Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12

An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed. Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage. This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism. The RFLP patterns indicate a burst of IS30 transposition to occur occasionally. Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied. A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence. Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants. These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.      

Bacterial genetic methods to explore the biology of <Emphasis Type="Italic">mariner</Emphasis> transposons

Mariners are small DNA mediated transposons of eukaryotes that fortuitously function in bacteria. Using bacterial genetics, it is possible to study a variety of properties of mariners, including transpositional ability, dominant-negative regulation, overexpresson inhibition, and the function of cis-acting sequences like the inverted terminal repeats. In conjunction with biochemical techniques, the structure of the transposase can be elucidated and the activity of the elements can be improved for genetic tool use. Finally, it is possible to uncover functional transposase genes directly from genomes given a suitable bacterial genetic screen.     

Multiple genetic switches spontaneously modulating bacterial mutability

Background

All life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations?

Results

To prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution.

Conclusions

MMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution.

     

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Background

Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders'' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers'' targets.

Results

We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.

Conclusions

We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.     

Bacterial transposon Tn5: evolutionary inferences

Transposable elements induce spontaneous mutations, promote genome rearrangements, regulate gene expression, and participate in the horizontal spread of genes encoding traits such as antibiotic resistance among bacterial genera too distantly related to undergo homologous recombination. Here we review the bacterial transposon Tn5 and focus on those aspects of its functional organization and transposition which provide insights into how it and other elements may have arisen, proliferated, and evolved.      

Stable propagation of ‘selfish’ genetic elements

Extrachromosomal or chromosomally integrated genetic elements are common among prokaryotic and eukaryotic cells. These elements exhibit a variety of ‘selfish’ strategies to ensure their replication and propagation during the growth of their host cells. To establish long-term persistence, they have to moderate the degree of selfishness so as not to imperil the fitness of their hosts. Earlier genetic and biochemical studies together with more recent cell biological investigations have revealed details of the partitioning mechanisms employed by low copy bacterial plasmids. At least some bacterial chromosomes also appear to rely on similar mechanisms for their own segregation. The 2 μm plasmid ofSaccharomyces cerevisiae and related yeast plasmids provide models for optimized eukaryotic selfish DNA elements. Selfish DNA elements exploit the genetic endowments of their hosts without imposing an undue metabolic burden on them. The partitioning systems of these plasmids appear to make use of a molecular trick by which the plasmids feed into the segregation pathway established for the host chromosomes.     

Identification,characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer from cell to cell by conjugation (like plasmids) and integrate into the chromosomes of bacterial hosts (like lysogenic phages or transposons). ICEs are prevalent in bacterial chromosomes and play a major role in bacterial evolution by promoting horizontal gene transfer. Exclusion prevents the redundant transfer of conjugative elements into host cells that already contain a copy of the element. Exclusion has been characterized mostly for conjugative elements of Gram‐negative bacteria. Here, we report the identification and characterization of an exclusion mechanism in ICEBs1 from the Gram‐positive bacterium Bacillus subtilis. We found that cells containing ICEBs1 inhibit the activity of the ICEBs1‐encoded conjugation machinery in other cells. This inhibition (exclusion) was specific to the cognate conjugation machinery and the ICEBs1 gene yddJ was both necessary and sufficient to mediate exclusion by recipient cells. Through a mutagenesis and enrichment screen, we identified exclusion‐resistant mutations in the ICEBs1 gene conG. Using genes from a heterologous but related ICE, we found that the exclusion specificity was determined by ConG and YddJ. Finally, we found that under conditions that support conjugation, exclusion provides a selective advantage to the element and its host cells.     

Shaping Neuronal Network Activity by Presynaptic Mechanisms

Neuronal microcircuits generate oscillatory activity, which has been linked to basic functions such as sleep, learning and sensorimotor gating. Although synaptic release processes are well known for their ability to shape the interaction between neurons in microcircuits, most computational models do not simulate the synaptic transmission process directly and hence cannot explain how changes in synaptic parameters alter neuronal network activity. In this paper, we present a novel neuronal network model that incorporates presynaptic release mechanisms, such as vesicle pool dynamics and calcium-dependent release probability, to model the spontaneous activity of neuronal networks. The model, which is based on modified leaky integrate-and-fire neurons, generates spontaneous network activity patterns, which are similar to experimental data and robust under changes in the model''s primary gain parameters such as excitatory postsynaptic potential and connectivity ratio. Furthermore, it reliably recreates experimental findings and provides mechanistic explanations for data obtained from microelectrode array recordings, such as network burst termination and the effects of pharmacological and genetic manipulations. The model demonstrates how elevated asynchronous release, but not spontaneous release, synchronizes neuronal network activity and reveals that asynchronous release enhances utilization of the recycling vesicle pool to induce the network effect. The model further predicts a positive correlation between vesicle priming at the single-neuron level and burst frequency at the network level; this prediction is supported by experimental findings. Thus, the model is utilized to reveal how synaptic release processes at the neuronal level govern activity patterns and synchronization at the network level.     

The nature of spontaneous and induced mutagenesis in a genetically unstable mutator strain of Drosophila melanogaster]

The spontaneous and induced frequencies of visible mutations by N-nitroso-N-ethylurea in male cells of Drosophila melanogaster genetically unstable mutator strain have been investigated. The spontaneous and induced by N-nitroso-N-ethylurea genetic instability in mutator strain have similar manifestation, that evidently testifies the existence of general mechanisms of the appearance of unstable mutations, namely the transpositions of the mobile genetic elements.     

甘肃省野生羊肚菌根际细菌群落与土壤环境因子相关性研究

[背景]羊肚菌是全球广泛分布的物种,具有重要的经济和科研价值,其根际微生态系统各要素间的相关性研究相对较少.[目的]探究甘肃省不同地区野生羊肚菌根际土壤中细菌群落-土壤理化性质及细菌群落-酶活性相关性.[方法]采用Illumina MiSeq高通量测序技术,对细菌群落组成进行测量,进而分析其多样性,最终揭示细菌群落-土...     

Indicators of spontaneous and stimulated nitroblue tetrazole test of polymorphous-nuclear leucocytes in acute dysentery patients

140 healthy individuals and 93 sick with acute dysentery were subjected to an examination by spontaneous and by bacterial preparations stimulated reaction with nitroblue tetrazole (NBT test). Indicators in healthy persons were normal in the spontaneous, and increased in the NBT test, stimulated by bacterial preparations. Indicators of the spontaneous NBT test in patients with acute dysentery were raised with a maximum in the period of early convalescence. Stimulation by a live shigella culture--the dysentery vaccine--revealed by means of Sonne diagnostic high, and when endotoxin from Serratia marcescens and dysenterin was used as an inductor, mild indicators of NBT test activity. When a polyvalent agglutinating dysentery serum was used as a stimulator, the activity increased considerably, and a simultaneous use of serum and vaccine had an inhibiting effect on the indicators of the stimulated NBT test. The obtained results testify the sufficient high reserve possibilities of leucocytes towards complete phagocytosis and the efficiency of the NBT test, stimulated by bacterial preparations for the study of functional and metabolic activity of leucocytes in the process of acute bacterial dysentery.     

Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.     

Sheltering Behavior and Locomotor Activity in 11 Genetically Diverse Common Inbred Mouse Strains Using Home-Cage Monitoring

Functional genetic analyses in mice rely on efficient and in-depth characterization of the behavioral spectrum. Automated home-cage observation can provide a systematic and efficient screening method to detect unexplored, novel behavioral phenotypes. Here, we analyzed high-throughput automated home-cage data using existing and novel concepts, to detect a plethora of genetic differences in spontaneous behavior in a panel of commonly used inbred strains (129S1/SvImJ, A/J, C3H/HeJ, C57BL/6J, BALB/cJ, DBA/2J, NOD/LtJ, FVB/NJ, WSB/EiJ, PWK/PhJ and CAST/EiJ). Continuous video-tracking observations of sheltering behavior and locomotor activity were segmented into distinguishable behavioral elements, and studied at different time scales, yielding a set of 115 behavioral parameters of which 105 showed highly significant strain differences. This set of 115 parameters was highly dimensional; principal component analysis identified 26 orthogonal components with eigenvalues above one. Especially novel parameters of sheltering behavior and parameters describing aspects of motion of the mouse in the home-cage showed high genetic effect sizes. Multi-day habituation curves and patterns of behavior surrounding dark/light phase transitions showed striking strain differences, albeit with lower genetic effect sizes. This spontaneous home-cage behavior study demonstrates high dimensionality, with a strong genetic contribution to specific sets of behavioral measures. Importantly, spontaneous home-cage behavior analysis detects genetic effects that cannot be studied in conventional behavioral tests, showing that the inclusion of a few days of undisturbed, labor extensive home-cage assessment may greatly aid gene function analyses and drug target discovery.     

Opsonic activity of ascitic fluids by a chemiluminescent method

Cirrhotic ascites are highly susceptible to spontaneous bacterial infection, whereas carcinogenic ascites are seldom infected. This difference may be explained by differences in their chemotactic, bactericidal and/or opsomic activities. We measured the chemotactic and opsonic activity of ascitic fluids from 35 alcoholic cirrhotic ascites and of 12 peritoneal carcinogenic ascites. Chemotactic activity was measured by the under-agarose technique and opsonic activity by a luminol-enhanced method. Ascitic fluids from alcoholic cirrhosis had low chemotactic (62 ± 24.5% that of N-formylated peptide) and opsonic (67 ± 50% of normal serum) activities on normal human neutrophils. In contrast, ascitic fluids from peritoneal carcinoma were found to possess high opsonic activity (114 ± 49% of normal serum) and chemotactic activity similar to that of N-formylated peptide. During a 3-month follow-up, 11 spontaneous bacterial infections were observed among the first group against none in the carcinogenic group.     

Exhaustive mutational analysis of severe acute respiratory syndrome coronavirus 2 ORF3a: An essential component in the pathogen's infectivity cycle

Detailed knowledge of a protein's key residues may assist in understanding its function and designing inhibitors against it. Consequently, such knowledge of one of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)'s proteins is advantageous since the virus is the etiological agent behind one of the biggest health crises of recent times. To that end, we constructed an exhaustive library of bacteria differing from each other by the mutated version of the virus's ORF3a viroporin they harbor. Since the protein is harmful to bacterial growth due to its channel activity, genetic selection followed by deep sequencing could readily identify mutations that abolish the protein's function. Our results have yielded numerous mutations dispersed throughout the sequence that counteract ORF3a's ability to slow bacterial growth. Comparing these data with the conservation pattern of ORF3a within the coronavirinae provided interesting insights: Deleterious mutations obtained in our study corresponded to conserved residues in the protein. However, despite the comprehensive nature of our mutagenesis coverage (108 average mutations per site), we could not reveal all of the protein's conserved residues. Therefore, it is tempting to speculate that our study unearthed positions in the protein pertinent to channel activity, while other conserved residues may correspond to different functionalities of ORF3a. In conclusion, our study provides important information on a key component of SARS-CoV-2 and establishes a procedure to analyze other viroporins comprehensively.     

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.     

Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes

The compositions of bacterial genomes can be changed rapidly and dramatically through a variety of processes including horizontal gene transfer. This form of change is key to bacterial evolution, as it leads to ‘evolution in quantum leaps’. Horizontal gene transfer entails the incorporation of genetic elements transferred from another organism—perhaps in an earlier generation—directly into the genome, where they form ‘genomic islands’, i.e. blocks of DNA with signatures of mobile genetic elements. Genomic islands whose functions increase bacterial fitness, either directly or indirectly, have most likely been positively selected and can be termed ‘fitness islands’. Fitness islands can be divided into several subtypes: ‘ecological islands’ in environmental bacteria and ‘saprophytic islands’, ‘symbiosis islands’ or ‘pathogenicity islands’ (PAIs) in microorganisms that interact with living hosts. Here we discuss ways in which PAIs contribute to the pathogenic potency of bacteria, and the idea that genetic entities similar to genomic islands may also be present in the genomes of eukaryotes.