Luria-Bertani Research Articles

Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain BD7100.

Coptidis rhizome (CR) is a widely used herbal medicine that contains protoberberine-type alkaloids. CR extract exhibits various pharmacologic activities. A previous study reported the isolation of Rhodococcus sp. strain BD7100 as a berberine (BBR)-utilizing bacterium, and the BBR-degradation pathway has been investigated. When we incubated strain BD7100 cells with CR extract, the number of viable cells declined with the degradation of components in the CR extract, and the culture broth exhibited antibacterial activity against strain BD7100. These results suggest that CR extract cultured in the presence of strain BD7100 contains one or more antibacterial agents. In this study, we isolated coptirhoquinone A (1) from CR extract incubated with strain BD7100 in Luria-Bertani (LB) medium, and the structure was elucidated using NMR and MS analysis. We also report the total synthesis and antimicrobial activities of 1 against bacteria, fungi, and Pythium sp.

Combining LC-MS/MS and hollow-fiber infection model for real-time quantitation of ampicillin to antimicrobial resistance.

Although a marked decrease in mortality associated with bacterial infections is attributed to the discovery of antibiotics, antibiotic resistance has become a global health concern due to their misuse. A dynamic in vitro hollow-fiber system was used to study antibiotic resistance in Escherichia coli using ampicillin. An LC–MS/MS assay was validated for quantitative analysis of ampicillin in Luria–Bertani broth. The assay was linear from 0.10–50.00 μg/ml. The assay met acceptance criteria for inter- and intra-assay precisions and accuracies across three quality controls. Stability of ampicillin was confirmed at three different storage conditions. In vitro data were similar to simulated plasma PK data further confirming the appropriateness of the experimental design to quantify antibiotics and study occurrence of antimicrobial resistance in real-time.

Molecular characterization of Plant Growth Promoting Rhizobacteria using 16S rRNA sequences in the organic rice field of Sukorejo Village, Central Java, Indonesia

Santosa S, Sutarno, Purwanto E, Suranto, Sajidan. 2018. Molecular characterization of Plant Growth Promoting Rhizobacteria using 16S rRNA sequences in the organic rice field of Sukorejo Village, Central Java, Indonesia. Biodiversitas 19: 2157-2162. Plant Growth Promoting Rhizobacteria (PGPR) are rhizosphere bacteria that can be utilized to increase plant growth and suppress plant diseases. PGPR concentrate on the concept of sustainable agriculture due to their role as biostimulant by synthesizing and regulating the concentration of various phytohormones and as biofertilizers. It is important to determine the original PGPR found in the roots of rice plants. This study aimed to identify PGPR based on 16S rRNA sequences. The bacterial strain was isolated from the rhizospheric soil of IR 64 organic and inorganic rice fields in the region of Sragen District, Central Java, Indonesia. The bacterial isolates were grown on the Luria Bertani (LB) Agar Medium. There were 10 colonies obtained with different morphological variations, nine of those were from the organic rice field. Molecular characterization using 16R rRNA sequences suggested the identity the nine isolates were of Pseudomonas aeruginosa strain RI-98-1, Stenotrophomonas maltophilia strain S431, Bacillus subtilis strain CEB2, Bacillus cereus strain ATCC 14579 clone EA195, S. maltophilia strain 5517, Exiguobacterium acetylicum strain SSA-3, Serratia nematodiphila strain HC4, Bacillus cereus strain ANP221, and Acinetobacter junii strain M. pstv. 21.4 Pseudomonas and Bassilus produce phytohormones (auxins) and convert inorganic phosphate to organic and Stenotropomonas can increase the growth of Acinetobacter and Exigobacterium as phosphate solvents in the soil.

Synergistic anticancer and antibacterial activities of cordycepin and selected natural bioactive compounds

Purpose: To investigate if cordycepin (Cor) exerts synergistic anticancer and antibacterial activities with curcumin (Cur), resveratrol (Res), genistein (Gen), and anthocyanidin (Ant) in order to improve its therapeutic effect. Methods: Antibacterial activity was assayed against Escherichia coli BL21 (DE3) cultured in the Luria-Bertani (LB) medium, while anticancer activity against Hela cells was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Immunofluorescence assay was used to measure the expressions of p53 and caspase-3, while apoptosis was assayed in Hela cells using Wright-Giemsa dye staining method. Results: The combination of Cor and Gen produced the most significant synergistic antibacterial activity, while combination of Cor, Cur and Gen exerted significant and synergistic anticancer activity against Hela cells. Among all three-compound combinations, Cor + Res + Gen had the highest synergistic anticancer activity. Immunofluorescence data indicate that Cor functioned by up-regulating p53-dependent apoptosis, while Res exerted its effect by p53- and caspase-3-dependent apoptosis, and Gen functioned by inhibiting adenosine deaminase and Cor degradation. Conclusion: The three compounds function via different mechanisms, and synergistically induce Hela cell apoptosis. This work provides a strong basis for the development of highly active and multicomponent anticancer drugs from natural sources. Keywords: Cordycepin, Natural phenolic compounds, Caspase, Anticancer, Synergy

Isolation and characterization of the Bacillus cereus BC7 strain, which is capable of zearalenone removal and intestinal flora modulation in mice

Zearalenone (ZEN) causes serious diseases in both animals and humans and thereby leads to substantial economic losses. The elimination of ZEN contamination from food and feed is an important concern worldwide. This study aimed to screen a bacterium that can efficiently detoxify ZEN both in vitro and in vivo. A bacterium (designated BC7) with high ZEN-removing capability was isolated from mouldy contaminated feeds and characterized as Bacillus cereus based on biochemical and 16S rRNA sequencing analyses. BC7 could remove 100% and 89.31% of 10 mg/L ZEN in Luria-Bertani (LB) medium and simulated gastric fluid (GSF), respectively, within 24 h at 37 °C. The effects of BC7 on ZEN detoxification and on the intestinal flora were further evaluated using four groups of mice that were intragastrically administered normal saline, BC7 culture (CFU = 3.45 × 108/mL), ZEN (10 mg/kg BW) or BC7 culture (CFU = 3.45 × 108/mL) + ZEN (10 mg/kg BW) for 2 weeks. ZEN showed distinct reproductive and hepatic toxicity, as characterized by increased weights of the uterus and liver, altered levels of oestradiol (E2) and luteinizing hormone (LH), increased secretion of the liver injury biomarkers alanine transaminase (ALT) and aspartate transaminase (AST), and abnormal histological phenotypes for the uterus, ovary and liver. However, BC7 could significantly reduce all the above-mentioned adverse effects caused by ZEN with no harmful effect on the reproductive system and liver in mice. Moreover, the addition of BC7 could efficiently renormalize the ZEN-induced perturbation of the gut microbiota and significantly increase the abundance of Lactobacillus to maintain the health of the intestinal flora in mice. In conclusion, Bacillus cereus BC7 could be used as a potential feed additive to efficiently remove ZEN in vitro or in vivo and to normalize the disordered gut microbiota in mice.

Role of pvdE Pyoverdine Synthesis in Pseudomonas aeruginosa Keratitis.

Pseudomonas aeruginosa produces pyoverdine, encoded by the pvdE gene, for high-affinity iron uptake from transferrin and lactoferrin. This study investigated the contribution of pyoverdine to P. aeruginosa keratitis pathogenesis using in vitro and in vivo models. The P. aeruginosa strains examined were parental strain PAO1 and isogenic mutant strain pvdE (ΔpvdE) defective in pyoverdine. Bacterial growth in vitro was determined by PAO1 and ΔpvdE optical densities in Luria-Bertani (LB) broth. PAO1 or ΔpvdE (10 colony-forming units/mL) was inoculated onto cultured human corneal epithelial cells (HCECs) for 1 hour. The monolayers were examined for bacterial adhesion and invasion. In addition, the corneas of C57BL/6 mice were infected with PAO1 or ΔpvdE. Corneal virulence was evaluated by determining clinical scores and bacterial counts during infection. The growth of PAO1 and ΔpvdE in LB broth was similar. Although adhesion of ΔpvdE onto HCECs was significantly increased compared with PAO1, the invasive capacity of ΔpvdE was significantly decreased. Clinical scores and bacterial numbers were significantly lower in ΔpvdE-infected eyes compared with PAO1-infected eyes at 6, 24, and 48 hours (P < 0.001). ΔpvdE was not detected in mouse corneas and did not induce corneal opacity at 6, 24, or 48 hours. ΔpvdE lost invasive ability toward HCECs. Moreover, ΔpvdE did not cause keratitis in vivo. Thus, pvdE pyoverdine synthesis has critical roles in proliferation and invasion on ocular surfaces and could be a target for prevention of P. aeruginosa keratitis.

Bacillus velezensis A2 fermentation exerts a protective effect on renal injury induced by Zearalenone in mice

Zearalenone (ZEN) is an estrogen-like mycotoxin occurring in food and feeds, and it can cause oxidative damage and apoptosis in the testis, liver, and kidney. A current concern for researchers is how to reduce the harm it causes to humans and animals. In this study, our aim was to isolate and identify a novel and efficient ZEN-detoxifying strain of bacteria, and we aimed to assess the protective effect of the isolated strain on kidney damage caused by ZEN in mice. Our results indicated that a strain of Bacillus velezensis (B. velezensis), named A2, could completely degrade ZEN (7.45 μg/mL) after three days of incubation at 37 °C in the Luria-Bertani (LB) medium. This fermentation broth of the B. velezensis A2 strain was given to mice. The histopathological analysis indicated that the fermentation broth from the B. velezensis A2 strain reduced the degree of renal injury that is induced by ZEN. Furthermore, it greatly reduced the increase in serum levels of creatinine (CRE), uric acid (UA), and urea nitrogen (BUN) caused by ZEN. In addition, B. velezensis A2 strain also significantly inhibited the increase of malonaldehyde (MDA) content, and reversed the decreases of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities caused by ZEN. Studies have shown that ZEN is involved in the regulation of mRNA and protein levels of genes involved in the ER stress-induced apoptotic pathway, such as heavy chain binding protein (BIP), C-/-EBP homologous protein (CHOP), cysteine Aspartate-specific protease-12 (Caspase-12), c-Jun N-terminal kinase (JNK), and BCL2-related X protein (Bcl-2 and Bax). However, when mice were administered the fermentation broth of the B. velezensis A2 strain, it significantly reversed the expressions of these genes in their kidney tissue. In conclusion, our results indicate that the newly identified strain of B. velezensis A2, has a protective effect from renal injury induced by ZEN in mice. This strain has a potential application in the detoxification of ZEN in feed and protects animals from ZEN poisoning.

Quantitative proteomics reveals the crucial role of YbgC for Salmonella enterica serovar Enteritidis survival in egg white

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a food-borne bacterial pathogen that can cause human salmonellosis predominately by contamination of eggs and egg products. However, its survival mechanisms in egg white are not fully understood, especially from a proteomic point of view. In this study, the proteomic profiles of S. Enteritidis in Luria-Bertani (LB) broth containing 50% and 80% egg white, and in whole egg white were compared with the profile in LB broth using iTRAQ technology to identify key proteins that were involved in S. Enteritidis survival in egg white. It was found that there were 303, 284 and 273 differentially expressed proteins in S. Enteritidis after 6 h exposure to whole, 80% and 50% egg white, respectively. Most of up-regulated proteins were primarily associated with iron acquisition, cofactor and amino acid biosynthesis, transporter, regulation and stress responses, whereas down-regulated proteins were mainly involved in energy metabolism, virulence as well as motility and chemotaxis. Three stress response-related proteins (YbgC, TolQ, TolA) of the tol-pal system responsible for maintaining cell membrane stability of Gram-negative bacteria were up-regulated in S. Enteritidis in response to whole egg white. Interestingly, deletion of ybgC resulted in a decreased resistance of S. Enteritidis to egg white. Compared with the wild type and complementary strains, a 3-log population reduction was observed in △ybgC mutant strain after incubation in whole egg white for 24 h. Cellular morphology of △ybgC mutant strain was altered from rods to spheres along with cell lysis in whole egg white. Furthermore, deletion of ybgC decreased the expression of tol-pal system-related genes (tolR, tolA). Collectively, these proteomic and mutagenic analysis reveal that YbgC is essential for S. Enteritidis survival in egg white.

COMPARATIVE STUDY OF GELATINASE ACTIVITY AND PELLICLE FORMATION AMONG EXTENDED-SPECTRUM BETA-LACTAMASE AND NON-EXTENDED-SPECTRUM BETA-LACTAMASE PRODUCING ACINETOBACTER BAUMANNII FROM DIABETIC FOOT ULCER INFECTIONS

Objective: The aim of the study was to assess and compare the gelatinase activity and pellicle formation in extended-spectrum beta-lactamase (ESBL) and non-ESBL producing Acinetobacter baumannii isolates from diabetic foot ulcer infection (DFI).Methods: A total of 42 isolates of A. baumannii recovered from patients of DFI from September 2016 to February 2018. Isolates were identified by the standard microbiological method and confirmed by the BD Phoenix 100 system. The antimicrobial susceptibility test was performed by the Kirby–Bauer disk diffusion method and ESBL was detected by double disk diffusion synergy test method. Gelatinase production was determined by the Luria Bertani agar supplemented with 30 g/L gelatin, and pellicle formation was determined by the Mueller-Hinton broth which is incubated at two different temperatures.Results: A total of 42 A. baumannii isolates were multidrug resistant. Among 21 isolates, each was ESBL and non-ESBL producers. Pellicle formation at 25°C in ESBL and non-ESBL producer isolates was 47.61% (10/21) and 28.57% (06/21). Pellicle formation at 37°C in ESBL and non-ESBL producer isolates was 57.14% (12/21) and 42.85% (09/21), respectively. Gelatinase production was present in 38.09% ESBL and 28.57% in non-ESBL producers. ESBL strains were more virulent compared to non-ESBL producers among patients of DFIs.Conclusion: This study showed that pellicle formation at 37°C was highly virulent due to ESBL producers. Gelatinase production was elevated in ESBL compared to non-ESBL producer isolates. This attribute of the isolates could render ESBL positive more pathogenic. Colistin and polymyxin B are the only choices of treatment for multidrug-resistant Acinetobacter baumannii infections.

The Construction of an Engineered Bacterial Strain and Its Application in Accumulating Mercury from Wastewater

To remove organic and inorganic mercury from wastewater, an engineered bacterial strain, BL21-7, was constructed that contained the artificial operon P16S-g10-merT-merP-merB1-merB2-ppk-rpsT. For BL21-7, the minimum inhibitory concentrations of mercuric chloride, methylmercury chloride and phenylmercury chloride in Luria-Bertani (LB) medium were 100 µmol/L, 60 µmol/L and 80 µmol/L, respectively. After being cultured in three media (liquid LB containing 80 µmol/L mercuric chloride, 40 µmol/L methylmercury chloride or 60 µmol/L phenylmercury chloride) for 72 h, the engineered bacteria accumulated up to 70.5 ± 1.5 µmol/L, 33.5 ± 3.2 µmol/L and 45.3 ± 3.7 µmol/L of mercury, respectively. In the presence of 10 µmol/L Cd2+, 10 µmol/L Pb2+ or 10 µmol/L Cu2+, the accumulation of mercurial derivatives by BL21-7 was not affected. BL21-7 could accumulate mercury well in media with pH values ranging from 5 to 8 and it could work well at temperatures from 25 °C to 37 °C. After BL21-7 was added to wastewater and cultured for 24 h, approximately 43.7% of the Hg in the wastewater was removed.

Recombinant Escherichia coli BL21-pET28a-egfp Cultivated with Nanomaterials in a Modified Microchannel for Biofilm Formation.

The application of whole cells as catalytic biofilms in microchannels has attracted increasing scientific interest. However, the excessive biomass formation and structure of biofilms in a reactor limits their use. A microchannel reactor with surface modification was used to colonize recombinant Escherichia coil BL21-pET28a-egfp rapidly and accelerated growth of biofilms in the microchannel. The segmented flow system of ‘air/culture medium containing nanomaterials’ was firstly used to modulate the biofilms formation of recombinant E. coil; the inhibitory effects of nanomaterials on biofilm formation were investigated. The results indicated that the segmental flow mode has a significant impact on the structure and development of biofilms. Using the channels modified by silane reagent, the culture time of biofilms (30 h) was reduced by 6 h compared to unmodified channels. With the addition of graphene sheets (10 mg/L) in Luria-Bertani (LB) medium, the graphene sheets possessed a minimum inhibition rate of 3.23% against recombinant E. coil. The biofilms cultivated by the LB medium with added graphene sheets were stably formed in 20 h; the formation time was 33.33% shorter than that by LB medium without graphene. The developed method provides an efficient and simple approach for rapid preparation of catalytic biofilms in microchannel reactors.

Biofilm-Producing Ability of Bovine Extraintestinal Pathogenic Escherichia coli and Its Correlation with Attachment Factors

Background: Escherichia coli is recognized as a common cause of infection. Long-lasting presence of bacteria on biotic and abiotic surfaces and failure of bacterial eradication are predicted by biofilm production. Some extracellular frills of E. coli can be implicated in productive events, leading to biofilm formation by surface colonization. Objectives: In the present study, correlation of csgA (encoding curli fimbriae) and fimA (encoding a large subunit of type I fimberiae) gene expression with biofilm formation of extraintestinal bovine pathogenic E. coli strains was evaluated in different enrichment media. Methods: The microtiter plate-based crystal violet method was applied to examine the biofilm production of 30 E. coli strains in Luria-Bertani (LB) and Brain-Heart Infusion (BHI) broth with 1% sucrose. PCR assay was performed to determine the presence of the studied genes. Results: According to the results, 100% of isolates contained csgA genes, and 96.7% contained fimA genes. Using the BHI medium with 1% sucrose, 53.3% and 16.6% of strains were average and strong biofilm producers, respectively. On the other hand, by using the LB medium, 66.6% of isolates were poor biofilm producers, whereas none were strong biofilm producers. Conclusions: The BHI medium containing 1% sucrose was better detected in biofilm production, compared to the LB medium. Since the studied genes were present in non-biofilm producing isolates, the correlation of these genes with biofilm-producing ability is questioned.

Biosorption of arsenic through bacteria isolated from Pakistan.

The aim of this study was to isolate arsenic-resistant bacteria and to further exploit it for remediation purposes. In the present study, we have isolated arsenic-resistant strain from ground water of Pakistan AT-01. The strain was cultivated at 37°C in Luria Bertani broth supplemented with different concentrations of arsenate and arsenite. The minimum inhibitory concentration of arsenic against the bacterial isolate was 7g/L (7000mg/L) for arsenate and 1.4g/L (1400mg/L) for arsenite salt. The bacterial isolate was also characterized both on molecular and biochemical basis. The isolated strain belonged to the Pseudomonas aeruginosa. The high resistance against arsenic offered by the bacteria was exploited further for bioremediation purposes. The bacterial biomass generated from AT-01 strain was able to efficiently remove arsenic with 98% efficiency. Arsenic contamination of ground water is a widespread worldwide problem. The present study shows the potential of high arsenic-resistant bacteria for efficient arsenic removal.

Regulation of virulence and motility by acetate in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli colonizes the human small intestine and causes severe diarrhea. Short-chain fatty acids are abundant in the intestine owing to the metabolic activity of the microflora and are important for intestinal health. Here, we found that acetate promotes the adherence of enteropathogenic E. coli O127:H6 to Caco-2 intestinal epithelial cells and its motility on semi-solid Luria–Bertani agar by activating the expression of locus of enterocyte effacement genes and flagellar genes, respectively. The effect of acetate on locus of enterocyte effacement gene expression is mediated by Ler, the master regulator of locus of enterocyte effacement genes, whereas the regulation of flagellar genes by acetate is dependent on the RNA polymerase sigma factor FliA. Conversely, formate, propionate, and butyrate had little or no effect on enteropathogenic E. coli O127:H6 adherence and motility. Finally, the acetate-mediated regulatory pathway was found to be a widespread mechanism used by a range of enteropathogenic E. coli to mediate bacterial virulence and motility. Therefore, upon entering the human small intestine, enteropathogenic E. coli may respond to the higher acetate level to enhance its virulence and motility, leading to efficient colonization of the target niche.

Expression and purification of a recombinant capsid protein of Semliki Forest virus and associate with a RNA of interest

Background: The Semliki Forest virus (SFV) is one of the most studied positive RNA viruses. The SFV has an icosahedric nucleocapsid composed of virus genomic RNA and the capsid protein (C). The nucleocapsid has 180 copies of C protein and is covered by a lipoproteic cell-derived membrane containing viral spike glycoproteins. The 267 amino acid C protein has two main functions in virus life cycle: it is responsible for the signalization of structural proteins, and also for RNA binding keeping it conserved. The objective this work is to express the C protein of SFV and associate it in vitro with a self-replicative RNA, containing Rabies Virus Glicoprotein (RVGP) gene. Methods & Materials: The C protein cDNA was cloned into pET28a vector fusioned with a histidine tag, obtaining the pET/C plasmid. BL21 (DE3) E. coli were transformed with pET/C by heat-shock and plated on agar-kanamycin medium. Colonies were cultivated in Luria-Bertani (LB) liquid medium, induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein of interest was purified using affinity and desalting columns. The protein was associated in vitro with a self-replicative RNA and BHK-21 cells were transfected with the complex and analyzed by immunofluorescence. Results: The expression and purification of the capsid protein were successfully performed. A partial purification was performed by affinity chromatography followed by a desalting step. The C protein efficiently bound the RNA and delivered it to the cells where the RVGP expression was detected by immunofluorescence. Conclusion: The expression of this protein in bacteria has some advantages over the utilization of eukaryotic systems, as the simple manipulation, high productivity and low cultivation cost.

Antimicrobial activity and mechanism of action of olive oil polyphenols extract against Cronobacter sakazakii

This study was conducted to analyze the antimicrobial activity of olive oil polyphenols extract (OOPE) against Cronobacter sakazakii. The antimicrobial effect was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, and survival of bacteria after treatments with OOPE in sterile normal saline (NS) and Luria-Bertani (LB) broth. The changes in intracellular ATP concentration, cell membrane potential, content of bacterial protein, and cell morphology were analyzed to reveal possible mechanism of action. The results showed that the MICs and MBCs of OOPE against C. sakazakii strains ranged from 0.625 to 1.25 mg/mL, and 1.25–2.50 mg/mL, respectively. Approximately 8 log CFU/mL of C. sakazakii was completely inactivated after a treatment with 1 MIC of OOPE in NS at 37 °C for 3 h, and in LB broth at 37 °C for 10 h. The mechanism of action of OOPE against C.sakazakii was associated with reduction of intracellular ATP concentrations, cell membranes depolarization, leakage of cytoplasm, decrease of bacterial protein content and leakage of cytoplasm. Therefore, our findings provided a theoretical possibility that OOPE can be used as a natural food preservative or bactericide to reduce the food safety problems caused by C. sakazakii strains in food industries as well as food processing environments.

Effect of carbonic anhydrase-containing endophytic bacteria on growth and physiological attributes of wheat under water-deficit conditions

ABSTRACTDrought is one of the major limitations to agricultural productivity, suppressing plant growth and yield of food crops throughout the world particularly in arid and semiarid regions. Drought-tolerant carbonic anhydrase (CA; EC 4.2.1.1)-containing endophytic bacteria may improve plant growth under stressed conditions. In the present study, effect of drought-tolerant CA-containing endophytic bacteria on growth and physiology of wheat under water-deficit conditions was studied. One hundred and fifty isolates were isolated from wheat plants and screened for their ability to tolerate polyethylene glycol (PEG) 6000-induced water-deficit stress (−0.31 to −3.20 MPa). Fifty isolates exhibiting intrinsic ability to tolerate stress were further screened for CA activity. Ten drought-tolerant isolates with higher CA activity were evaluated for improving wheat growth under water-deficit conditions (−0.04, −1.09, −1.23 MPa). Results showed that PEG-mediated water-deficit stress significantly reduced growth of wheat. However, inoculation with isolates WR2, WS11 and WL19 significantly enhanced seedling growth by improving maximum root length, shoot length, root and shoot dry weight under non-stressed as well as stressed conditions. These isolates were identified by 16S rRNA as Bacillus marisflavi (WR2) Bacillus thuringiensis (WS11) and Bacillus subtilis (WL19). Isolate WL19 also improved chlorophyll content, photosynthetic rate, CA activity and relative water content compared to uninoculated control plants. Overall, our findings suggest that endophytic bacterial isolates WR2, WS11 and WL19 with CA activity can enhance photosynthesis and biomass of wheat seedlings under water-deficit conditions.Abbreviations: CA: Carbonic anhydrase; PEG: Polyethylene glycol; CO2: Carbon dioxide; HCO3–: Bicarbonate; TSA: Tryptic Soy Agar; LB: Luria Bertani; A: CO2 assimilation rate; E: Transpiration rate; gs: Stomatal conductance; Ci: Substomatal CO2 concentration; RWC: Relative water content; EL: Electrolyte leakage

Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments

Burkholderia pseudomallei is a flagellated, gram-negative environmental bacterium that causes melioidosis, a severe infectious disease of humans and animals in tropical areas. We hypothesised that B. pseudomallei may undergo phenotypic adaptation in response to an increase in growth temperature. We analysed the growth curves of B. pseudomallei strain 153 cultured in Luria–Bertani broth at five different temperatures (25 °C–42 °C) and compared the proteomes of bacteria cultured at 37 °C and 42 °C. B. pseudomallei exhibited the highest growth rate at 37 °C with modest reductions at 30 °C, 40 °C and 42 °C but a more marked delay at 25 °C. Proteome analysis revealed 34 differentially expressed protein spots between bacterial cultures at 42 °C versus 37 °C. These were identified as chaperones (7 spots), metabolic enzymes (12 spots), antioxidants (10 spots), motility proteins (2 spots), structural proteins (2 spots) and hypothetical proteins (1 spot). Of the 22 down-regulated proteins at 42 °C, redundancy in motility and antioxidant proteins was observed. qRT-PCR confirmed decreased expression of fliC and katE. Experiments on three B. pseudomallei strains demonstrated that these had the highest motility, greatest resistance to H2O2 and greatest tolerance to salt stress at 37 °C. Our data suggest that temperature affects B. pseudomallei motility and resistance to stress.

Induction of Escherichia coli into a VBNC state through chlorination/chloramination and differences in characteristics of the bacterium between states

Many pathogens can enter into a viable but nonculturable (VBNC) state in response to harsh environmental stresses. Bacteria in this state can retain certain features of viable cells, such as cellular integrity, metabolic activity, or virulence and may present health risks associated with drinking water. In this study, we investigated the ability of chlorination and chloramination, which are widely used methods to disinfect drinking water, to induce Escherichia coli into a VBNC state. After treatment with chlorine and chloramine at concentrations of 1, 2, 3, and 4 mg/L, the counts of culturable E. coli cells decreased from 106 CFU/mL to 0 CFU/mL at 5–60 min post treatment. Meanwhile, viable cell counts were still approximately 103–105 cells/mL. These viable E. coli cells may be induced into a VBNC state by chlorination and chloramination. Scanning electron microscopy and laser confocal microscopy showed that some bacteria maintained cellular integrity, but the average length of VBNC cells was less than that of culturable cells. Respiratory activity of VBNC cells decreased approximately 50% relative to that of culturable cells. We also used heavy water (D2O) combined with Raman microspectroscopy to show that E. coli in a VBNC state retained metabolic activity involving water (e.g. condensation reactions) at the single-cell level. Furthermore, soxR, gadA, and katG genes remained highly expressed, suggesting that VBNC cells were physiologically active. Finally, resuscitation of VBNC cells induced by chlorine in Luria-Bertani (LB) broth was identified by calculating the generation time. Results of this study will facilitate a better understanding of the health risks associated with VBNC bacteria and the development of more effective strategies for drinking water disinfection.

Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis

The trans-translation system is recognized as an excellent target for developing new drugs to rapidly sterilize Mycobacterium tuberculosis (TB) infection and significantly shorten TB treatment duration. As a vital component of the trans-translation system for rescuing stalled ribosomes, the SmpB protein from Mycobacterium tuberculosis (MtbSmpB, 1-160 a. a.) mediates tmRNA binding to stalled ribosomes through forming a complex with tmRNA. So far, few works have been conducted to prepare, characterize biophysical properties and determine three-dimensional structure for the full-length MtbSmpB protein. In the present work, we successfully expressed and purified the His-tagged full-length MtbSmpB protein in Escherichia coli with a yield of 26.9 mg from 1 L of Luria Bertani medium. We also obtained MtbSmpB with a yield of 18.5 mg from 1 L of M9 minimal medium. The MtbSmpB protein showed a single band in SDS-PAGE with a molecular weight of ∼20 kDa consistent with the measurement from MALDI-TOF-mass spectrometry. The dynamic light scattering experiment indicated that MtbSmpB existed in a monomeric form. Moreover, both circular dichroism and nuclear magnetic resonance (NMR) experiments exhibited that MtbSmpB was well structured, suggesting that it could be feasible to determine its solution structure by NMR spectroscopy. NMR titration experiments showed that MtbSmpB specifically bound to tmRNA. This work lays the essential basis for further determining the solution structure and dynamics of the full-length MtbSmpB protein.