Development of a microbial test suite and data integration method for assessing microbial health of contaminated soil.

There is no standard methodology or guideline for assessing soil microbial health for the purposes of contaminant risk assessments. Here we propose a laboratory-based test suite and novel data integration method for evaluating soil microbial health using site-specific contaminated and reference soil. The test suite encompasses experiments for evaluating microbial biomass, activity, and diversity. The results from the tests are then integrated so that a Soil Microbial Health Score (SMHS) may be assigned. This test suite and data integration method was tested on soils from 3 different contaminated sites in Canada. The soil microbial health of a petroleum hydrocarbon (PHC) contaminated site was found to be 'Mildly Impacted' and 'Moderately Impacted' for two soil horizons at a boreal forest site. The soil microbial health of the mixed metal/PHC and mixed metal sites were both found to be 'Not Impacted'. Continued use of this test suite and data integration method will help create guidelines for assessing soil microbial health in ecological risk assessments.

The Molecularly Crowded Cytoplasm of Bacterial Cells: Dividing Cells Contrasted with Viable but Non-culturable (VBNC) Bacterial Cells.

In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but nonculturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell division. In contrast, nutrient starving bacteria in the physiological VBNC state are struggling to survive, as essential nutrients are not available or limiting. The cytoplasm is not as molecularly crowded as gene expression is minimal (e.g., ribosome, transcript, tRNA and protein numbers are decreased), energy pools are depleted, cells may exhibit leakage, and DNA is not being replicated for cell division.

Comparative bacterial genomics: defining the minimal core genome.

A comparative genomics analysis revealed 702 genes present in the bacterial Gram-negative core gene set (92 species analyzed) and 959 genes in the Gram-positive core gene set (93 species analyzed). Mycoplasma genitalium, which has the smallest known genome (517 genes) of a non-symbiont, was used in a three-way reciprocal analysis with the Gram-negative core genes and the Gram-positive core genes, and 151 common bacterial core genes were found. Of these 151 core genes, 39 were putative genes encoding the 30S and 50S ribosomal subunits, whilst among recognized cell division genes, only one gene, the major ftsZ, was present. In addition, 86 reciprocal matches were identified between the 151 common bacterial genes and a previously determined 2,723 common eukaryotic core gene set. An analysis was also done to optimize the threshold bit score used to declare that genes were homologous, and a bit score cutoff of 40 was selected.

Non-terrestrial origin of life: a transformative research paradigm shift.

Theories and hypotheses in science are continually subject to verification, critical re-evaluation, revision and indeed evolution, in response to new observations and discoveries. Theories of the origin of life have been more constrained than other scientific theories and hypotheses in this regard, through the force of social and cultural pressures. There has been a tendency to adhere too rigidly to a class of theory that demands a purely terrestrial origin of life. For nearly five decades evidence in favour of a non-terrestrial origin of life and panspermia has accumulated which has not been properly assessed. A point has now been reached that demands the serious attention of biologists to a possibly transformative paradigm shift of the question of the origin of life, with profound implications across many disciplines.

Physical and biochemical nature of the bacterial cytoplasm: movement and localization of mRNA and the 30S subunits of ribosomes.

There is a paucity of knowledge on how mRNA transcripts in the spatially crowded, but molecularly organized bacterial cytoplasm contact the 30S ribosomal subunits. Does simple diffusion in the cytoplasm account for transcript-ribosome interactions given that a large number of ribosomes (e.g., about 72,000 in Escherichia coli during exponential growth) can be present in the cytoplasm? Or are undiscovered mechanisms present where specific transcripts are directed to specific ribosomes at specific cytoplasmic locations, while others are mobilized in a random manner? Moreover, is it possible that cytoplasmic mobilization occurs in bacteria, driven possibly by thermal infrared (IR) radiation and the generation of exclusion zone (EZ) water? These aspects will be discussed in this article and hypotheses presented.

Origin of life: hypothesized roles of high-energy electrical discharges, infrared radiation, thermosynthesis and pre-photosynthesis.

The hypothesis is proposed that during the organization of pre-biotic bacterial cell(s), high-energy electrical discharges, infrared radiation (IR), thermosynthesis and possibly pre-photosynthesis were central to the origin of life. High-energy electrical discharges generated some simple organic molecules available for the origin of life. Infrared radiation, both incoming to the Earth and generated on the cooling Earth with day/night and warming/cooling cycles, was a component of heat engine thermosynthesis before enzymes and the genetic code were present. Eventually, a primitive forerunner of photosynthesis and the capability to capture visible light emerged. In addition, the dual particle-wave nature of light is discussed from the perspective that life requires light acting both as a wave and particle.

Transformative research: definitions, approaches and consequences.

In this perspective a definition of transformative research (TR) is proposed and discussed. We define TR as that which "transforms" or causes a major change in thought patterns concerning an area of scientific endeavor. This type of research is often elusive, requires different approaches and sometimes depends on a bit of luck. TR concerns intangibles such as human intuition, serendipity, unpredictable events, implausible hypotheses, a well-prepared mind and often interpersonal communications. Examples are provided to illustrate how TR may unfold. Contributions it makes to basic and applied humanistic knowledge are highlighted.

Can dead bacterial cells be defined and are genes expressed after cell death?

There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division.

Bacterial gene expression at low temperatures.

Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.

A perspective on the mobilization, localization and delivery of molecules in the crowded bacterial cytoplasm.

It has been assumed that diffusion of molecules in the bacterial cytoplasm is the mechanism that moves molecules in the absence of cytoplasmic streaming. However, is there an undiscovered mechanism present that mobilizes cytoplasm and its molecular contents, and delivers tRNAs to specific ribosomes at specific bacterial cytoplasmic locations? Mobilization of specific tRNA (and also mRNA transcripts and ribosomes) and cell division proteins to specific intracellular locations may suggest that instructions and/or mechanism(s) are needed. The alternative is that molecular crowding in the cytoplasm is sufficient for gentle contact between mRNA, ribosomes and tRNA. Or is it plausible that the bacterial cytoplasm (and its contents) are mobilized with the outcome being more gentle collisions between molecules than by a diffusion only mechanism? One hypothesis is that cytoplasmic and molecule mobilization and spatial organization are possibly driven by the photons in thermal infrared (IR) radiation and generation of exclusion zone (EZ) water in the cytoplasm.

Origin of microbial life hypothesis: a gel cytoplasm lacking a bilayer membrane, with infrared radiation producing exclusion zone (EZ) water, hydrogen as an energy source and thermosynthesis for bioenergetics.

The hypothesis is proposed that pre-biotic bacterial cell(s) and the first cells capable of growth/division did not require a cytoplasmic membrane. A gel-like microscopic structure less than a cubic micrometer may have had a dual role as both an ancient pre-cytoplasm and a boundary layer to the higher-entropy external environment. The gel pre-cytoplasm exposed to radiant energy, especially in the infrared (IR) region of the EM spectrum resulted in the production of an exclusion zone (EZ) with a charge differential (-100 to -200 mV) and boundary that may have been a possible location for the latter organization of the first cytoplasmic membrane. Pre-biotic cells and then-living cells may have used hydrogen as the universal energy source, and thermosynthesis in their bioenergetic processes. These components will be discussed as to how they are interconnected, and their hypothesized roles in the origin of life.

Microbial processes in the Athabasca Oil Sands and their potential applications in microbial enhanced oil recovery.

The Athabasca Oil Sands are located within the Western Canadian Sedimentary Basin, which covers over 140,200 km(2) of land in Alberta, Canada. The oil sands provide a unique environment for bacteria as a result of the stressors of low water availability and high hydrocarbon concentrations. Understanding the mechanisms bacteria use to tolerate these stresses may aid in our understanding of how hydrocarbon degradation has occurred over geological time, and how these processes and related tolerance mechanisms may be used in biotechnology applications such as microbial enhanced oil recovery (MEOR). The majority of research has focused on microbiology processes in oil reservoirs and oilfields; as such there is a paucity of information specific to oil sands. By studying microbial processes in oil sands there is the potential to use microbes in MEOR applications. This article reviews the microbiology of the Athabasca Oil Sands and the mechanisms bacteria use to tolerate low water and high hydrocarbon availability in oil reservoirs and oilfields, and potential applications in MEOR.

Viable but non-culturable (VBNC) bacteria: Gene expression in planktonic and biofilm cells.

Viable but non-culturable (VBNC) bacteria are common in nutrient poor and/or stressed environments as planktonic cells and biofilms. This article discusses approaches to researching VBNC bacteria to obtain knowledge that is lacking on their gene expression while in the VBNC state, and when they enter into and then recover from this state, when provided with the necessary nutrients and environmental conditions to support growth and cell division. Two-dimensional gel electrophoresis of proteins, global gene expression, reverse-transcription polymerase chain reaction (PCR) analysis and sequencing by synthesis coupled with data on cell numbers, viability and species present are central to understanding the VBNC state.

Quantum microbiology.

During his famous 1943 lecture series at Trinity College Dublin, the reknown physicist Erwin Schrodinger discussed the failure and challenges of interpreting life by classical physics alone and that a new approach, rooted in Quantum principles, must be involved. Quantum events are simply a level of organization below the molecular level. This includes the atomic and subatomic makeup of matter in microbial metabolism and structures, as well as the organic, genetic information code of DNA and RNA. Quantum events at this time do not elucidate, for example, how specific genetic instructions were first encoded in an organic genetic code in microbial cells capable of growth and division, and its subsequent evolution over 3.6 to 4 billion years. However, due to recent technological advances, biologists and physicists are starting to demonstrate linkages between various quantum principles like quantum tunneling, entanglement and coherence in biological processes illustrating that nature has exerted some level quantum control to optimize various processes in living organisms. In this article we explore the role of quantum events in microbial processes and endeavor to show that after nearly 67 years, Schrödinger was prophetic and visionary in his view of quantum theory and its connection with some of the fundamental mechanisms of life.

Origin of microbial life: Nano- and molecular events, thermodynamics/entropy, quantum mechanisms and genetic instructions.

Currently, there are no agreed upon mechanisms and supporting evidence for the origin of the first microbial cells on the Earth. However, some hypotheses have been proposed with minimal supporting evidence and experimentation/observations. The approach taken in this article is that life originated at the nano- and molecular levels of biological organization, using quantum mechanic principles that became manifested as classical microbial cell(s), allowing the origin of microbial life on the Earth with a core or minimal, organic, genetic code containing the correct instructions for cell(s) for growth and division, in a micron dimension environment, with a local entropy range conducive to life (present about 4 billion years ago), and obeying the laws of thermodynamics. An integrated approach that explores all encompassing factors necessary for the origin of life, may bring forth plausible hypotheses (and mechanisms) with much needed supporting experimentation and observations for an origin of life theory.

How much cytoplasm can a bacterial genome control?

In this perspective we discuss that bacterial genomes have optimized during evolution to control a range of cytoplasm, from immediately after cell division to a maximum amount/volume present just prior to DNA replication and subsequent cell division. The genetic expansion of bacteria via evolution may be limited to a genome size:cytoplasm amount/volume ratios and energetics that have been selected for during 3.6-4 billion years of evolution on the Earth. The optimal genome size is one that is relatively constant, but also has some plasticity for evolutionary change (via gene transfer) and mutational events, and can control a range of cytoplasm during the cell cycle.

Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans.

Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu(2+) and Ni(2+) concentrations (1-200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility.