Stability Of Bacteria Research Articles

Plasmid segregation and stability in bacteria

Plasmid segregation and stability in bacteria

Construction and Rescue of a DNA-Launched DENV2 Infectious Clone.

Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, the production of Zika virus has been successful from CMV promoter-driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued viruses maintained the ability to form plaques and replicate efficiently in commonly used cell lines. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

Molecular dissection of RbpA-mediated regulation of fidaxomicin sensitivity in mycobacteria

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RPo). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RPo stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σA holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RPo complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RPo-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RPo stability and Fdx activity, we find that another factor that promotes RPo stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.

Evolution of the spontaneous sourdoughs microbiota prepared with organic or conventional whole wheat flours from South Brazil

The purpose of this study was to compare the composition and stability of bacteria and fungi communities during the propagation of sourdoughs prepared with organic or conventional whole wheat (Triticum aestivum) flours from South Brazil. Sourdoughs were prepared and samples were collected during different fermentation times (0 to 216 h). Total DNA of sourdough samples were extracted and the 16S rRNA gene and Internal Transcribed Spacer region were sequenced by MiSeq-Illumina. A total of 43 and 56 OTUs were identified and defined as core taxa in the bacterial and fungal communities, respectively. The analysis revealed increases in the relative abundances of the lactic acid (Pediococcus pentosaceus, Weissella hellenica and Limosilactobacillus pontis) and acetic acid bacteria (Gluconobacter frateurii and Acetobacter tropicalis) during the sourdough propagation. The filaments fungi, Alternaria tenuissima, Fusarium culmorum, Fusarium petersiae and Microdochium seminicola remained more stable in organic than conventional during propagation cycles. After 216 h of fermentation, both sourdoughs were dominated by acid- and salt-tolerant yeast Issatchenkia orientalis (syn Pichia kudriavzevii, and Candida glycerinogenes). In conclusion, there were no significant differences in microbial communities among the sourdough samples. This study revealed that both flours contain autochthonous LAB, AAB, and yeasts with biotechnological applications in sourdough bread-making.

A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.

Archaeal and bacterial communities assembly and co-occurrence networks in subtropical mangrove sediments under Spartina alterniflora invasion

BackgroundMangrove ecosystems are vulnerable due to the exotic Spartina alterniflora (S. alterniflora) invasion in China. However, little is known about mangrove sediment microbial community assembly processes and interactions under S. alterniflora invasion. Here, we investigated the assembly processes and co-occurrence networks of the archaeal and bacterial communities under S. alterniflora invasion along the coastlines of Fujian province, southeast China.ResultsAssembly of overall archaeal and bacterial communities was driven predominantly by stochastic processes, and the relative role of stochasticity was stronger for bacteria than archaea. Co-occurrence network analyses showed that the network structure of bacteria was more complex than that of the archaea. The keystone taxa often had low relative abundances (conditionally rare taxa), suggesting low abundance taxa may significantly contribute to network stability. Moreover, S. alterniflora invasion increased bacterial and archaeal drift process (part of stochastic processes), and improved archaeal network complexity and stability, but decreased the network complexity and stability of bacteria. This could be attributed to S. alterniflora invasion influenced microbial communities diversity and dispersal ability, as well as soil environmental conditions.ConclusionsThis study fills a gap in the community assembly and co-occurrence patterns of both archaea and bacteria in mangrove ecosystem under S. alterniflora invasion. Thereby provides new insights of the plant invasion on mangrove microbial biogeographic distribution and co-occurrence network patterns.

The activity of the disinfectant “Enzidez” against bacteria in biofilms

Currently, the pharmaceutical industry is constantly working on the creation of ideal disinfectants that would have a wide spectrum of antimicrobial action in minimal concentrations, did not result in the formation of stability in bacteria, were not toxic, not corrosive, not allergenic, cheap. The purpose of the work was to investigate the activity of the “Enzidesis” disidissance to influence microbial biofilms in vitro conditions for various sampling parameters. The density of microbial biofilms and the effects of disinfectants were determined on sterile stainless steel plates, which were placed in a Petri dish. For this purpose, biofilms were grown on plates, washed out biofilms from planktonic cells, acted as a disguise for a certain concentration and for a period of 5–30 minutes, fixed and painted biofilms and determined the optical density of the flushing solution from biofilms spectrometrically. It has been established that the disinfectant “Enzidesis” destroys the biofilms taken in the experiment of museum test cultures S. aureus, E. coli and P. aeruginosa. In particular, for the influence of the lowest concentration in experiment, 0.075 % optical density of washing solutions from S. aureus biofilmes decreased by 2.6 times, E. coli and P. aeruginosa biofilm in 2.9 times, respectively, comparing with biofilms after treatment with water. Under the actions of such a concentration of the “Enzidesis” means, the biofilm though significantly degraded, but they were still a medium density, more than 0.5 units. Increasing the concentration of a means of 0.075 % to 0.5 % contributed to the intensity of degradation of test-cultures, an average of 3.0 times (P < 0.05) and they became a weak density (0.24–0.20 s). Increasing the concentration of the “Enzidesis” means up to 1.0 % or more, did not significantly destroy the matrix of the biofilm of microorganisms, since the optical density of the washing solutions was as in control. In determining the influence of the temperature of the working solutions of the “Enzidosis” means on its film-building activity, it has been found that with an increase in the disinfection agent “Enzidesis” with +20 to +60 °С there is an increase in the degradation of the biofilm formed S. aureus, E. coli and P. aeruginosa. Dezilities can be effectively used in 0.5 % concentration at room temperature of solutions. When substantiating the exposure time of the “Enzidesis” exhibition time, it has been found that to remove the S. aureus, E. coli and P. aeruginosa 0.5 % means at a solution temperature of +20 ± 1 °C, it is necessary that the action time is from 15 to 30 minutes.

Structural Analysis of Glycosylglycerolipids Using NMR Spectroscopy.

Glycosylglycerolipids are essential components of plant and bacterial membranes. These lipids exert central roles in physiological processes such as photosynthesis in plants or to maintain membrane stability in bacteria. They are composed of a glycerol backbone esterified with two fatty acids at the sn-1 and sn-2 positions, and carbohydrate moieties connected via a glycosidic bond at the sn-3 position. Nuclear magnetic resonance (NMR) spectroscopy is a state-of-the-art technique to determine the nature of the bound carbohydrates as well as their anomeric configurations. Here we describe the analysis of intact glycosylglycerolipids by NMR spectroscopy to determine structural details of their sugar head groups without the need of chemical derivatization.

Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.

Regulation of mRNA Stability During Bacterial Stress Responses.

Bacteria have a remarkable ability to sense environmental changes, swiftly regulating their transcriptional and posttranscriptional machinery as a response. Under conditions that cause growth to slow or stop, bacteria typically stabilize their transcriptomes in what has been shown to be a conserved stress response. In recent years, diverse studies have elucidated many of the mechanisms underlying mRNA degradation, yet an understanding of the regulation of mRNA degradation under stress conditions remains elusive. In this review we discuss the diverse mechanisms that have been shown to affect mRNA stability in bacteria. While many of these mechanisms are transcript-specific, they provide insight into possible mechanisms of global mRNA stabilization. To that end, we have compiled information on how mRNA fate is affected by RNA secondary structures; interaction with ribosomes, RNA binding proteins, and small RNAs; RNA base modifications; the chemical nature of 5′ ends; activity and concentration of RNases and other degradation proteins; mRNA and RNase localization; and the stringent response. We also provide an analysis of reported relationships between mRNA abundance and mRNA stability, and discuss the importance of stress-associated mRNA stabilization as a potential target for therapeutic development.

Immobilization of Bacterial Cells and Chitinolytic Activity ofStreptomycessp. (PB 2)

This study was aimed to immobilize the chitinolytic bacteriaStreptomycessp. PB 2 using sodium alginate as immobilization agent.Streptomycessp. PB 2 was reported have a good chitinolytic activity and immobilization is known to increase the stability of bacteria during repeated used. Sodium alginate has been used on several methods of immobilization and sodium alginate are reported as a good agent. Cell immobilization was done by growing the cell on nutrient broth (NB), mixed with 1% sodium alginate to form the beads, then the mixture were put to a syringe and droped into CaCl2 solution. The beads were washed with 0.85% NaCl solution and filtered. To test the bacterial activity, beads were applied into chitin liquid medium and the chitinolytic activity was observed every 24 hours for 5 days. To test its stability, after the 5thday, the beads was filtered and put in to chitin liquid medium and tested the chitinolytic activity. This experiment was repeated twice. The highest chitinolytic activity ofStreptomycessp. PB2 was observed on the 4thday with the value of 0.00014 U/ml and the concentration of NAG of 5.42087 µg/ml with the treatment of immobilized NB medium without the addition of chitin.

The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA

The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification of Vibrio cholerae tRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiI strain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence of thiI Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.

Building Blocks of the Outer Membrane: Calculating a General Elastic Energy Model for β-Barrel Membrane Proteins.

The outer membranes of Gram negative bacteria are the first points of contact these organisms make with their environment. Understanding how composition determines the mechanical properties of this essential barrier is of paramount importance. Therefore, we developed a new computational method to measure the elasticity of transmembrane proteins found in the outer membrane. Using all-atom molecular dynamics simulations of these proteins, we apply a set of external forces to mechanically stress the transmembrane β-barrels. Our results from four representative β-barrels show that outer membrane proteins display elastic properties that are approximately 70 to 190 times stiffer than neat lipid membranes. These findings suggest that outer membrane β-barrels are a significant source of mechanical stability in bacteria. Our all-atom approach further reveals that resistance to radial stress is encoded by a general mechanism that includes stretching of backbone hydrogen bonds and tilting of β-strands with respect to the bilayer normal. This computational framework facilitates an increased theoretical understanding of how varying lipid and protein amounts affect the mechanical properties of the bacterial outer membrane.

Visualization of tandem repeat mutagenesis in Bacillus subtilis

Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation.

The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression.

Changing mRNA stability is a major post-transcriptional way of controlling gene expression, particularly in newly encountered conditions. As the concentration of mRNA is the result of an equilibrium between transcription and degradation, it is generally assumed that at constant transcription, any change in mRNA concentration is the consequence of mRNA stabilization or destabilization. However, the literature reports many cases of opposite variations in mRNA concentration and stability in bacteria. Here, we analyzed the causal link between the concentration and stability of mRNA in two phylogenetically distant bacteria Escherichia coli and Lactococcus lactis. Using reporter mRNAs, we showed that modifying the stability of an mRNA had unpredictable effects, either higher or lower, on its concentration, whereas increasing its concentration systematically reduced stability. This inverse relationship between the concentration and stability of mRNA was generalized to native genes at the genome scale in both bacteria. Higher mRNA turnover in the case of higher concentrations appears to be a simple physical mechanism to regulate gene expression in the bacterial kingdom. The consequences for bacterial adaptation of this control of the stability of an mRNA by its concentration are discussed.

The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

Freeze-drying of lactic acid bacteria.

Lactic acid bacteria are of great importance for the food and biotechnology industry. They are widely used as starters for manufacturing food (e.g., yogurt, cheese, fermented meats, and vegetables) and probiotic products, as well as for green chemistry applications. Freeze-drying or lyophilization is a convenient method for preservation of bacteria. By reducing water activity to values below 0.2, it allows long-term storage and low-cost distribution at suprazero temperatures, while minimizing losses in viability and functionality. Stabilization of bacteria via freeze-drying starts with the addition of a protectant solution to the bacterial suspension. Freeze-drying includes three steps, namely, (1) freezing of the concentrated and protected cell suspension, (2) primary drying to remove ice by sublimation, and (3) secondary drying to remove unfrozen water by desorption. In this chapter we describe a method for freeze-drying of lactic acid bacteria at a pilot scale, thus allowing control of the process parameters for maximal survival and functionality recovery.

Probiotic stability of yoghurts during refrigerated storage

Probiotic strains were isolated from the cheese, turkey and dimity and fermented products (rayb, zeer milk,kariesh cheese), screened for clotting time of skim milk. The seven effective isolates which clotting skim milk were identified as Bifidobacterium spp (RC1 b8, RC4 b2, FC1b1, SC1 a4, LZ1b8 and LZ1a7)and Lactobacillus spp (S4b1). The seven effective strains of probiotic bacteria were used in the production of probiotic yoghurt 100%, yoghurt of 50% probiotic bacteria with 50% yoghurt bacteria and compared with the normal yoghurt with 100% normal yoghurt bacteria. The changes in pH, stability of bacteria, and physical properties in probiotic yoghurt 100%, yoghurt of 50% probiotic bacteria with 50% yoghurt bacteria and the normal yoghurt with 100% normal yoghurt bacteria during product’s shelf life at 4C for four weaks were also studied.

Bacteria distribution and dynamics in constructed wetlands based on modelling results

Bacteria communities growing in constructed wetlands play a major role on the removal of pollutants from wastewater and the presence of a stable community is a critical factor affecting their performance. With this work we aimed at finding how long it takes for bacterial communities to stabilise in constructed wetlands and at answering specific questions regarding their abundance, spatial distribution and their relative importance on the treatment processes. To this end the numerical model BIO_PORE was used to simulate the dynamics of 6 functional bacteria groups (heterotrophic, autotrophic nitrifying, fermenting, acetotrophic methanogenic, acetotrophic sulphate reducing and sulphide oxidising bacteria) within a wetland for a period of 3years. Three indicators of bacterial stabilisation were used: 1) total biomass; b) effluent pollutant concentrations and c) Shannon's diversity index. Results indicate that aerobic bacteria dominated the wetland until the 80th day of operation. Anaerobic bacteria dominated the wetland from that moment and until the end of the studied period. Bacteria stability was reached between 400 and 700days after starting operation. Once the wetland reached stability, sulphate reducing bacteria accounted for the highest biomass of all bacterial groups (46%). The distribution of bacterial communities obtained after bacterial stability is consistent with available experimental results, and was clearly controlled by dissolved oxygen (SO) concentrations and H2S toxicity. After stability, the progressive accumulation of inert solids pushed the location of the active bacteria zone towards the outlet section.

Aptazyme-Mediated Regulation of 16S Ribosomal RNA

Developing artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria.