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The effects of Calcium (Ca+²) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+² on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+² on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.(AU)

Os efeitos do cálcio (Ca+²) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+² sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+² na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.(AU)

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The effects of Calcium (Ca+²) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+² on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+² on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.

Os efeitos do cálcio (Ca+²) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+² sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+² na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.

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Bacterial chitinases are a subject of intense scientific research due to their biotechnological applications, particularly their use as biological pesticides against phytopathogenic fungi as a green alternative to avoid the use of synthetic pesticides. Bacillus cereus sensu lato B25 is a rhizospheric bacterium that is a proven antagonist of Fusarium verticillioides, a major fungal pathogen of maize. This bacterium produces two chitinases that degrade the fungal cell wall and inhibit its growth. In this work, we used a heterologous expression system to purify both enzymes to investigate their biochemical traits in terms of Km, Vmax, optimal pH and temperature. ChiA and ChiB work as exochitinases, but ChiB exhibited a dual substrate activity and it is also an endochitinase. In this work, the direct addition of these chitinases inhibited fungal conidial germination and therefore they may play a major role in the antagonism against F. verticillioides.

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Bacteria of the Bacillus cereus group colonize several ecological niches and infect different hosts. Bacillus cereus, a ubiquitous species causing food poisoning, Bacillus thuringiensis, an entomopathogen, and Bacillus anthracis, which is highly pathogenic to mammals, are the most important species of this group. These species are closely related genetically, and their specific toxins are encoded by plasmids. The infectious cycle of B. thuringiensis in its insect host is regulated by quorum-sensing systems from the RNPP family. Among them, the Rap-Phr systems, which are well-described in Bacillus subtilis, regulate essential processes, such as sporulation. Given the importance of these systems, we performed a global in silico analysis to investigate their prevalence, distribution, diversity and their role in sporulation in B. cereus group species. The rap-phr genes were identified in all selected strains with 30% located on plasmids, predominantly in B. thuringiensis. Despite a high variability in their sequences, there is a remarkable association between closely related strains and their Rap-Phr profile. Based on the key residues involved in RapH phosphatase activity, we predicted that 32% of the Rap proteins could regulate sporulation by preventing the phosphorylation of Spo0F. These Rap are preferentially located on plasmids and mostly related to B. thuringiensis. The predictions were partially validated by in vivo sporulation experiments suggesting that the residues linked to the phosphatase function are necessary but not sufficient to predict this activity. The wide distribution and diversity of Rap-Phr systems could strictly control the commitment to sporulation and then improve the adaptation capacities of the bacteria to environmental changes.

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A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39 ± 5.15 U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45 °C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2-11.0 and 25-50 °C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76 kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.

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The NprR protein and NprRB signaling peptide comprise a bifunctional quorum-sensing system from the Bacillus cereus group that is involved in transcriptional activation through DNA-binding and in sporulation initiation by binding to Spo0F. We characterized in vitro the direct interactions established by NprR that may be relevant for performing its two functions. Apo-NprR interacted with Spo0F, but not with the target DNA. The NprRB signaling peptide SSKPDIVG that binds strongly to Apo-NprR, failed to bind and disrupt the NprR-Spo0F complex. Finally, the NprR-NprRB complex bound both to Spo0F and the target DNA with similar affinity. Based on our findings, we propose that rather than a switch triggered by NprRB, the NprR/NprRB ratio and the availability of Spo0F binding sites define the function of NprR.

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This study presents a system for expanded bed adsorption for the purification of chitosanase from broth extract in a single step. A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01 and used to produce chitosanases. The expanded bed adsorption conditions for chitosanase purification were optimized statistically using STREAMLINE(TM) DEAE and a homemade column (2.6 × 30.0 cm). Dependent variables were defined by the quality criteria purification factor (P) and enzyme yield to optimize the chromatographic process. Statistical analyses showed that the optimum conditions for the maximum P were 150 cm/h load flow velocity, 6.0 cm settled bed height, and 7.36 cm distributor height. Distributor height had a strong influence on the process, considerably affecting both the P and enzyme yield. Optimizing the purification variables resulted in an approximately 3.66-fold increase in the P compared with the value under nonoptimized conditions. This system is promising for the recovery of chitosanase from B. cereus C-01 and is economically viable because it promotes the reduction steps.

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Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.

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At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named desA (Δ5 desaturase) and desB (Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus.

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The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar-Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-α-benzoyl-L-Arg-p-nitroanilide (L-BApNA) and N-α-p-tosyl-L-Arg methyl ester (L-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-tosyl-L-lysine chloromethyl ketone (TLCK)] as well as CaCl2, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.

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Bacillus sp. are specific producers of peptidase amongst bacteria and peptidase enzymes and are of significant ones due to their multifarious applications. Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial by-products for many biochemical reactions. Due to their rich organic nature, they can serve as an ideal substrate for the production of different value added products like peptidases. In the present work, an attempt was made to optimize different variables by Taguchi methodology for the production of peptidase using agro-industrial by-products hydrolyzed by a Bacillus cereus strain, resulting in brewer's spent grain (BSG) being the optimal organic substrate. Subsequently, operative variables for the BSG were investigated using Taguchi methodology in order to maximize the enzyme production. Additionally, the main medium components were optimized using a mixture design. Finally, the production of peptidase by B. cereus was investigated; also the possible interaction with other proteolytic microbial strains was evaluated. A notorious synergistic effect was observed when B. cereus was inoculated with Pseudomonas sp. These brought a triple benefit, first, opening the possibility to produce technical enzymes at low cost, second, giving greater value to a food industry by-product, and third, reducing the environmental impact caused by the product removal directly into the environment.

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A chemical study of acyl-homoserine lactones (acyl-HSLs) produced by Enterobacter sakazakii resulted in the identification of three molecules: (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL. Mixed cultures of E. sakazakii and Bacillus cereus depleted E. sakazakii acyl-HSLs, suggesting acyl-HSL degradation by B. cereus hydrolases (hydrolysis of the lactone or amide moiety). The expression of B. cereus acyl-HSL lactonase and acyl-homoserine acylase was confirmed by monitoring the biotransformation of (S)-N-dodecanoyl-HSL into (S)-N-dodecanoyl-homoserine, dodecanoic acid and homoserine in the presence of B. cereus whole cells, using electrospray-mass spectrometry (ESI-MS).

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An important way to reuse agroindustrial by-products and to produce added-value products consists of the production of protein hydrolysates. In the current study, we used Brewer's spent grain (BSG), mainly because of its availability and cost, as a substrate for the enzymatic hydrolysis by Bacillus cereus. First, the physicochemical and microbiological characterization of BSG batches from three varieties was carried out. Furthermore, the optimal fermentation upstream processes for enzymatic hydrolysis by B. cereus were defined. Finally, the ability of B. cereus to hydrolyze different fractions of BSG was analyzed and possible synergistic effects of this bacterium along with other proteolytic bacteria were also investigated. Results showed that the naturally associated microflora was predominantly thermophilic aerobic bacteria and the drying process was the better alternative for BSG preservation. Water, lipids, and ash content differed significantly among the three varieties; however, no statistically significant differences were found in the protein content among them. After BSG characterization studies, the following protocol was set to obtain the fermentation substrate (FS): drying at 60°C for 24-48 H; sieving, grinding, and polyphenol extraction with an alcohol-water solution; and finally autoclaving. A synergistic effect was observed when B. cereus was inoculated with Pseudomonas strains in FS.

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Subclass B1 beta-lactamases are Zn(II)-dependent hydrolases that confer bacterial resistance to most clinically useful beta-lactam antibiotics. The enzyme BcII from Bacillus cereus is a prototypical enzyme that belongs to this group, the first Zn(II)-dependent beta-lactamase to be discovered. Crucial aspects of the BcII catalytic mechanism and metal binding mode have been assessed mostly on the Co(II)-substituted surrogate. Here we report a high-resolution structure of Co(II)-BcII, revealing a metal coordination geometry identical to that of the native zinc enzyme. In addition, a high-resolution structure of the apoenzyme, together with structures with different degrees of metal occupancy and oxidation levels of a conserved Cys ligand, discloses a considerable mobility of two loops containing four metal ligands (namely, regions His116-Arg121 and Gly219-Cys221). This flexibility is expected to assist in the structural rearrangement of the metal sites during catalytic turnover, which, along with the coordination geometry adaptability of Zn(II) ions, grants the interaction with a variety of substrates, a characteristic feature of B1 metallo-beta-lactamases.

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Metallo-beta-lactamases are enzymes capable of hydrolyzing all known classes of beta-lactam antibiotics, rendering them ineffective. The design of inhibitors active against all classes of metallo-beta-lactamases has been hampered by the heterogeneity in metal content in the active site and the existence of two different mononuclear forms. BcII is a B1 metallo-beta-lactamase which is found in both mononuclear and dinuclear forms. Despite very elegant studies, there is still controversy on the nature of the active BcII species. We carried out a non-steady-state study of the hydrolysis of penicillin G catalyzed by Co(II)-substituted BcII, and we followed the modifications occurring at the active site of the enzyme. Working at different metal/enzyme ratios we demonstrate that both mono-Co(II) and di-Co(II) BcII are active metallo-beta-lactamases. Besides, we here present evidence that during penicillin G hydrolysis catalyzed by mono-Co(II) BcII the metal is localized in the DCH site (the Zn2 site in B1 enzymes). These conclusions allow us to propose that both in mono-Co(II) and di-Co(II) BcII the substrate is bound to the enzyme through interactions with the Co(II) ion localized in the DCH site. The finding that the DCH site is able to give rise to an active lactamase suggests that the Zn2 site is a common feature to all subclasses of metallo-beta-lactamases and would play a similar role. This proposal provides a starting point for the design of inhibitors based on transition-state analogs, which might be effective against all MbetaLs.

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Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the beta-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-beta-lactamases.

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Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MbetaL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.

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Metallo-beta-lactamases are zinc-dependent hydrolases that inactivate beta-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-beta-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the beta-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule.

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Biological activity and presence of DNA sequences related to virulence genes were studied in 21 strains of the Bacillus cereus group. The activity of spent culture supernatants and the effect of infection by vegetative bacterial cells were assessed on cultured human enterocytes (Caco-2 cells). The effect of extracellular factors on the detachment, necrosis and mitochondrial dehydrogenase activity of cultured human enterocytes was studied. Hemolytic activity on rabbit red blood cells was also evaluated and the effect of direct procaryotic-eucaryotic interactions was assessed in infection assays with vegetative bacterial cells. Concerning virulence genes, presence of the DNA sequences corresponding to the genes entS, entFM, nhe (A, B and C), sph, hbl (A, B, C and D), piplC and bceT was assessed by PCR. Ribopatterns were determined by an automated riboprinting analysis after digestion of the DNA with EcoRI. Principal component analysis and biplots were used to address the relationship between variables. Results showed a wide range of biological activities: decrease in mitochondrial dehydrogenase activity, necrosis, cell detachment and hemolytic activity. These effects were strain-dependent. Concerning the occurrence of the DNA sequences tested, different patterns were found. In addition, ribotyping showed that strains under study grouped into two main clusters. One of these clusters includes all the strains that were positive for all the DNA sequences tested. Positive and negative correlations between variables under study were evidenced. Interestingly, high detaching strains were positively correlated with the presence of the sequences entS, nheC and sph. Within gene complexes, high correlation was found between sequences of the hbl complex. In contrast, sequences of the nhe complex were not correlated. Some strains clustered together in the biplots. These strains were positive for all the DNA sequences tested and they were able to detach enterocytes upon infection. Our results highlight the multifactorial character of the virulence of the B. cereus group and show the correlation between ribopatterns, occurrence of toxin genes and biological activity of the strains under study.

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Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most beta-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.

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