Photochemical Synthesis of Silver Hydrosol Stabilized by Carbonate Ions and Study of Its Bactericidal Impact on Escherichia coli: Direct and Indirect Effects

The great attention paid to silver nanoparticles is largely related to their antibacterial and antiviral effects and their possible use as efficient biocidal agents. Silver nanoparticles are being widely introduced into various areas of life, including industry, medicine, and agriculture. This leads to their spreading and entering the environment, which generates the potential risk of toxic effect on humans and other biological organisms. Proposed paper describes the preparation of silver hydrosols containing spherical metal nanoparticles by photochemical reduction of Ag+ ions with oxalate ions. In deaerated solutions, this gives ~10 nm particles, while in aerated solutions, ~20 nm particles with inclusion of the oxide Ag2O are obtained. Nanoparticles inhibit the bacterium Escherichia coli and suppress the cell growth at concentrations of ~1 × 10−6–1 × 10−4 mol L−1. Silver particles cause the loss of pili and deformation and destruction of cell membranes. A mechanism of antibacterial action was proposed, taking into account indirect suppressing action of Ag+ ions released upon the oxidative metal dissolution and direct (contact) action of nanoparticles on bacterial cells, resulting in a change in the shape and destruction of the bacteria.

Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

Targeting of Pseudomonas aeruginosa cell surface via GP12, an Escherichia coli specific bacteriophage protein

Bacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.

Features of the Bioconversion of Pentacyclic Triterpenoid Oleanolic Acid Using Rhodococcus Actinobacteria

The ability of actinobacteria of the genus Rhodococcus to transform oleanolic acid (OA), a plant pentacyclic triterpenoid, was shown for the first time using bioresources of the Regional Specialized Collection of AlkanotrophicMicroorganisms (IEGM; WDCM #768;www.iegmcol.ru). The most promising strains (R.opacus IEGM 488 and R.rhodochrousIEGM 285) were selected, and these catalyzed80% bioconversion of OA (0.5 g/L) in the presence of n-hexadecane (0.1% v/v) for seven days. The process of OA bioconversion was accompanied by a gradual decrease in the culture medium pH. Adaptive responses of bacterial cells to the OA effects included the formation of compact cellular aggregates, a marked change in the surface-to-volume ratio of cells, and a significant increase in the Zeta potential values. The results demonstrated that the process of OA bioconversion was catalyzed by membrane-bound enzyme complexes. Participation of cytochrome P450-dependent monooxygenases in the oxidation of the OA moleculewas confirmedusing specific inhibitors. The obtained data expand our knowledge on the catalytic activity of actinobacteria of the genus Rhodococcus and their possible use as biocatalysts for the bioconversion of complex hydrophobic compounds. The results can also be used inthe searchfor promising OA derivatives to be used in the synthesis of biologically active agents. Keywords: bioconversion, oleanolic acid, Rhodococcus, biologically active compounds

A small RNA that cooperatively senses two stacked metabolites in one pocket for gene control

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.

Antibacterial Activity and Multi-Targeting Mechanism of Dehydrocorydaline From Corydalis turtschaninovii Bess. Against Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen, with relatively low incidence but high case-fatality. Phytochemicals have been recognized as a promising antimicrobial agent as an alternative to synthetic chemicals due to their safety and high efficacy with multi-target sites. This study identified and characterized a novel antibacterial agent, dehydrocorydaline, in the Corydalis turschaninovii rhizome using HPLC-LTQ-Orbitrap-HRMS, and its antibacterial effect with lowest MIC (1 mg/mL) and MBC (2 mg/mL) values. In addition, an in vitro growth kinetic assay, cytoplasmic nucleic acid and protein leakage assay, and observation of morphological changes in bacterial cells supported the strong antibacterial activity. Dehydrocorydaline also displayed effective inhibitory effects on biofilm formation and bacterial motility. In order to investigate the potential antibacterial mechanism of action of dehydrocorydaline against L. monocytogenes, label-free quantitative proteomics was used, demonstrating that dehydrocorydaline has multiple targets for combating L. monocytogenes including dysregulation of carbohydrate metabolism, suppression of cell wall synthesis, and inhibition of bacterial motility. Overall, this study demonstrated that dehydrocorydaline has potential as a natural and effective antibacterial agent with multi-target sites in pathogenic bacteria, and provides the basis for development of a new class of antibacterial agent.

Isolation, characterization, molecular analysis and application of bacteriophage DW-EC to control Enterotoxigenic Escherichia coli on various foods

Among food preservation methods, bacteriophage treatment can be a viable alternative method to overcome the drawbacks of traditional approaches. Bacteriophages are naturally occurring viruses that are highly specific to their hosts and have the capability to lyse bacterial cells, making them useful as biopreservation agents. This study aims to characterize and determine the application of bacteriophage isolated from Indonesian traditional Ready-to-Eat (RTE) food to control Enterotoxigenic Escherichia coli (ETEC) population in various foods. Phage DW-EC isolated from Indonesian traditional RTE food called dawet with ETEC as its host showed a positive result by the formation of plaques (clear zone) in the bacterial host lawn. Transmission electron microscopy (TEM) results also showed that DW-EC can be suspected to belong to the Myoviridae family. Molecular characterization and bioinformatic analysis showed that DW-EC exhibited characteristics as promising biocontrol agents in food samples. Genes related to the lytic cycle, such as lysozyme and tail fiber assembly protein, were annotated. There were also no signs of lysogenic genes among the annotation results. The resulting PHACTS data also indicated that DW-EC was leaning toward being exclusively lytic. DW-EC significantly reduced the ETEC population (P ≤ 0.05) in various food samples after two different incubation times (1 day and 6 days) in chicken meat (80.93%; 87.29%), fish meat (63.78%; 87.89%), cucumber (61.42%; 71.88%), tomato (56.24%; 74.51%), and lettuce (46.88%; 43.38%).

The Effect of Bacteria on Early Age Strength of CEM I and CEM II Cementitious Composites

Despite being associated with lower carbon emissions, CEM II cementitious materials exhibit reduced early age strength compared to that of CEM I. Several studies have demonstrated early age strength improvements by incorporating bacterial cells in concrete. In this study, live vegetative bacteria and dead bacteria killed in two different ways were used to explore whether changes in strength are related to the bacteria’s viability or their surface morphology. Compressive and flexural strength tests were performed at mortars with and without bacteria for both CEM I and CEM II cement. Their microstructure, porosity and mineralogy were also examined. No net strength gain was recorded for either CEM I or CEM II bacterial mortars compared to non-bacterial controls, although changes in the porosity were reported. It is proposed that two phenomena, one causing strength-reduction and one causing strength-gain, took place in the bacterial specimens, simultaneously. It is suggested that each phenomenon is dependent on the alkalinity of the cement matrix, which differs between CEM I and CEM II mortars at early age. Nevertheless, in neither case could it be recommended that the addition of bacteria is an effective way of increasing the early age strength of mortars.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .