN leads to a small reduction in competence efficiency. We found that ComN promotes the accumulation of its target comE mRNA to septal and polar sites. Thus, we speculate that localized translation of ComE proteins may be required for efficient competence development. Our results underscore the versatility of DivIVA as a promoter of the differentiation of bacterial poles and demonstrate that the repertoire of polarly localized molecules in B. subtilis is broad, including a regulator of gene expression and its target mRNA. Moreover, our findings suggest that mRNA localization may play a role in the subcellular organization of bacteria.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Vectorial signalling mechanism required for cell-cell communication during sporulation in Bacillus subtilis

Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cellcell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor sE to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of sE activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cellcell communication during development.

Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis

Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short beta-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable "open" conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.

Bindeprotein-abhängige DctS-Sensorkinasen aus Bacillus subtilis und Geobacillus kaustophilus

Das Enterobakterium Escherichia coli sowie das Bodenbakterium Bacillus subtilis können C4-Dicarbonsäuren als aerobe Kohlenstoffquelle zur Energiekonservierung nutzen. Die Regulation des C4-Dicarboxylatstoffwechsels erfolgt in E. coli und B. subtilis durch das Zweikomponentensystem DcuSREc bzw. DctSRBs, bestehend aus einer Sensorkinase und einem Responseregulator. Diese kontrollieren die Expression des C4-Dicarboxylat-Transporters DctA. Der Sensor DcuSEc benötigt für seine Funktion im aeroben Stoffwechsel den Transporter DctA als Cosensor. Für das DctSRBs-System gibt es Hinweise aus genetischen Untersuchungen, dass DctSBs das Bindeprotein DctBBs und möglicherweise auch DctABs als Cosensoren für seine Funktion benötigt. In dieser Arbeit sollte ein direkter Nachweis geführt werden, ob DctBBs und DctABs gemeinsam oder nur jeweils eine der Komponenten als Cosensoren für DctSBs fungieren. Sowohl für DctBBs als auch für DctABs wurde eine direkte Protein-Protein-Interaktion mit DctSBs durch zwei in vivo Interaktionsmethoden nachgewiesen. Beide Methoden beruhen auf der Co-Reinigung der Interaktionspartner mittels Affinitätschromatographie und werden je nach Affinitätssäule als mSPINE oder mHPINE (Membrane Strep/His-Protein INteraction Experiment) bezeichnet. Die Interaktion von DctSBs mit DctBBs wurde zusätzlich über ein bakterielles Two-Hybrid System nachgewiesen. Nach Coexpression mit DctSBs interagieren DctABs und DctBBs in mSPINE-Tests gleichzeitig mit der Sensorkinase. DctSBs bildet somit eine sensorische DctS/DctA/DctB-Einheit in B. subtilis und das Bindeprotein DctBBs agiert nur als Cosensor, nicht aber als Transport-Bindeprotein. Eine direkte Interaktion zwischen dem Transporter DctABs und dem Bindeprotein DctBBs besteht nicht. Transportmessungen belegen, dass der DctA-vermittelte Transport von [14C]-Succinat unabhängig ist von DctBBs. Außerdem wurde untersucht, ob Zweikomponentensysteme aus anderen Bakteriengruppen nach einem ähnlichen Schema wie DcuSREc bzw. DctSRBs aufgebaut sind. Das thermophile Bakterium Geobacillus kaustophilus verfügt über ein DctSR-System, welches auf genetischer Ebene mit einem Transporter des DctA-Typs und einem DctB-Bindeprotein geclustert vorliegt. Die Sensorkinase DctSGk wurde in E. coli heterolog exprimiert und gereinigt. Diese zeigt in einer E. coli DcuS-Insertionsmutanten Komplementation der DcuS-Funktion und besitzt dabei Spezifität für die C4-Dicarbonsäuren Malat, Fumarat, L-Tartrat und Succinat sowie für die C6-Tricarbonsäure Citrat. In Liposomen rekonstituiertes DctSGk zeigt Autokinase-Aktivität nach Zugabe von [γ-33P]-ATP. Der KD-Wert für [γ-33P]-ATP der Kinasedomäne von DctSGk liegt bei 43 μM, die Affinität für ATP ist damit etwa 10-fach höher als in DcuSEc.

Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis

BACKGROUND/AIMS: Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. METHODS: Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. RESULTS: Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. CONCLUSIONS: Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

The role of the amino terminus of Bacillus subtilis sigmaK in transcription initiation

The sigma (σ) subunit of eubacterial RNA polymerase (RNAP) is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of bacterial gene expression can be achieved by modulating a factor activity. The Bacillus subtilis sporulation a σ factor, σ K, controls gene expression of the late sporulation regulon. σ K is synthesized as an inactive precursor protein, pro-σ K, with a 20 amino acid pro sequence. Proteolytic processing of the pro sequence produces the active form, σK, which is able to bind to the core subunits of RNAP to direct gene expression. Thus, the pro sequence renders σK inactive in vivo. After processing, the amino terminus of σK consists of region 1.2, which is conserved among various σ factors. To understand the role of the amino terminus of σK, namely the pro sequence and region 1.2, mutagenesis of both regions was pursued. NH 2-terminal truncations of pro-σK were constructed to address how the pro sequence silences σK activity. The work described here shows that the pro sequence inhibits the ability of σ K to associate with the core subunits and that a deletion of only six amino acids of the pro sequence is sufficient to activate pro-σ K for DNA binding and transcription initiation to levels similar to σ K. Additionally, site directed mutagenesis was used to obtain single amino acid substitutions in region 1.2 to address the role of region 1.2 in σ K transcriptional activity. Two mutations were isolated, converting a lysine (K) to an alanine (A) at position three, and an asparagine (N) to a tyrosine (Y) at position five, both of which alter the efficiency of transcription initiation by RNAP containing the mutant σKs. Surprisingly, σ KK3A increased transcript production when compared to wild type. This increase is due to improvement in DNA affinity and increased stability of RNAP-DNA promoter open complexes. σKN5Y showed a decrease in transcription activity that is related to defects in the ability of RNAP to make the transition from the closed to open RNAP-DNA complex. Results of both the pro sequence and region 1.2 analyses indicate that the amino terminus of σK is important for transcription activity and this work adds to the increasing body of evidence that the amino termini of many σ factors modulate transcription initiation by RNAP. ^