Unveiling the structure-emulsifying function relationship of truncated recombinant forms of the SA01-OmpA protein opens up a new vista in bioemulsifiers.

The emulsifying ability of SA01-OmpA (outer membrane protein A from Acinetobacter sp. SA01) was found to be constrained by challenges like low production efficiency and high costs associated with protein recovery from E. coli inclusion bodies, as described in our previous study. The present study sought to benefit from the advantages of the targeted truncating of SA01-OmpA protein, taking into account the reduced propensity of protein expression as inclusion bodies and cytotoxicity. Here, the structure and activity relationship of two truncated recombinant forms of SA01-OmpA protein was unraveled through a hybrid approach based on experimental data and computational methodologies, representing an innovative bioemulsifier with advantageous emulsifying activity. The recombinant truncated SA01-OmpA variants were cloned and heterologously expressed in E. coli host cells and subsequently purified. The results showed increased emulsifying activity of N-terminally truncated SA01-OmpA (NT-OmpA) compared to full-length SA01-OmpA. Molecular dynamics (MD) simulations analysis demonstrated a direct correlation between the C-terminally truncated SA01-OmpA (CT-OmpA) and its expression as inclusion bodies. Analysis of the structure-activity relationship of truncated variants of SA01-OmpA revealed that, compared to the full-length protein, deletion of the ß-barrel portion from the N-terminal of SA01-OmpA increased the emulsifying activity of NT-OmpA while lowering its expression as inclusion bodies. Contrary to the full-length protein, the N-terminally truncated SA01-OmpA was not as cytotoxic, according to the MTT assay, FCM analysis, and AO/EB staining. The findings of this extensive study advance our knowledge of SA01-OmpA at the molecular level as well as the design and development of efficient bioemulsifiers.IMPORTANCEPrevious research (Shahryari et al. 2021, mSystems 6: e01175-20) introduced and characterized the SA01-OmpA protein as a multifaceted protein with a variety of functions, including maintaining cellular homeostasis under oxidative stress conditions, biofilm formation, outer membrane vesicles (OMV) biogenesis, and beneficial emulsifying capacity. By truncating the SA01-OmpA protein, the current study presents a unique method for developing protein-type bioemulsifiers. The findings indicate that the N-terminally truncated SA01-OmpA (NT-OmpA) has the potential to fully replace full-length SA01-OmpA as a novel bioemulsifier with significant emulsifying activity. This study opens up a new frontier in bioemulsifiers, shedding light on a possible relationship between the structure and activity of SA01-OmpA truncated forms.

Structure and activity of a novel robust peroxidase from Alkanna frigida cell culture.

Demands for peroxidases (POX)s with diverse physicochemical properties have steadily grown as more applications of POXs are demonstrated. Plants are among the best sources of versatile POXs, and plant biotechnology, as an agricultural hassle-free technology, promises to circumvent the limitations of natural resource exploitation and to address the demands. Following this trend, it was shown that POX production steadily increased during the 31-day subculture of Alkanna frigida (from Boraginaceae) callus on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The purified cationic enzyme (POXalf) maintained its optimal activity over pH 4-7 for 2 years. It was resistant to H2O2 high concentrations (IC50 = 543.7 mM) and showed high specific activity in the reaction with phenol (4320.5 AU mg-1 > 20-fold of HRP AU). Furthermore, the specificity constant ratio of guaiacol to phenol indicated a 100 times faster reaction of POXalf with guaiacol. However, in contrast to HRP, it had little effect on diazo derivatives of aniline and meta-diaminobenzene. Based on the resulting primary structure from the tandem mass analysis, the POXalf 3D structure was constructed via homology modelling. Despite the high topological similarity between the HRP and POXalf structures, there were important differences between the active site pockets that could explain the observed differences in the corresponding substrate spectra and the specific activities. Considering the dynamics of POXalf production, its inactivity towards IAA and its high affinity for guaiacol, POXalf may have associated roles with A. frigida cell wall construction and monolignol metabolism.

Growth Optimization of Lactobacillus acidophilus for Production of Antimicrobial Peptide Acidocin 4356: Scale up from Flask to Lab-Scale Fermenter.

BACKGROUND: Antibiotic-resistant bacteria are a major threat to global health. Older antibiotics have become more or less ineffective as a result of widespread microbial resistance and an urgent need has emerged for the development of new antimicrobial strategies. Acidocin 4356 is a novel antimicrobial bacteriocin peptide produced by Lactobacillus acidophilus ATCC 4356 and capable of confronting the Pseudomonas aeruginosa ATCC 27853 infection challenges. According to our previous studies, the production of Acidocin 4356 is in parallel with cellular biomass production. OBJECTIVES: Given the costly production of Acidocin 4356, the development of a beneficial approach for increasing productivity of the cellular biomass has been targeted in the lab-scale fermenter for scale-up production of this bacteriocin. Therefore, in this study, we developed an inexpensive optimal culture medium based on the whey feedstock, evaluating this medium for scaling-up of the bacteriocin production from flask to fermenter. MATERIAL AND METHODS: In the first step, the optimization of the process parameters and medium components was carried out using the Plackett-Burman (PB) design and Response surface methodology (RSM) in flask culture. After optimization of the medium, bacteriocin production in the optimum culture medium was compared with de Man, Rogosa and Sharpe (MRS) medium by analyzing the intensity of the peptide band. Intensity analysis has been conducted on the PAGE band of the peptide using Image J software. Finally, the scale- up of bacteriocin production in the optimum culture medium was evaluated by batch fermentation in a 3-liter fermenter. RESULTS: In this study, a medium containing whey (40 g.L-1) and sodium acetate (5 g.L-1) was used as basal medium, and the effect of other factors were then evaluated. According to the PB design, three factors of peptone concentration, yeast extract concentrations and cultivation temperature were selected as the most effective factors which improve the growth of L. acidophilus. The condition providing the highest growth capacity for bacteriocin production were predicted based on the results of RSM as following: temperature 40 ° C, yeast (4 g.L-1), and peptone (8 g.L-1). Finally, the dry cell weight was obtained after incubation for 12 h as 2.25 g.L-1. Comparison of cell growth and bacteriocin production between MRS medium and optimized medium confirmed the efficacy of these optimal conditions for the cost-effective production of Acidocin 4356 in the flask. Besides, the scale- up of bacteriocin production has made under optimal condition in the 3-L fermenter. CONCLUSIONS: In this study, for the first time, scale- up production of Acidocin 4356 was presented by using a low-cost method based on whey feedstock to tackle P. aeruginosa infections.

Mucoid and coccoid Helicobacter pylori with fast growth and antibiotic resistance.

BACKGROUND: In this study, one Helicobacter pylori isolate, from gastric biopsy of a dyspeptic patient that turned into mucoid-coccoid (MC) form upon consecutive subcultures, was identified. The culturability, antibiotic resistance, and lipid contents of MC were compared with those of non-mucoid (NM) spiral H pylori. MATERIALS AND METHODS: Mucoid-coccoid and NM H pylori were subcultured on Brucella blood agar (BBA) and incubated under aerobic and microaerobic atmospheres at 37°C. Cultures were examined for colony characteristics and bacterial morphology after 1-3 days. The isolates were identified by biochemical tests and detection of H pylori-16S rDNA. Antibiogram was performed with currently used antibiotics for H pylori eradication. Cellular lipid contents were extracted and analyzed by gas chromatography. RESULTS: Compared with pin-pointed and glistening colonies of NM H pylori that appeared under microaerobic conditions, MC H pylori grew well in consecutive subcultures under aerobic and microaerobic atmospheres and produced white patches of mucoid colonies. MC exhibited coccoid and NM spiral morphology. Both isolates were catalase, oxidase, and urease positive and contained 16S rDNA. Compared with NM that was susceptible to almost all the antibiotics, MC was resistant to all the antibiotics. Lipid analyses showed high frequency of unsaturated fatty acids and cholesterol in MC. CONCLUSIONS: Coccoid forms with high fatty acid and cholesterol contents that show resistance to antibiotics might resist against other stressful conditions such as gastric acidity and immune response. Moreover, mucoid property may enhance resistance of coccoids to stresses. With mucoid-coccoid lifestyle, H pylori may establish a chronic infection refractory to antimicrobial therapy.

Overproduction of lipopeptide biosurfactant by Aneurinibacillus thermoaerophilus HAK01 in various fed-batch modes under thermophilic conditions.

An efficient lipopeptide biosurfactant (BS) producer, Aneurinibacillus thermoaerophilus HAK01, was isolated from municipal landfill sites. The strain was able to produce about 4.9 g L-1 lipopeptide at a thermophilic temperature of 45 °C. After optimization of culture component concentrations using the response surface method, the main focus is to find the most appropriate fed-batch strategy to enhance lipopeptide production by the HAK01 strain. For this purpose, four fed-batch strategies including (a) pH-stat mode, (b) constant feeding rate strategy, (c) DO-stat mode, and (d) combined feeding strategy were designed. The production of BS was increased systematically from 4.9 g L-1 in batch mode to 5.9, 7.1, 8.8 and 11.2 g L-1 in each fed-batch mode, respectively. While poor results were obtained in the pH-stat mode, the DO-stat mode showed excellent results in the production of BS. The results of the study confirmed the importance of operational mode, oxygen supply and the kind of feeding strategy in BS production.

Biosurfactants from probiotic bacteria: A review.

Among microorganisms, bacteria are the main group of biosurfactant-producing organisms. Different types of bacteria including Pseudomonas sp., Acinetobacter sp., Bacillus sp., and Arthrobacter sp. are among the most commonly studied bacteria in the realm of scientific research. However, due to the pathogenic nature of the producing organisms, the application of these compounds is restricted, therefore, not suitable for use in food-related industries. Given that probiotic bacteria impact human health, applying probiotics as nonpathogenic and safe organisms have gained much attention for the production of biosurfactants in recent years. Most biosurfactants obtained from probiotic bacteria are related to a number of lactic acid bacteria (LAB). These types of biosurfactants are classified based on their structures as protein-carbohydrate complexes, lipids, or fatty acids. The present paper seeks to provide comprehensive and useful information about the production of various kinds of biosurfactants by different probiotic bacteria. In addition, we have extensively reviewed their potential for possible future applications.

A cold-adapted endoglucanase from camel rumen with high catalytic activity at moderate and low temperatures: an anomaly of truly cold-adapted evolution in a mesophilic environment.

Endoglucanases are important enzymes in plant biomass degradation. They have current and potential applications in various industrial sectors including human and animal food processing, textile, paper, and renewable biofuel production. It is assumed that the cold-active endoglucanases, with high catalytic rates in moderate and cold temperatures, can improve the cost-effectiveness of industrial processes by lowering the need for heating and, thus, energy consumption. In this study, the endoglucanase CelCM3 was procured from a camel rumen metagenome via gene cloning and expression in Escherichia coli BL21 (DE3). The maximum activity of the enzyme on carboxymethyl cellulose (CMC) was obtained at pH 5 and 30 °C with a Vmax and Km of 339 U/mg and 2.57 mg/ml, respectively. The enzyme with an estimated low melting temperature of 45 °C and about 50% activity at 4 °C was identified to be cold-adapted. A thermodynamic analysis corroborated that CelCM3 with an activation energy (Ea), enthalpy of activation (ΔH), and Gibb's free energy (ΔG) of, respectively, 18.47 kJ mol-1, 16.12 kJ mol-1, and 56.09 kJ mol-1 is a cold-active endoglucanase. In addition, CelCM3 was tolerant of metal ions, non-ionic detergents, urea, and organic solvents. Given these interesting characteristics, CelCM3 shows promise to meet the requirements of industrial applications.

Development of a bifunctional xylanase-cellulase chimera with enhanced activity on rice and barley straws using a modular xylanase and an endoglucanase procured from camel rumen metagenome.

The camel rumen metagenome is an untapped source of glycoside hydrolases. In this study, novel genes encoding for a modular xylanase (XylC) and a cellulase (CelC) were isolated from a camel rumen metagenome and expressed in Escherichia coli BL21 (DE3). XylC with xylanase (Xyn), CBM, and carbohydrate esterase (CE) domains was characterized as a ß-1,4-endoxylanase with remarkable catalytic activity on oat-spelt xylan (K cat = 2919 ± 57 s-1). The implication of XylC's modular structure in its high catalytic activity was analyzed by truncation and fusion construction with CelC. The resulting fusions including Cel-CBM, Cel-CBM-CE, and Xyn-CBM-Cel showed remarkable enhancement in CMCase activity with K cat values of 742 ± 12, 1289 ± 34.5, and 2799 ± 51 s-1 compared to CelC with a K cat of 422 ± 3.5 s-1. It was also shown that the bifunctional Xyn-CBM-Cel with synergistic xylanase/cellulase activities was more efficient than XylC and CelC in hydrolysis of rice and barley straws.

Analysis of Pseudomonas aeruginosa PAO1 Biofilm Protein Profile After Exposure to n-Butanolic Cyclamen coum Extract Alone and in Combination with Ciprofloxacin.

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

Immuno-physiological alterations from AFB1 in rats counteracted by treatments with Lactobacillus paracasei BEJ01 and montmorillonite clay mixture.

High contamination by aflatoxin B1 (AFB1) has been detected in Beja province (Tunisia) in many dairy products and animal feed, which has resulted in many tons of cereals and cereals being removed from the market, causing economic loss. While removal represents a means of reducing risk, exposures still occur. Studies have increasingly focused on means of AFB1 biodegradation/elimination using lactic acid bacteria and clay mineral. In the study here, Lactobacillus paracasei BEJ01 (LP) and montmorilonite clay (MT) were used to reduce the physio-/immunotoxicologic disorders that could develop in rats that underwent AFB1 exposures for a total of 7 consecutive days. The results indicated that rats treated with AFB1 (80 µg/kg BW) alone had significant decreases in lymphocytes in their blood (including B-lymphocytes, CD3(+), CD4(+), and CD8(+) T-lymphocyte subtypes, and NK cells), immunoglobulins (IgA and IgG) and pro-inflammatory cytokines; these rats also had altered oxidative stress status. In contrast, in rats treated with LP + MT (2 × 10(9) cfu/ml [∼ 2 mg/kg] + 0.5 mg MT/kg BW) for a total of 7 days before, concurrent with or after AFB1 treatment, there was a significant blockade/mitigation of each AFB1-impacted parameter. Moreover, treatment with the mixture at any point in relation to AFB1 treatment expectedly caused enhanced TNFα and IL-1ß expression relative to control values; all other parameters were comparable to values noted in control rats. Alone, the mixture had no impact on host parameters. From the results here it may be concluded the the LP + MT mixture was effective in protecting these hosts against AFB1-induced immunologic/physiologic disorders and that LP + MT could prevent and/or mitigate AFB1 toxicities in vivo.

Cadmium uptake capacity of an indigenous cyanobacterial strain, Nostoc entophytum ISC32: new insight into metal uptake in microgravity-simulating conditions.

Among nine cyanobacterial strains isolated from oil-contaminated regions in southern Iran, an isolate with maximum cadmium uptake capacity was selected and identified on the basis of analysis of morphological criteria and 16S rRNA gene sequence similarity as Nostoc entophytum (with 99% similarity). The isolate was tentatively designated N. entophytum ISC32. The phylogenetic affiliation of the isolates was determined on the basis of their 16S rRNA gene sequence. The maximum amount of Cd(II) adsorbed by strain ISC32 was 302.91 mg g(-1) from an initial exposure to a solution with a Cd(II) concentration of 150 mg l(-1). The cadmium uptake by metabolically active cells of cyanobacterial strain N. entophytum ISC32, retained in a clinostat for 6 days to simulate microgravity conditions, was examined and compared with that of ground control samples. N. entophytum ISC32 under the influence of microgravity was able to take up cadmium at amounts up to 29% higher than those of controls. The activity of antioxidant enzymes including catalase and peroxidase was increased in strain ISC32 exposed to microgravity conditions in a clinostat for 6 days, as catalase activity of the cells was more than three times higher than that of controls. The activity of the peroxidase enzyme increased by 36% compared with that of the controls. Membrane lipid peroxidation was also increased in the cells retained under microgravity conditions, up to 2.89-fold higher than in non-treated cells. Images obtained using scanning electron microscopy showed that cyanobacterial cells form continuous filaments which are drawn at certain levels, while the cells placed in a clinostat appeared as round-shaped, accumulated together and distorted to some extent.

Antibacterial activity of Pseudonocardia sp. JB05, a rare salty soil actinomycete against Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that causes many harmful and life-threatening diseases. Some strains of this bacterium are resistant to available antibiotics. This study was designed to evaluate the ability of indigenous actinomycetes to produce antibacterial compounds against S. aureus and characterize the structure of the resultant antibacterial compounds. Therefore, a slightly modified agar well diffusion method was used to determine the antibacterial activity of actinomycete isolates against the test microorganisms. The bacterial extracts with antibacterial activity were fractionated by silica gel and G-25 sephadex column chromatography. Also, the active fractions were analyzed by thin layer chromatography. Finally, the partial structure of the resultant antibacterial compound was characterized by Fourier transform infrared spectroscopy. One of the isolates, which had a broad spectrum and high antibacterial activity, was designated as Pseudonocardia sp. JB05, based on the results of biochemical and 16S rDNA gene sequence analysis. Minimum inhibitory concentration for this bacterium was 40 AU mL(-1) against S. aureus. The antibacterial activity of this bacterium was stable after autoclaving, 10% SDS, boiling, and proteinase K. Thin layer chromatography, using anthrone reagent, showed the presence of carbohydrates in the purified antibacterial compound. Finally, FT-IR spectrum of the active compound illustrated hydroxyl groups, hydrocarbon skeleton, and double bond of polygenic compounds in its structure. To the best of our knowledge, this is the first report describing the efficient antibacterial activity by a local strain of Pseudonocardia. The results presented in this work, although at the initial stage in bioactive product characterization, will possibly contribute toward the Pseudonocardia scale-up for the production and identification of the antibacterial compounds.

Eradication of Pseudomonas aeruginosa Biofilms Using the Combination of n-butanolic Cyclamen coum Extract and Ciprofloxacin.

BACKGROUND: Biofilm formation is a major pathogenic factor in different bacteria such as Pseudomonas aeruginosa. A number of studies have reported that bacterial biofilms show different levels of antibiotic resistance. In order to re-sensitize the bacterial biofilms to antibiotics, biofilms should be dispersed. OBJECTIVES: In this study, the effect of n-butanolic Cyclamen coum extract in combination with ciprofloxacin was examined on one, three and five day old P. aeruginosa biofilms. The synergistic effect of n-butanolic C. coum extract and ciprofloxacin towards dispersing pre-established P. aeruginosa biofilms was also studied. MATERIALS AND METHODS: The ability of biofilm formation by six different P. aeruginosa strains was confirmed by microtiter plate method and PCR assay for the cupA gene. The extraction of C. coum tubers was achieved by fractionation method using different solvents. The minimum inhibitory concentration (MIC) of n-butanolic C. coum extract and ciprofloxacin against planktonic cells was evaluated using agar well diffusion and microdilution methods. The microdilution chequerboard method was used to determine the fractional biofilm eradication concentration index (FBCI), when the combination of n-butanolic C. coum extract and ciprofloxacin were used against P. aeruginosa biofilms. RESULTS: The ability of biofilm formation by P. aeruginosa strains was quantitatively confirmed. The PCR method confirmed the existence of cup A gene (172 bp) in all studied strains. Saponin content of the n-butanolic C. coum extract was 156 µg/mL. The extract revealed antibacterial activity against planktonic cells of P. aeruginosa strains. The results showed that one and three day old biofilms are affected by either ciprofloxacin or n-butanolic C. coum extract. However, n-butanolic C. coum extract in combination with ciprofloxacin was significantly more effective against P. aeruginosa biofilms. CONCLUSIONS: Using n-butanolic C. coum extract in combination with ciprofloxacin offers a novel strategy to control biofilm-based infections caused by P. aeruginosa.

A novel ∼34-kDa α-amylase from psychrotroph Exiguobacterium sp. SH3: production, purification, and characterization.

An amylase-producing psychrotroph bacterium was isolated from soil and identified as belonging to the genus Exiguobacterium. A novel cold-adapted α-amylase, Amy SH3, was purified from culture medium of this bacterium using acetone precipitation and DEAE-Sepharose anion-exchange chromatography. The molecular mass of the enzyme was estimated about 34 kDa using SDS-PAGE. Biochemical characterization of Amy SH3 revealed that the optimum temperature for maximum activity of Amy SH3 was 37°C. However, Amy SH3 was also active at cold temperatures, showing 13% and 39% activity at 0 and 10°C, respectively. The optimum pH for maximum activity of Amy SH3 was pH 7, whereas the amylase was active over a pH range of 5 to 10. The activity of Amy SH3 was enhanced by Co²âº but decreased by Mg²âº, Mn²âº, Zn²âº, Fe²âº, and Ca²âº. Amy SH3 was able to retain 76% of its activity in the presence of 0.5% SDS. The K(m) and V(max) of the enzyme were calculated to be 0.06 mg/mL and 4,010 U/mL, respectively. The cold-adapted Amy SH3 seems very promising for applications at ambient temperature.

Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

Antibacterial activity and probiotic potential of Lactobacillus plantarum HKN01: a new insight into the morphological changes of antibacterial compound-treated Escherichia coli by electron microscopy.

Among several bacteria examined, an antibacterial-producing Lactobacillus strain with probiotic characteristics was selected and identified based on 16S rRNA gene sequencing. Subsequent purification and mode of action of the antibacterial compounds on target cells including E. coli were investigated. Maximum production of the antibacterial compound was recorded at 18 h incubation at 30 degrees C. Interestingly, antibacterial activity remained unchanged after heating at 121 degrees C for 45 min, 24 h storage in temperature range of 70 degrees C to room temperature, and 15 min exposure to UV light, and it was stable in the pH of range 2-10. The active compounds were inactivated by proteolytic enzymes, indicating their proteinaceous nature, and, therefore, referred to as bacteriocin-like inhibitory substances. Isolation and partial purification of the effective agent was done by performing ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the GFC-purified active compound (~3 kDa) was determined by Tris-Tricine SDS-PAGE. To predict the mechanisms of action, transmission electron microscopy (TEM) analysis of ultrathin sections of E. coli before and after antibacterial treatment was carried out. TEM analysis of antibacterial compounds-treated E. coli demonstrated that the completely altered bacteria appear much darker compared with the less altered bacteria, suggesting a change in the cytoplasmic composition. There were also some membrane-bound convoluted structures visible within the completely altered bacteria, which could be attributed to the response of the E. coli to the treatment with the antibacterial compound. According to the in vivo experiments oral administration of L. plantarum HKN01 resulted in recovery of infected BALB/c mice with Salmonella enterica ser. Typhimurium.

In silico modeling of the type 2 IDI enzymes of Bacillus licheniformis, Pseudomonas stutzeri, Streptococcus pyogenes, and Staphylococcus aureus for virtual screening of potential inhibitors of this therapeutic target.

Isopentenyl diphosphate isomerase is an essential enzyme in those living organisms such as pathogenic strains of Streptococcus and Staphylococcus genera which rely on the Mevalonate pathway for the production of isoprenoids. The pathogens contain type 2 IDI in contrast to human that contains type 1 IDI. Therefore, the type 2 IDI may be a potential target for the therapy of some infectious diseases. In the current study, a virtual screening by docking was performed among 2000 chemicals from CoCoCo library to find a specific inhibitor for type 2 IDIs. To this end, the structures of the type 2 IDIs of Bacillus licheniformis, Pseudomonas stutzeri, Streptococcus pyogenes, and Staphylococcus aureus were molded using comparative modeling and Hidden Markov Model (HMM) based prediction. The predicted models were evaluated based on Q-mean and Prosa score. Molegro Virtual Docker with MolDock scoring function was used for measuring the binding affinity of the found inhibitor to the active site of the models. Also the inhibition effect of the compound was virtually tested on the crystallography-solved structures of the Sulfolobus shibatae and Thermus thermophilus type 2 IDIs as well as the Escherichia coli type 1 IDI. Finally, the inhibition effect of the found inhibitor was virtually tested on the human type 1 IDI. Interestingly, the results suggest that the inhibitor efficiently binds to and inhibits the bacterial IDIs especially the type 2 IDIs of pathogens while it is not inhibiting the human IDI.

Antibacterial Activity of Probiotic Lactobacillus plantarum HK01: Effect of Divalent Metal Cations and Food Additives on Production Efficiency of Antibacterial Compounds.

One hundred and sixty lactic acid bacteria, isolated from Iranian traditional dairy products, were screened for antibacterial potential. Among them, an isolate showing remarkable antibacterial activity against both Staphylococcus aureus (PTCC 1112) and Escherichia coli (PTCC 1338) was selected based on minimum inhibitory concentration (AU/mL). The morphological and biochemical characteristics of the isolate matched the literature description about genus Lactobacillus. Partial sequencing of 16S rRNA gene and its alignment with other Lactobacillus strains revealed that the isolate was closely related to the Lactobacillus plantarum. The isolate also exhibited the highest similarity (>99 %) to L. plantarum. We thus tentatively classified the bacterial isolate as L. plantarum HK01. The antibacterial active compound from HK01 strain remained stable for 45 min at 121 °C, and it reached a maximum activity at the end of log phase and the early part of stationary phase. The antibacterial activity of the test isolate, its probiotic properties and production efficacy through addition of some divalent metal cations and food additives were studied as well. The study of bile salt hydrolase (BSH) activity as a function of growth revealed that HK01 strain hydrolysing up to 5 % of sodium salt of glycodeoxycholic acid, correlated with the presence of bsh gene in the isolate. HK01 strain showed high resistance to lysozyme, good adaptation to simulated gastric juice and a moderate bile tolerance. Results obtained from simulated gastric juice conditions showed no significant difference occured during the 70 min. HK01 strain was classified as a strain with low hydrophobicity (34.2 %). Addition of trisodium citrate dehydrates as a food-grade chelator of divalent cations restored antibacterial compound production in MRS broth. Antibacterial compounds of L. plantarum HK01 endured treatment with 10 g/L of SDS, Tween 20, Tween 80 and urea. Concerning food additives, the results demonstrated that antibacterial compound production by L. plantarum HK01 was influenced by the presence of surfactants, EDTA, KCl and sodium citrate.

Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws.

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.

Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters.