Evolutionary analysis and quality assessment of ζ-carbonic anhydrase sequences from environmental microbiome.

Carbonic anhydrase (CA) is one of the most vital enzymes in living cells. This study has been performed due to the significance of this metalloenzyme for life and the novelty of some CA families like ζ-CA to evaluate evolutionary processes and quality check their sequences. In this study, bioinformatics methods revealed the presence of ζ-CA in some eukaryotic and prokaryotic microorganisms. Notably, it has not been previously reported in prokaryotes. The coexistence of ß- and ζ-CAs in some microorganisms is also a novel finding as well. Also, our analysis identified several CA proteins with 6-14 amino acid intervals between histidine and cysteine in the second highly conserved motif, which can be classified as the novel ζ-CA subfamily members that emerged under the Zn deficiency of aquatic ecosystems and selection pressure in these environments. There is also a possibility that the achieved results are rooted in the contamination of samples from the environmental microbiome genome with genomes of diatom species and the occurrence of errors was observed in the DNA sequencing outcomes. Combining of all results from evolutionary analysis to quality control of ζ-CA DNA sequences is the incentive motivation to explore more the hidden aspects of ζ-CAs.

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.

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.

Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates.

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λmax = 390 nm, ɛ = 32 880 M(-1) cm(-1) at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL(-1) was achieved for peroxidase.

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.

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.

Genome Study of α-, ß-, and γ-Carbonic Anhydrases from the Thermophilic Microbiome of Marine Hydrothermal Vent Ecosystems.

Carbonic anhydrases (CAs) are metalloenzymes that can help organisms survive in hydrothermal vents by hydrating carbon dioxide (CO2). In this study, we focus on alpha (α), beta (ß), and gamma (γ) CAs, which are present in the thermophilic microbiome of marine hydrothermal vents. The coding genes of these enzymes can be transferred between hydrothermal-vent organisms via horizontal gene transfer (HGT), which is an important tool in natural biodiversity. We performed big data mining and bioinformatics studies on α-, ß-, and γ-CA coding genes from the thermophilic microbiome of marine hydrothermal vents. The results showed a reasonable association between thermostable α-, ß-, and γ-CAs in the microbial population of the hydrothermal vents. This relationship could be due to HGT. We found evidence of HGT of α- and ß-CAs between Cycloclasticus sp., a symbiont of Bathymodiolus heckerae, and an endosymbiont of Riftia pachyptila via Integrons. Conversely, HGT of ß-CA genes from the endosymbiont Tevnia jerichonana to the endosymbiont Riftia pachyptila was detected. In addition, Hydrogenovibrio crunogenus SP-41 contains a ß-CA gene on genomic islands (GIs). This gene can be transferred by HGT to Hydrogenovibrio sp. MA2-6, a methanotrophic endosymbiont of Bathymodiolus azoricus, and a methanotrophic endosymbiont of Bathymodiolus puteoserpentis. The endosymbiont of R. pachyptila has a γ-CA gene in the genome. If α- and ß-CA coding genes have been derived from other microorganisms, such as endosymbionts of T. jerichonana and Cycloclasticus sp. as the endosymbiont of B. heckerae, through HGT, the theory of the necessity of thermostable CA enzymes for survival in the extreme ecosystem of hydrothermal vents is suggested and helps the conservation of microbiome natural diversity in hydrothermal vents. These harsh ecosystems, with their integral players, such as HGT and endosymbionts, significantly impact the enrichment of life on Earth and the carbon cycle in the ocean.

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.

New Provisional Function of OmpA from Acinetobacter sp. Strain SA01 Based on Environmental Challenges.

An outer membrane protein A (OmpA) from Acinetobacter sp. strain SA01 was identified and characterized in-depth based on the structural and functional characteristics already known of its homologues. In silico structural studies showed that this protein can be a slow porin, binds to peptidoglycan, and exhibits emulsifying properties. Characterization of the recombinant SA01-OmpA, based on its emulsifying properties, represented its promising potentials in biotechnology. Also, the presence of SA01-OmpA in outer membrane vesicles (OMV) and biofilm showed that this protein, like its homologues in Acinetobacter baumannii, can be secreted into the extracellular environment through OMVs and play a role in the formation of biofilm. After ensuring the correct selection of the protein of interest, the role of oxidative stress induced by cell nutritional parameters (utilization of specific carbon sources) on the expression level of OmpA was carefully studied. For this purpose, the oxidative stress level of SA01 cell cultures in the presence of three nonrelevant carbon sources (sodium acetate, ethanol, and phenol) was examined under each condition. High expression of SA01-OmpA in ethanol- and phenol-fed cells with higher levels of oxidative stress than acetate suggested that oxidative stress could be a substantial factor in the regulation of SA01-OmpA expression. The significant association of SA01-OmpA expression with the levels of oxidative stress induced by cadmium and H2O2, with oxidative stress-inducing properties and lack of nutritional value, confirmed that the cells tend to harness their capacities with a possible increase in OmpA production. Collectively, this study suggests a homeostasis role for OmpA in Acinetobacter sp. SA01 under oxidative stress besides assuming many other roles hitherto attributed to this protein.IMPORTANCE Acinetobacter OmpA is known as a multifaceted protein with multiple functions, including emulsifying properties. Bioemulsifiers are surface-active compounds that can disperse hydrophobic compounds in water and help increase the bioavailability of hydrophobic hydrocarbons to be used by degrading microorganisms. In this study, an OmpA from Acinetobacter sp. SA01 was identified and introduced as an emulsifier with a higher emulsifying capacity than Pseudomonas aeruginosa rhamnolipid. We also showed that the expression of this protein is not dependent on the nutritional requirements but is more influenced by the oxidative stress caused by stressors. This finding, along with the structural role of this protein as a slow porin or its role in OMV biogenesis and biofilm formation, suggests that this protein can play an important role in maintaining cellular homeostasis under oxidative stress conditions. Altogether, the present study provides a new perspective on the functional performance of Acinetobacter OmpA, which can be used both to optimize its production as an emulsifier and a target in the treatment of multidrug-resistant strains.

Lactobacillus salivarius NK02: a Potent Probiotic for Clinical Application in Mouthwash.

A specific strain of naturally occurring oral lactobacilli was isolated and identified based on morphological, biochemical, and 16S rRNA gene sequencing. The phylogenetic affiliation of the isolate confirmed that the NK02 strain had close association with the Lactobacillus salivarius. An effective mouthwash was developed for treatment of periodontitis and suppression of the indicator bacterium Aggregatibacter actinomycetemcomitans which is an obvious pathogen of periodontal disease. The mouthwash containing L. salivarius NK02 was tested at a dose level of 108 (colony forming units (CFU) ml-1), monitoring over a period of 4 weeks. The study was a randomized double-blind placebo control trial, and the patients were treated in two groups of control and test by using scaling and root planing (SRP) + placebo and scaling and root planing (SRP) + probiotic, respectively. It appeared that the probiotic mouthwash was able to inhibit the bacterial growth on both saliva and sub-gingival crevice and exhibited antibacterial activity against A. actinomycetemcomitans. The results also showed that SRP+ probiotic treatment led to a significant decrease of gingival index (GI) and bleeding on probing (BOP) compared with that of SRP + placebo for the probiotic group. The rate of decrease in pocket depth was displayed in the group with SRP + probiotic treatment equal to 1/2 mm, and probing pocket depth (PPD) value was decreased in the probiotic bacteria treatment group that can explain the decrease in inflammation in gingiva. Our findings suggest that probiotic mouthwash is healthy for daily use as an alternative for maintaining dental and periodontal health.

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.

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate.

BACKGROUND: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). METHODS: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. RESULTS: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 µg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. CONCLUSION: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .

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.

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.

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.