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Introduction: Microbial extracellularalkaline proteases are one of the most important industrial enzymes with a wide range of applications in various industries. In the present study, the production of an alkaline protease by Bacillus pseudofirmus MSB4 was evaluated using response surface methodology (RSM). Materials and methods: Four significant independent variables were selected, based on the results of one-factor-at-a-time (OFAT) approach (xylose concentration, beef extract concentration, pH and temperature), for rotatable central composite design (RCCD) to analyze the response pattern and to establish a model. The design consisted of total 30 runs, at five levels for each factor, with four replications of the center points. In addition, the presence of extracellular alkaline protease genes was evaluated by using PCR. Results: Maximum protease production (185.397 U/ml), 2.2 times higher than that of OFAT method and 3.7 times higher than the unoptimized conditions, was obtained by using 3% w/v beef extract, at pH 9 and 37ºC according to the results of RCCD. Furthermore, the genes sub I, II and apr with the expected size (319, 486 and 194 bp, respectively) corresponding to the extracellular alkaline serine protease and metalloprotease were detected in MSB4 by using PCR. Discussion and conclusion:The experimental data well fitted the model (Adj R2: 0.9982) and the established quadratic model has a great ability to predict responses for new observations (Pred R2: 0.9967). The results showed that the OFAT and RSM strategies were a useful screening and optimization method for enhancing protease production of MSB4, respectively.

The pathogenic influences of uterine bacteria on endometrial carcinogenesis remain unclear. The aim of this pilot study was to compare the microbiota composition of endometrial lavage samples obtained from women with either endometrial hyperplasia (EH) or endometrial cancer (EC) versus those with benign uterine conditions. We hypothesized that specific microbiota signatures would distinguish between the two groups, possibly leading to the identification of bacterial species associated with endometrial tumorigenesis. A total of 35 endometrial lavage specimens (EH, n = 18; EC, n = 7; metastatic EC, n = 2; benign endometrial lesions, n = 8) were collected from 32 women who had undergone office hysteroscopy. Microbiota composition was determined by sequencing the V3−V4 region of 16S rRNA genes and results were validated by real-time qPCR in 46 patients with EC/EH and 13 control women. Surprisingly, we found that Bacillus pseudofirmus and Stenotrophomonas rhizophila – two plastic-degrading bacterial species – were over-represented in endometrial lavage specimens collected from patients with EC/EH. Using computational analysis, we found that the functional profile of endometrial microbiota in EC/EH was associated with fatty acid and amino acid metabolism. In summary, our hypothesis-generating data indicate that the plastic-degrading bacteria Bacillus pseudofirmus and Stenotrophomonas rhizophila are over-represented within the endometrial lavage microbiota of women with EC/EH living in Taiwan. Whether this may be related to plastic pollution deserves further investigation.

Soda lime is a by-product of the Solvay soda process for the production of sodium carbonate from limestone and sodium chloride. Due to a high salt concentration and alkaline pH, the lime is considered as a potential habitat of haloalkaliphilic and haloalkalitolerant microbial communities. This artificial and unique environment is nutrient-poor and devoid of vegetation, due in part to semi-arid, saline and alkaline conditions. Samples taken from the surface layer of the lime and from the depth of 2 m (both having pH ~11 and EC e up to 423 dS m −1 ) were investigated using culture-based (culturing on alkaline medium) and culture-independent microbiological approaches (microscopic analyses and pyrosequencing). A surprisingly diverse bacterial community was discovered in this highly saline, alkaline and nutrient-poor environment, with the bacterial phyla Proteobacteria (representing 52.8% of the total bacterial community) and Firmicutes (16.6%) showing dominance. Compared to the surface layer, higher bacterial abundance and diversity values were detected in the deep zone, where more stable environmental conditions may occur. The surface layer was dominated by members of the genera Phenylobacterium , Chelativorans and Skermanella , while in the interior layer the genus Fictibacillus was dominant. The culturable aerobic, haloalkaliphilic bacteria strains isolated in this study belonged mostly to the genus Bacillus and were closely related to the species Bacillus pseudofirmus , B. cereus , B. plakortidis , B. thuringensis and B. pumilus .

In bacteria, the sodium ion (Na(+)) cycle plays a critical role in negotiating the challenges of an extremely alkaline and sodium-rich environment. Alkaliphilic bacteria that grow optimally at high pH values use Na(+) for solute uptake and flagellar rotation because the proton (H(+)) motive force is insufficient for use at extremely alkaline pH. Only three types of electrically driven rotary motors exist in nature: the F-type ATPase, the V-type ATPase, and the bacterial flagellar motor. Until now, only H(+) and Na(+) have been reported as coupling ions for these motors. Here, we report that the alkaliphilic bacterium Bacillus alcalophilus Vedder 1934 can grow not only under a Na(+)-rich and potassium ion (K(+))-poor condition but also under the opposite condition in an extremely alkaline environment. In this organism, swimming performance depends on concentrations of Na(+), K(+) or Rb(+). In the absence of Na(+), swimming behavior is clearly K(+)- dependent. This pattern was confirmed in swimming assays of stator-less Bacillus subtilis and Escherichia coli mutants expressing MotPS from B. alcalophilus (BA-MotPS). Furthermore, a single mutation in BA-MotS was identified that converted the naturally bi-functional BA-MotPS to stators that cannot use K(+) or Rb(+). This is the first report that describes a flagellar motor that can use K(+) and Rb(+) as coupling ions. The finding will affect the understanding of the operating principles of flagellar motors and the molecular mechanisms of ion selectivity, the field of the evolution of environmental changes and stresses, and areas of nanotechnology.

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c ₁₂ rings compared with the WT c ₁₃. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c ₁₂ mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c ₁₂ or c ₁₃ can support ATP synthesis, and also underscore the critical importance of an alanine motif with c ₁₃ ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.

Alkaliphilic bacteria typically grow well at pH 9, with the most extremophilic strains growing up to pH values as high as pH 12-13. Interest in extreme alkaliphiles arises because they are sources of useful, stable enzymes, and the cells themselves can be used for biotechnological and other applications at high pH. In addition, alkaline hydrothermal vents represent an early evolutionary niche for alkaliphiles and novel extreme alkaliphiles have also recently been found in alkaline serpentinizing sites. A third focus of interest in alkaliphiles is the challenge raised by the use of proton-coupled ATP synthases for oxidative phosphorylation by non-fermentative alkaliphiles. This creates a problem with respect to tenets of the chemiosmotic model that remains the core model for the bioenergetics of oxidative phosphorylation. Each of these facets of alkaliphilic bacteria will be discussed with a focus on extremely alkaliphilic Bacillus strains. These alkaliphilic bacteria have provided a cogent experimental system to probe adaptations that enable their growth and oxidative phosphorylation at high pH. Adaptations are clearly needed to enable secreted or partially exposed enzymes or protein complexes to function at the high external pH. Also, alkaliphiles must maintain a cytoplasmic pH that is significantly lower than the pH of the outside medium. This protects cytoplasmic components from an external pH that is alkaline enough to impair their stability or function. However, the pH gradient across the cytoplasmic membrane, with its orientation of more acidic inside than outside, is in the reverse of the productive orientation for bioenergetic work. The reversed gradient reduces the trans-membrane proton-motive force available to energize ATP synthesis. Multiple strategies are hypothesized to be involved in enabling alkaliphiles to circumvent the challenge of a low bulk proton-motive force energizing proton-coupled ATP synthesis at high pH.

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na super(+). Little motility is observed at pH values < similar to 8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na super(+), but significant motility at pH 7 at much higher Na super(+) concentrations. At pH 8-10, the swimming speed increased with the increase of Na super(+) concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.

l-Tyrosine is a versatile compound used in the fine chemical, pharmaceutical, and functional food industries. Here, we report a bi-enzymatic cascade involving alanine racemase (ALR) and d-amino acid oxidase (DAAO) to produce pyruvate, as co-substrate for l-tyrosine production, from the cheap substrate l-alanine. The BpALR (ALR from Bacillus pseudofirmus) was used as a whole-cell biocatalyst, converting l-alanine to d, l-alanine. The FsDAAO (DAAO from Fusarium solani) was immobilized to oxidize the d-alanine generated in the first step to pyruvate. Both systems were combined as a continuous-flow reactor for maximized l-alanine-to-pyruvate conversion rates. The optimal parameters and appropriate conditions for FsDAAO immobilization were investigated. The pyruvate concentration of 86.6 g/L was achieved within 17 h. Subsequently, a whole-cell biocatalyst system for l-tyrosine production, catalyzed by the tyrosine phenol-lyase (TPL) from Erwinia herbicola (EhTPL), was developed, and a fed-batch approach was applied with phenol and the pyruvate produced with the ALR/DAAO system mentioned above. The concentration of phenol and pyruvate in the reactor should not exceed 7.5 g/L and 10 g/L, respectively. Significantly, the l-tyrosine concentration of 152.5 g/L was achieved within 10 h, demonstrating the great potential for high-efficiency production of l-tyrosine through the approach we established in this paper.Graphical abstractKey points• A specific bioreactor system for pyruvate produced froml-alanine was developed• The appropriate condition for immobilization of FsDAAO was investigated• A fed-batch process was established to producel-tyrosine with recombinant E. coli• The bi-enzymatic cascade was successfully used forl-tyrosine production at low cost

Cracks affect the durability of concrete by increasing its permeability. Self-healing materials can begin repairing themselves immediately after creating a crack. This is a big advantage of self-healing materials. In this study, effect of self-healing agents based on calcium carbonate precipitation for concrete is monitored for three months. Bacillus pseudofirmus was chosen as a self-healing agent and was tested on old cement pastes. Calcium precipitation was analyzed by scanning electron microscope with Energy-dispersive X-ray spectroscopy. The effect of added spontaneous calcination, culture media, bacteria and Ca2+ was monitored.

The prokaryotic voltage-gated Na super(+) channel, NaChBac, is one of a growing channel superfamily of unknown function. Here we show that Na sub(V)BP, the NaChBac homologue encoded by ncbA in alkaliphilic Bacillus pseudofirmus OF4, is a voltage-gated Na super(+) channel potentiated by alkaline pH. Na sub(V)BP has roles in motility, chemotaxis, and pH homeostasis at high pH. Reduced motility of bacteria lacking functional Na sub(V)BP was reversed by restoration of the native channel but not by a mutant Na sub(V)BP engineered to be Ca super(2+)-selective. Motile ncbA mutant cells and wild-type cells treated with a channel inhibitor exhibited behavior opposite to the wild type in response to chemoeffectors. Mutants lacking functional Na sub(V)BP were also defective in pH homeostasis in response to a sudden alkaline shift in external pH under conditions in which cytoplasmic [Na super(+)] is limiting for this crucial process. The defect was exacerbated by mutation of motPS, the motility channel genes. We hypothesize that activation of Na sub(V)BP at high pH supports diverse physiological processes by a combination of direct and indirect effects on the Na super(+) cycle and the chemotaxis system.