Laboratory Growth Research Articles

Plant Root Templated Irrigation

AbstractA new method is explored for subsurface irrigation where the plant root structure is used as the subsurface water distribution system. Plants are severed at the stem and the stem stump is attached to a pressurized water source such that water may permeate into surrounding soil through the existing buried root system. This pressurized root system serves as a well‐ distributed buried irrigation water source to the other nearby growing plants taking up water. The feasibility of using an existing root structure in this manner to supply water at rates necessary to satisfy other growing plants was determined by conducting laboratory water hydraulic conductance studies. Laboratory growth studies were used to directly test the proposed root templated irrigation (RTI) approach. Modelling simulations were also utilized to extend laboratory findings to other scenarios and compare expected water use efficiency benefits associated with this proposed irrigation method with surface irrigation (SI) and subsurface drip irrigation (SDI) practices. Irrigation study measurements showed that RTI provides more efficient water use as compared to SI. Modelling calculations were in qualitative agreement with the experimental studies, indicating that RTI yields improvement in root water uptake over the SI and also SDI unless SDI source spacing is narrow. Copyright © 2016 John Wiley & Sons, Ltd.

Extracellular Polymeric Substance (EPS) Production by <i>Nostoc minutum</i> under Different Laboratory Conditions

A study of the influence of different laboratory growth conditions on the biomass and EPS production by Nostoc minutum, a diazothophic cyanobacterium locally isolated, was carried out. Two culture media were tested, with or without NaNO3 addition, and three luminous intensities: low (4530 lux), intermediate (7300 lux) and high (9860 lux). BW3 medium was better than BG11 for N. minutum growth, with maximal values of biomass concentration (4.98 DO) and the highest growth rate (0.019 h-1) at 9860 lux of light intensity. A progressive increase in culture viscosity of N. minutum cultures was observed, for stirred condition and non-diazotrophic growth in BG11 medium, together with the production of maximal EPS concentration (2.485 g/L). On the other hand, the EPS production in BW3 medium was maximal in diazotrophic conditions, both for still (1.66 g/L) and stirred (2.56 g/L) cultures. The different yields of EPS reported for each condition, results in the requirement of a species-specific optimization of the cultivation conditions for the exploitation of an efficient technology for the production of N. minutum EPS.

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach.

With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

Algal recycling enhances algal productivity and settleability in Pediastrum boryanum pure cultures

Recycling a portion of gravity harvested algae (i.e. algae and associated bacteria biomass) has been shown to improve both algal biomass productivity and harvest efficiency by maintaining the dominance of a rapidly-settleable colonial alga, Pediastrum boryanum in both pilot-scale wastewater treatment High Rate Algal Ponds (HRAP) and outdoor mesocosms. While algal recycling did not change the relative proportions of algae and bacteria in the HRAP culture, the contribution of the wastewater bacteria to the improved algal biomass productivity and settleability with the recycling was not certain and still required investigation. P. boryanum was therefore isolated from the HRAP and grown in pure culture on synthetic wastewater growth media under laboratory conditions. The influence of recycling on the productivity and settleability of the pure P. boryanum culture was then determined without wastewater bacteria present. Six 1 L P. boryanum cultures were grown over 30 days in a laboratory growth chamber simulating New Zealand summer conditions either with (Pr) or without (Pc) recycling of 10% of gravity harvested algae. The cultures with recycling (Pr) had higher algal productivity than the controls (Pc) when the cultures were operated at both 4 and 3 d hydraulic retention times by 11% and 38% respectively. Furthermore, algal recycling also improved 1 h settleability from ∼60% to ∼85% by increasing the average P. boryanum colony size due to the extended mean cell residence time and promoted formation of large algal bio-flocs (>500 μm diameter). These results demonstrate that the presence of wastewater bacteria was not necessary to improve algal productivity and settleability with algal recycling.

Latitudinal variation in the response of tidepool copepods to mean and daily range in temperature

Understanding the evolutionary potential of organisms to adapt to a changing climate, and the fitness consequences of temperature fluctuations, are critical to forecasting the future of biodiversity. Geographic variation among populations in life history response to temperature mean and variability offers one view of the potential for local adaptation to broaden the thermal niche. We used laboratory growth experiments to examine the effects of temperatures between 13 degrees C and 30 degrees C on five life history traits and the intrinsic rate of increase for 15 Tigriopus californicus populations distributed over 17 degrees of latitude. Different life history stages showed distinct latitudinal shifts in thermal response, while the temperature of peak population growth consistently declined with increasing latitude. In addition, high-latitude populations grew faster at optimal temperatures but showed steeper fitness declines at high temperature. To test geographic population variation in response to the amplitude of daily thermal fluctuations, we grew three northern and three southern populations and manipulated nightly low and daily high temperatures. We found the lowest fitness overall in the treatment with the highest mean temperature, and the treatment with the greatest variability showed high fitness despite an 80C greater daily range. Population responses to daily thermal variability were unrelated to latitude of origin. Our results indicate that trade-offs between adaptation to high vs. low temperature, and between growth and maturation vs. survival and fecundity, govern local adaptation along the latitudinal gradient. They also indicate that, T. californicus populations can maintain fitness over a wide range of daily variability but are more sensitive to small changes in the mean temperature.

Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter.

ABSTRACTNucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus.IMPORTANCE Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory effect on the expression of the kdp genes encoding a potassium transporter. c-di-AMP binds to the USP domain of KdpD, thus providing for the first time evidence for the ability of such a domain to bind a cyclic dinucleotide.

Spontaneous Deletion of an "ORFanage" Region Facilitates Host Adaptation in a "Photosynthetic" Cyanophage.

Viruses have been suggested to be the largest source of genetic diversity on Earth. Genome sequencing and metagenomic surveys reveal that novel genes with unknown functions are abundant in viral genomes. Yet few observations exist for the processes and frequency by which these genes are gained and lost. The surface waters of marine environments are dominated by marine picocyanobacteria and their co-existing viruses (cyanophages). Recent genome sequencing of cyanophages has revealed a vast array of genes that have been acquired from their cyanobacterial hosts. Here, we re-sequenced the cyanophage S-PM2 genome after 10 years of near continuous passage through its marine Synechococcus host. During this time a spontaneous mutant (S-PM2d) lacking 13% of the S-PM2 ORFs became dominant in the cyanophage population. These ORFs are found at one loci and are not homologous to any proteins in any other sequenced organism (ORFans). We demonstrate a fitness cost to S-PM2WT associated with possession of these ORFs under standard laboratory growth. Metagenomic surveys reveal these ORFs are present in various aquatic environments, are likely of cyanophage origin and appear to be enriched in environments from the extremes of salinity (freshwater and hypersaline). We posit that these ORFs contribute to the flexible gene content of cyanophages and offer a distinct fitness advantage in freshwater and hypersaline environments.

Potential Application of Bacterial Inoculum toIncrease Different Mechanical Strength in thePrepared Concrete

Cracking in the surface layer of concrete mainly reduces its durability, since cracks are responsible for the transport of liquids and gasses that could potentially contain deleterious substances. So the objective of the present investigation is to study the potential application of bacteria, Bacillus sphaericus to increase different mechanical strength of the concrete. Conventional and bacterial concrete was prepared and its strength was evaluated using standard Indian Specifications. A significant increase of compressive strength, split-tensile strength and flexural strength was observed for respective B1 (100ml) and B2 (150ml) cell concentrations after 28 days of bio-curing. The Ultra-sonic pulse velocity value of B2 concrete revealed the self-healing properties when compared with the conventional concrete. The present study concludes that inexpensive alternative for laboratory growth media would potentially bring down the cost of the bacteria based self-healing sustainable concrete.

RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates.

Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis.

Co-aggregation of Pseudomonas fluorescens and Bacillus subtilis in culture and co-colonization in black gram (Vigna mungo L.) roots

Abstract Many plant growth promoting rhizobacteria (PGPR), biocontrol agents and biofertilizer bacteria are used in agriculture. They are applied to crops by seed or soil application which colonizes in the rhizosphere. Although there are extensive reports on the output (growth, yield, disease resistance, phenotypes, etc) of the combined inoculations of these bacteria the companionship among these bacteria and the relative contribution to overall output is largely unknown. The starting point to unravel this would be to study the bacterial companionship in terms of ability to co-aggregate, stability of co-aggregation and developing molecular evidences for co-colonization in crop roots. Our work mainly aimed at understanding the extent of co-aggregation between Pseudomonas fluorescens and Bacillus subtilis and their co-colonization abilities in plant roots. This was verified by conducting several experiments of co-aggregation and effect of various factors like pH and growth phase, effect of various treatments like urea, protease K, EDTA, heat, thermal stability, sonication and desiccation on the extent of the co-aggregation. Co-aggregation between these two bacteria was found to be pH and growth phase dependent. Protease and EDTA treatment reduced the co-aggregation whereas sonication treatment improved the same. Talc based formulation of the bacteria was prepared separately and used for seed treatment in black gram. The seeds were germinated under laboratory conditions and growth parameters were observed. DNA from roots of germinated seedlings was used as template to amplify bacterial DNA with species specific primers. PCR analysis results indicated that P. fluorescens and B. subtilis are compatible in colonizing black gram.

A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium.

The Mycoplasma genitalium MG428 protein shows homology to members of the sigma-70 family of sigma factors. Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function. Deletion of MG_428 or some of the up-regulated unknown genes led to severe recombination defects. Single cell analyses revealed that activation of the MG428-regulon is a rare event under laboratory growth conditions. A conserved sequence with sigma-70 promoter architecture (TTGTCA-N18/19-ATTWAT) was identified in the upstream region of all of the MG428-regulated genes or operons. Primer extension analyses demonstrated that transcription initiates immediately downstream of this sigma70-type promoter in a MG428-dependent manner. Furthermore, mutagenesis of the conserved −10 and −35 elements corroborated the requirement of these regions for promoter function. Therefore, a new mycoplasma promoter directs transcription of a unique recombination regulon. Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor. Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism. Since recombination is an important mechanism to generate antigenic variation, MG428 emerges as a novel factor contributing to M. genitalium virulence.

Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment.

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as 'mazei-T' and 'mazei-WC'. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.

Auxin Binding Protein 1 (ABP1): a matter of fact.

Science is a global community enterprise.Collectively,we contribute to building the knowledge tower by using bricks and mortar that should be strong,solid,and long lasting.A brick with defects may lead this tower,or a part there of,to collapse.When that happens,the community suffers,laying waste countless dedicated hours of work,especially by students and postdoctoral scholars.This is the reason ary

Induction of growth and proliferation of fibroblast cells in magnetic field

Background: Tissue engineering is generally defined as developing and changing the laboratory growth of molecules and cells in tissues or organs to replace and repair the damaged part of body. This study was carried out to stimulate the growth of cultured fibroblast cells by a physical electromagnetic method. Methods: First, an air-core coil was prepared and the cell culture plate was placed comfortably into the mold, then the plate containing the culture medium and human fibroblast cell along with air-core coil were placed in an incubator and then connected to the power supply. Thus, the sample underwent electromagnetic field at different times, and cell proliferation was studied by MTTassay. Results: Microscopic images indicated that the cells undergoing electromagnetic field (0.35 amps) had a significant growth compared to the cells in control group in a definit range of stimulation. Conclusion: In conclusion, electromagnetic stimulation in a definite range led to cell proliferation and could be used as a positive factor in tissue engineering.

Thermally adaptive tradeoffs in closely related marine bacterial strains.

Time series studies have shown that some bacterial taxa occur only at specific times of the year while others are ubiquitous in spite of seasonal shifts in environmental variables. Here, we ask if these ubiquitous clades are generalists that grow over a wide range of environmental conditions, or clusters of strain-level environmental specialists. To answer this question, vibrio strains isolated at a coastal time series were phylogenetically and physiologically characterized revealing three dominant strategies within the vibrio: mesophiles, psychrophiles and apparently generalist broad thermal range clades. Thermal performance curves from laboratory growth rate experiments help explain field observations of relative abundances: the mesophilic clade grows optimally at temperatures 16°C higher than the psychrophilic clade. Strains in the broad thermal range clade all have similar optimal growth temperatures but also exhibit temperature-related tradeoffs with faster growth rates for warm temperature strains and broader growth ranges for strains from cool temperatures. Moreover, the mechanisms of thermal adaptation apparently differ based on evolutionary time scales: shifts in the temperature of maximal growth occur between deeply branching clades but thermal performance curve shape changes on shorter time scales. Thus, apparently ubiquitous clades are likely not generalists, but contain subclusters with distinct environmental preferences.

Modelling and Simulation of Particle Size Distribution of Precipitates in Continuous Tubular Crystallizers

This paper presents a population balance model for describing the temporal evolution of the particle size distribution of a precipitate produced in a laboratory scale tubular crystallizer, including nucleation, growth and agglomeration of crystals. The quadrature method of moments is used to calculate the moments of the crystal size distribution. The gamma probability density function with the sixth order Laguerre polynomials are used to reproduce the particle size distribution from moments. A sensitivity analyses is carried out by simulation that can be helpful when designing a crystallizer. Influence of the design and process parameters on the particle size distribution of product is analysed.

Microarray transposon tracking for the mapping of conditionally essential genes in Campylobacter jejuni.

Although whole genome approaches to the study of bacteria have grown substantially in the past decade, there is still a need for quick and easy methods for the determination of which genes are essential for the growth of these bacteria under specific growth conditions. There are numerous methods to accomplish this depending on the resources and equipment available, each with their own advantages and disadvantages. Here we describe a method we successfully employed to map the essential genes of Campylobacter jejuni using a microarray transposon tracking approach where we constructed a saturated transposon mutant library in the C. jejuni strain NCTC11168 and used a genomic microarray approach to identify genes lacking transposon insertions under standard laboratory growth conditions. With a fully saturated library, the absence of transposon insertions can be used as an indicator of a gene essential for the survival and growth for the conditions used for the mutant library.

NextGen microbial natural products discovery.

NextGen microbial natural products discovery.

Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution.

Acinetobacter baylyi ADP1 has the potential to be a versatile bacterial host for synthetic biology because it is naturally transformable. To examine the genetic reliability of this desirable trait and to understand the potential stability of other engineered capabilities, we propagated ADP1 for 1,000 generations of growth in rich nutrient broth and analyzed the genetic changes that evolved by whole-genome sequencing. Substantially reduced transformability and increased cellular aggregation evolved during the experiment. New insertions of IS1236 transposable elements and IS1236-mediated deletions led to these phenotypes in most cases and were common overall among the selected mutations. We also observed a 49-kb deletion of a prophage region that removed an integration site, which has been used for genome engineering, from every evolved genome. The comparatively low rates of these three classes of mutations in lineages that were propagated with reduced selection for 7,500 generations indicate that they increase ADP1 fitness under common laboratory growth conditions. Our results suggest that eliminating transposable elements and other genetic failure modes that affect key organismal traits is essential for improving the reliability of metabolic engineering and genome editing in undomesticated microbial hosts, such as Acinetobacter baylyi ADP1.