Description: Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, resulting in severe economic losses worldwide. Coliphages represent alternative antibacterial substitutes based on high lytic efficiency and few side-effects. However, the complete genome sequences information of APEC phages are limited, and knowledge of undesired genes and the narrow host range restrict their applications. In this study, we isolated a virulent phage QL01, with a relatively broad lytic spectrum (41 of 78 APEC strains). Transmission electron micrography showed it belonged to the family Myoviridae with an elongated head and a contractile tail. Whole genome sequencing revealed a linear double-stranded DNA (170,527 kb; GC content, 39.6%) with 275 possible ORFs. Comparative genome analysis revealed high homology between QL01 and other T4-like phages. However, it also showed some unique features, for example, ORF142 and ORF143, which encode IP9 and IP8, respectively, and may counteract host resistance only exist in a few T4-like phages such as IME08 and vB_EcoM_VR5. Furthermore, phage therapy in artificially infected ducks showed a 26.67% decrease in mortality compared with the untreated group. Our study indicates the potential antibacterial function of phage QL01 against APEC infections and highlights unique molecular features underlying the relatively broad host range.

Description: Biosynthesis of silver nanoparticles (AgNPs) is an eco-friendly approach by using different biological sources; for example, plants and microorganisms such as bacteria, fungi, and actinobacteria. In this report, we present the biological synthesis of silver nanoparticles (AgNPs) by acidophilic actinomycetes SL19 and SL24 strains isolated from pine forest soil (pH 〈 4.0). The isolates based on 16S rRNA gene sequence were identified as Pilimelia columellifera subsp. pallida . The synthesized AgNPs were characterized by visual observations of colour change from light-yellow to dark-brown. The UV-vis spectra of AgNPs were recorded at 425 and 430 nm. The AgNPs were further characterized by Nanoparticle tracking analysis (NTA), Zeta potential, Fourier transform infrared spectroscopy (FTIR) and Transmission electron microscopy (TEM). FTIR analysis revealed the presence of proteins as a capping agent. TEM analysis confirmed the formation of spherical and polydispersed NPs of 12.7 and 15.9 nm sizes. The in vitro antibacterial activity of AgNPs alone and in combination with antibiotics was evaluated against clinical bacteria viz., Staphylococcus aureus , Bacillus subtilis , Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa , and uropathogens such as Enterobacter , S. aureus , P. aeruginosa , K. pneumoniae , and E. coli . The lowest MIC (40 μg ml −1 ) was demonstrated by AgNPs synthesized from SL24 against E. coli . However, the AgNPs of SL19 showed lowest MIC (70 μg ml −1 ) against S. aureus . The activity of antibiotic was enhanced, when tested in combination with silver nanoparticles synthesized from both actinobacterial strains.

Description: A uranium (U)-resistant and -accumulating Pseudomonas aeruginosa strain was characterized to assess the response of toxic metals toward its growth and expression of metal resistance determinants. The bacterium showed MIC (minimum inhibitory concentration) values of 6, 3, and 2 mM for Zn, Cu, and Cd, respectively; with resistance phenotype conferred by periplasmic Cu sequestering copA and RND type heavy metal efflux czcA genes. Real-time PCR-based expression analysis revealed significant upregulation of both these genes upon exposure to low concentrations of metals for short duration, whereas the global stress response gene sodA encoding superoxide dismutase enzyme was upregulated only at higher metal concentrations or longer exposure time. It could also be inferred that copA and czcA are involved in providing resistance only at low metal concentrations, whereas involvement of “global stress response” phenomenon (expression of sodA ) at higher metal concentration or increased exposure was evident. This study provides significant understanding of the adaptive response of bacteria surviving in metal and radionuclide contaminated environments along with the development of real-time PCR-based quantification method of using metal resistance genes as biomarker for monitoring relevant bacteria in such habitats.

Description: We have investigated the biotechnological potential of Arctic marine bacteria for their ability to produce a broad spectrum of cold-active enzymes. Marine bacteria exhibiting these features are of great interest for both fundamental research and industrial applications. Macrobiota, water and sediment samples have been collected during 2010 and 2011 expeditions around the Lofoten and Svalbard islands. Bacteria were isolated from this material and identified through 16S rRNA gene sequence analysis for the purpose of establishing a culture collection of marine Arctic bacteria. Herein, we present the functional screening for different extracellular enzymatic activities from 100 diversely chosen microbial isolates incubated at 4 and 20 °C. The production of esterase/lipase, DNase, and protease activities were revealed in 67, 53, and 56% of the strains, respectively, while 41, 23, 9, and 7% of the strains possessed amylase, chitinase, cellulase, and xylanase activities, respectively. Our findings show that phylogenetically diverse bacteria, including many new species, could be cultured from the marine arctic environment. The Arctic polar environment is still an untapped reservoir of biodiversity for bioprospecting.

Description: Quorum sensing is used by bacteria to coordinate gene expression in response to population density and involves the production, detection and response to extracellular signaling molecules known as autoinducers (AIs). Salmonella does not synthesize the AI-1, acyl homoserine lactone (AHL) common to gram-negative bacteria; however, it has a receptor for AI-1, the SdiA protein. The effect of SdiA in modulating phenotypes of Salmonella has not been elucidated. In this report, we provide evidence that the AIs-1 affect Salmonella enterica serovar Enteritidis behavior by enhancing the biofilm formation and expression of virulence genes under anaerobic conditions. Biofilm formation by Salmonella was detected by the crystal violet method and by scanning electron microscopy. The presence of AHLs, particularly C12-HSL, increased biofilm formation and promoted expression of biofilm formation genes ( lpfA , fimF , fliF , glgC ) and virulence genes ( hilA , invA , invF ). Our results demonstrated that AHLs produced by other organisms played an important role in virulence phenotypes of Salmonella Enteritidis.

Description: α- l -Fucosidases are key enzymes for the degradation of intestinal glycans by gut microbes. In this work, three putative α- l -fucosidases (Afc1, Afc2, and Afc3) genes from Clostridium perfringens ATCC 13124 were cloned and expressed in Escherichia coli . Afc1 had the α- l -fucosidase domain of glycoside hydrolase (GH) 29 family but showed no enzyme activity toward all the substrates examined. The putative acid/base residue of Afc1, Ser205, was replaced by a glutamic acid which is conserved in GH29-B α- l -fucosidases. However, the mutant Afc1-S205E still failed to show enzyme activity. Afc2 and Afc3 were determined to be 1,3-1,4-α- l -fucosidase of GH29-B subfamily and 1,2-α- l -fucosidase of GH95 family, respectively, and both of them could release fucose from porcine gastric mucin (PGM). When C. perfringens ATCC 13124 grew with the presence of PGM, the transcription of afc 1 decreased slightly, while those of afc 2 and afc 3 increased to 2.2-fold and 1.4-fold, respectively, and the enzyme activities of Afc2 and Afc3 in the culture increased to 2.2-fold and 2.6-fold, respectively. These results suggest that Afc2 and Afc3 are involved in the degradation of intestinal fucosyl glycans by C. perfringens ATCC 13124.

Description: Penicilliopsis clavariiformis AP, a rare salt tolerant fungus reported for the first time from India was identified through polyphasic taxonomy. Scanning electron microscopy showed that the fungus has unique features such as biverticillate penicilli bearing masses of oval to ellipsoidal conidia. The fungus has been characterized for salt tolerance and to understand the relevance of central carbon metabolism in salt stress adaptation. It showed optimal growth at 24 °C and able to tolerate up to 10% (w/v) NaCl. To understand the mechanism of adaptation to high salinity, activities of the key enzymes regulating glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle were investigated under normal (0% NaCl) and saline stress environment (10% NaCl). The results revealed a re-routing of carbon metabolism away from glycolysis to the pentose phosphate pathway (PPP), served as a cellular stress-resistance mechanism in fungi under saline environment. The detection and significant expression of fungus genes ( Hsp98 , Hsp60 , HTB , and RHO ) under saline stress suggest that these halotolerance conferring genes from the fungus could have a role in fungus protection and adaptation under saline environment. Overall, the present findings indicate that the rearrangement of the metabolic fluxes distribution and stress related genes play an important role in cell survival and adaptation under saline environment.

Description: Many ecosystems are currently co-contaminated with heavy metals such as cadmium (Cd 2+ ) and pesticides such as chlorpyrifos (CP) and γ-hexachlorocyclohexane (γ-HCH). A feasible approach to remediate the combined pollution of heavy metals and pesticides is the use of γ-HCH degrading bacteria endowed with CP hydrolysis and heavy metal biosorption capabilities. In this work, a recombinant microorganism capable of simultaneously detoxifying Cd 2+ , CP, and γ-HCH was constructed by display of synthetic phytochelatins (EC20) and methyl parathion hydrolase (MPH) fusion protein on the cell surface of the γ-HCH degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC–EC20–MPH was verified by cell fractionation, Western blot analysis, immunofluorescence microscopy, and proteinase accessibility experiment. Expression of EC20 on the cell surface not only improved Cd 2+ binding but also alleviated the cellular toxicity of Cd 2+ . As expected, the rates of CP and γ-HCH degradation were reduced in the presence of Cd 2+ for cells without EC20 expression. However, expression of EC20 (higher Cd 2+ accumulation) significantly restored the levels of CP and γ-HCH degradation. These results demonstrated that surface display of EC20 enhanced not only Cd 2+ accumulation but also protected the recombinant strain against the toxic effects of Cd 2+ on CP and γ-HCH degradation.

Description: This study evaluated ethanol fermentation and its correlation with glutathione (GSH) synthesis under various cadmium-conditions with different metal ions and nitrogen sources. We found that corn steep liquor (CSL) and yeast extract have differential roles to play in GSH accumulation in cell even though both of them could alleviate the inhibition by cadmium. The different GSH accumulation in cell resulted from the different contents of metal ions in CSL and yeast extract. Intracellular GSH decreased with increasing calcium concentrations, and high calcium concentrations rendered the yeast more tolerant to cadmium stress than the nitrogen sources did. When the mole ratio of calcium to cadmium was 100:1, yeast tolerated 1000 µmol/L cadmium with no decrease in efficiency in ethanol production. As a result, the use of calcium allowed a significant saving of high-cost nutrient yeast extract with an efficient ethanol production, making the bioconversion of cadmium-containing biomass into ethanol possible.

Description: Double-stranded RNA (dsRNA) is discovered to participate in the regulation of gene expression in both bacterial and eukaryotic cells. Members of ribonuclease III (RNase III) family recognize RNA motifs and cleave substrates at specific sites in a divalent-metal-ion-dependent manner. In this study, we find the RNase III from Bacillus Calmette Guerin (BCG-RNase III) cleaves small hairpin RNA based on the conserved stem structure associated with Mycobacterium 16S ribosomal RNA precursor at specific sites which are not determined. To evaluate the influence of remnant endogenous ribonucleases from expression host on RNA cleavage assays for RNase III, we use E44A and D48A mutant of the enzyme to perform RNA cleavage assays and find that remnant ribonucleases have no effect on cleavage assays. The RNA cleavage activity of the enzyme can be supported by Mg 2+ , Mn 2+ , and Co 2+ and enhanced with the increasing salt concentration. The catalytic activity of the enzyme is exhibited when the temperature of the reaction buffer ranges from 30 to 55 °C and the pH of the buffer from 7.0 to 10.0. Two major cleavage sites in RNA substrate are identified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE).