Suitable microscopic entropy for the origin of microbial life: Microbiological methods are challenges

A hypothesis is proposed that the first living microbial cell(s) on Earth assembled about 3.6-4 billion years ago when an environmental microscopic entropy (balance between order and disorder; suitable amount of randomness) was within a range suitable for the origin of microbial cell(s) in a hydrogel environment. An earlier origin of microbial life was not possible as the elements, molecules and entropy conditions necessary for life were not available at the microscopic level. Methodology limitations to study postulated past origin of microbial life events and to mimic these events in the laboratory, are still obstacles to understanding the origin of life.     

Genetic variation: molecular mechanisms and impact on microbial evolution

On the basis of established knowledge of microbial genetics one can distinguish three major natural strategies in the spontaneous generation of genetic variations in bacteria. These strategies are: (1) small local changes in the nucleotide sequence of the genome, (2) intragenomic reshuffling of segments of genomic sequences and (3) the acquisition of DNA sequences from another organism. The three general strategies differ in the quality of their contribution to microbial evolution. Besides a number of non-genetic factors, various specific gene products are involved in the generation of genetic variation and in the modulation of the frequency of genetic variation. The underlying genes are called evolution genes. They act for the benefit of the biological evolution of populations as opposed to the action of housekeeping genes and accessory genes which are for the benefit of individuals. Examples of evolution genes acting as variation generators are found in the transposition of mobile genetic elements and in so-called site-specific recombination systems. DNA repair systems and restriction-modification systems are examples of modulators of the frequency of genetic variation. The involvement of bacterial viruses and of plasmids in DNA reshuffling and in horizontal gene transfer is a hint for their evolutionary functions. Evolution genes are thought to undergo biological evolution themselves, but natural selection for their functions is indirect, at the level of populations, and is called second-order selection. In spite of an involvement of gene products in the generation of genetic variations, evolution genes do not programmatically direct evolution towards a specific goal. Rather, a steady interplay between natural selection and mixed populations of genetic variants gives microbial evolution its direction.     

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.     

Pyrroloquinoline scaffold-based 5-HT6R ligands: Synthesis,quantum chemical and molecular dynamic studies,and influence of nitrogen atom position in the scaffold on affinity

Based on pyrroloquinoline scaffold bearing 5-HT_2C agonists, a series of arylsulfonamide derivatives of 1H-pyrrolo[2,3-f]quinoline and 1H-pyrrolo[3,2-h]quinoline, substituted at position 3 with tetrahydropyridine, were synthesized and evaluated in vitro for their affinity for 5-HT₆ receptors. A structure–activity relationship study showed that the 1H-pyrrolo[3,2-h]quinoline scaffold was more favorable for 5-HT₆R binding than the 1H-pyrrolo[2,3-f]quinoline one, suggesting dependence upon the type of condensation of the pyrrole and quinoline rings. As revealed by quantum-chemical calculations and molecular dynamic studies, position of the quinoline nitrogen atom in the planar pyrroloquinoline skeleton might affect the spatial orientation of the arylsulfonyl fragment, as a result of structure stabilization by internal hydrogen bonds.     

Perspective: Researching the transition from non-living to the first microorganisms: Methods and experiments are major challenges

Methods to research the origin of microbial life are limited. However, microorganisms were the first organisms on the Earth capable of cell growth and division, and interactions with their environment, other microbial cells, and eventually with diverse eukaryotic organisms. The origin of microbial life and the supporting scientific evidence are both an enigma and a scientific priority. Numerous hypotheses have been proposed, scenarios imagined, speculations presented in papers, insights shared, and assumptions made without supporting experimentation, which have led to limited progress in understanding the origin of microbial life. The use of the human imagination to envision the origin of life events, without supporting experimentation, observation and independently replicated experiments required for science, is a significant constraint. The challenge remains how to better understand the origin of microbial life using observations and experimental methods as opposed to speculation, assumptions, scenarios, envisioning events and un-testable hypotheses. This is not an easy challenge as experimental design and plausible hypothesis testing are difficult. Since past approaches have been inconclusive in providing evidence for the origin of microbial life mechanisms and the manner in which genetic instructions was encoded into DNA/RNA, it is reasonable and logical to propose that progress will be made when testable, plausible hypotheses and methods are used in the origin of microbial life research, and the experimental observations are, or are not reproduced in independent laboratories. These perspectives will be discussed in this article as well as the possibility that a pre-biotic film preceded a microbial biofilm as a possible micro-location for the origin of microbial cells capable of growth and division.     

Leaving the meristem behind: The genetic and molecular control of leaf patterning and morphogenesis

Leaves, which play an essential role in plant photosynthesis, share common features such as being flat structures, but also show an impressive variability in their sizes and shapes. Following its initiation in the meristems, leaf development is patterned along three polarization axes to establish its basic architecture. This process is further complicated in the case of compound leaves with the formation of new growth axes. Growth and differentiation must be properly coordinated to regulate the size and the flatness of the leaf. This review provides an overview of the genetic and molecular regulatory networks underlying leaf development, with an emphasis on leaf polarity and the comparison of simple and compound leaves.     

Minimum population size,genetic diversity,and social structure of the Asian elephant in Cat Tien National Park and its adjoining areas,Vietnam, based on molecular genetic analyses

Vietnam’s elephant population that has suffered severe declines during the past three decades is now believed to number 60–80 individuals in the wild. Cat Tien National Park is thought to be one of the key areas for the recovery of Vietnam’s elephants. We carried out a molecular genetic study of elephants in Cat Tien National Park and its adjoining areas with the objectives of estimating minimum population size, assessing genetic diversity, and obtaining insights into social organization. We obtained a minimum population size of 11 elephants based on a combination of unique nuclear microsatellite genotypes and mitochondrial haplotypes. While mitochondrial diversity based on a 600-base pair segment was high in this small sample of individuals, the six microsatellite loci examined showed low diversity and the signature of a recent population bottleneck. Along with nuclear genetic depauperation of Cat Tien’s elephants, we also report disruption of normal social organization, with different matrilines having coalesced into a single social group because of anthropogenic disturbance. The results emphasize the critical condition of this elephant population and the need for urgent conservation measures if this population is to be saved.     

Mycobacterium tuberculosis mutants with multidrug resistance: history of origin, genetic and molecular mechanisms of resistance, and emerging challenges

The review summarizes the data on the Mycobacterium tuberculosis mutations that lead to multidrug resistance (MDR) to various antibiotics. MDR strains arose over the past 30 years as a variety of antituberculosis drugs were introduced in medicine, and they largely discount the results of chemotherapy for tuberculosis. The most dangerous of them are strains with extensive drug resistance (XDR), which are resistant to four or five different drugs on average. The molecular mechanisms that make a strain resistant are considered. XDR and MDR strains result from successive and usually independent resistance mutations, which arise in various regions of the mycobacterial genome. In addition, the formation of resistant strains is affected by the phenomenon of tolerance and mycobacterial latency in infected tissues.     

Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H₂O₂ (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme.     

Control of reduction thermodynamics in [2Fe-2S] ferredoxins: Entropy-enthalpy compensation and the influence of surface mutations

The reaction thermodynamics for the one-electron reduction of the [2Fe-2S] cluster of both human ferredoxin and various surface point mutants, in which each of the negatively charged residues Asp72, Glu73, Asp76, and Asp79 were converted to Ala, have been determined by variable temperature spectroelectrochemical measurements. The above are conserved residues that have been implicated in interactions between the vertebrate-type ferredoxins and their redox partners. In all cases, and similar to other 2Fe-ferredoxins, the reduction potentials are negative as a result of both an enthalpic and entropic stabilization of the oxidized state. Although all Hs Fd mutants, with the exception of Asp72Ala, show slightly higher E°′ values than that of wild type Hs Fd, according to expectations for a purely electrostatic model, they exhibit changes in the ?H°′_rc values that are electrostatically counter-intuitive. The observation of enthalpy-entropy compensation within the protein series indicates that the mutation-induced changes in ?H°′_rc and ?S°′_rc are dominated by reduction-induced solvent reorganization effects. Protein-based entropic effects are likely to be responsible for the low E°′ value of D72A.     

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

 An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM. Received: 26 March 1998 / Accepted: 30 April 1998     

Cellular and molecular mechanisms of autosomal dominant form of progressive hearing loss, DFNA2

Despite advances in identifying deafness genes, determination of the underlying cellular and functional mechanisms for auditory diseases remains a challenge. Mutations of the human K(+) channel hKv7.4 lead to post-lingual progressive hearing loss (DFNA2), which affects world-wide population with diverse racial backgrounds. Here, we have generated the spectrum of point mutations in the hKv7.4 that have been identified as diseased mutants. We report that expression of five point mutations in the pore region, namely L274H, W276S, L281S, G285C, and G296S, as well as the C-terminal mutant G321S in the heterologous expression system, yielded non-functional channels because of endoplasmic reticulum retention of the mutant channels. We mimicked the dominant diseased conditions by co-expressing the wild-type and mutant channels. As compared with expression of wild-type channel alone, the blend of wild-type and mutant channel subunits resulted in reduced currents. Moreover, the combinatorial ratios of wild type:mutant and the ensuing current magnitude could not be explained by the predictions of a tetrameric channel and a dominant negative effect of the mutant subunits. The results can be explained by the dependence of cell surface expression of the mutant on the wild-type subunit. Surprisingly, a transmembrane mutation F182L, which has been identified in a pre-lingual progressive hearing loss patient in Taiwan, yielded cell surface expression and functional features that were similar to that of the wild type, suggesting that this mutation may represent redundant polymorphism. Collectively, these findings provide traces of the cellular mechanisms for DFNA2.     

Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria,bacteri-polymer mixtures and biofilms

Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis for freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at ～ 1740 cm^?1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggests that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled to composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum (amide I/carbohydrate C-O-～ 1150 cm^?1) and bacteria-poly-β-hydroxybutyrate (amide I/carbonyl ～ 1740 cm^?1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR sepectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at ～ 1740 cm^?1 was seen in FT-IR spectra of V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactioni within adherent microbial consortia.     

Tellurite: history, oxidative stress, and molecular mechanisms of resistance

The perceived importance of tellurium (Te) in biological systems has lagged behind selenium (Se), its lighter sister in the Group 16 chalcogens, because of tellurium's lower crustal abundance, lower oxyanion solubility and biospheric mobility and the fact that, unlike Se, Te has yet to be found to be an essential trace element. Te applications in electronics, optics, batteries and mining industries have expanded during the last few years, leading to an increase in environmental Te contamination, thus renewing biological interest in Te toxicity. This chalcogen is rarely found in the nontoxic, elemental state (Te⁰), but its soluble oxyanions, tellurite (TeO₃²⁻) and tellurate (TeO₄²⁻), are toxic for most forms of life even at very low concentrations. Although a number of Te resistance determinants (Tel^R) have been identified in plasmids or in the bacterial chromosome of different species of bacteria, the genetic and/or biochemical basis underlying bacterial TeO₃²⁻ toxicity is still poorly understood. This review traces the history of Te in its biological interactions, its enigmatic toxicity, importance in cellular oxidative stress, and interaction in cysteine metabolism.     

Nutritional enrichment of a microbial community: The effects on activity, elemental composition, community structure and virus production

Abstract Viruses are active members of the microbial community in natural waters but little is known about the factors that regulate their activity and production. In this study we have investigated the effects of increased availability of organic nutrients and inorganic phosphate on activity, elemental composition, community structure and virus production in a natural bacterial community. The fraction of active cells in the community as estimated from microautoradiography of cells assimilating ³H-labeled thymidine ranged from 0–22%, but changes in the elemental composition of the cells indicated that more than 90% of the cells were active. The increase in carbon and energy availability stimulated virus production more than bacterial biomass production, while the increase in phosphate availability stimulated biomass production rather than virus production. A decrease in morphological diversity of the bacterial community was paralleled by a reduction in the virus-to-bacteria ratio (VBR) but the relationship between bacterial diversity and viral activity is uncertain. Our general conclusion is that nutrient availability, in addition to the bacterial activity, also affects the viral activity, and that both of these may affect the structure and diversity of the bacterial community.     

Origins of gene, genetic code, protein and life: comprehensive view of life systems from a GNC-SNS primitive genetic code hypothesis

We have investigated the origin of genes, the genetic code, proteins and life using six indices (hydropathy, α-helix, β-sheet and β-turn formabilities, acidic amino acid content and basic amino acid content) necessary for appropriate three-dimensional structure formation of globular proteins. From the analysis of microbial genes, we have concluded that newly-born genes are products of nonstop frames (NSF) on antisense strands of microbial GC-rich genes [GC-NSF(a)] and from SNS repeating sequences [(SNS)_n] similar to the GC-NSF(a) (S and N mean G or C and either of four bases, respectively). We have also proposed that the universal genetic code used by most organisms on the earth presently could be derived from a GNC-SNS primitive genetic code. We have further presented the [GADV]-protein world hypothesis of the origin of life as well as a hypothesis of protein production, suggesting that proteins were originally produced by random peptide formation of amino acids restricted in specific amino acid compositions termed as GNC-, SNS and GC-NSF(a)-0th order structures of proteins. The [GADV]-protein world hypothesis is primarily derived from the GNC-primitive genetic code hypothesis. It is also expected that basic properties of extant genes and proteins could be revealed by considerations based on the scenario with four stages This review is a modified English version of the paper, which was written in Japanese and published inViva Origino 2001 29 66–85.     

Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches

Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol. Besides this, they are also efficient in various reactions such as esterification, transesterification and aminolysis in organic solvents. Therefore, those enzymes are nowadays extensively studied for their potential industrial applications. Examples in the literature are numerous concerning their use in different fields such as resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oils and fats bioconversion and detergency applications. However, the drawbacks of the extensive use of lipases (and biocatalysts in general) compared to classical chemical catalysts can be found in the relatively low stability of enzyme in their native state as well as their prohibitive cost. Consequently, there is a great interest in methods trying to develop competitive biocatalysts for industrial applications by improvement of their catalytic properties such as activity, stability (pH or temperature range) or recycling capacity. Such improvement can be carried out by chemical, physical or genetical modifications of the native enzyme. The present review will survey the different procedures that have been developed to enhance the properties of lipases. It will first focus on the physical modifications of the biocatalysts by adsorption on a carrier material, entrapment or microencapsulation. Chemical modifications and methods such as modification of amino acids residues, covalent coupling to a water-insoluble material, or formation of cross-linked lipase matrix, will also be reviewed. Finally, new and promising methods of lipases modifications by genetic engineering will be discussed.