Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments. - Related Documents




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437301.0000Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments. Extremely cold environments are a challenge for all organisms. They are mostly inhabited by psychrophilic and psychrotolerant bacteria, which employ various strategies to cope with the cold. Such harsh environments are often highly vulnerable to the influence of external factors and may undergo frequent dynamic changes. The rapid adjustment of bacteria to changing environmental conditions is crucial for their survival. Such "short-term" evolution is often enabled by plasmids-extrachromosomal replicons that represent major players in horizontal gene transfer. The genomic sequences of thousands of microorganisms, including those of many cold-active bacteria have been obtained over the last decade, but the collected data have yet to be thoroughly analyzed. This report describes the results of a meta-analysis of the NCBI sequence databases to identify and characterize plasmids of psychrophilic and psychrotolerant bacteria. We have performed in-depth analyses of 66 plasmids, almost half of which are cryptic replicons not exceeding 10 kb in size. Our analyses of the larger plasmids revealed the presence of numerous genes, which may increase the phenotypic flexibility of their host strains. These genes encode enzymes possibly involved in (i) protection against cold and ultraviolet radiation, (ii) scavenging of reactive oxygen species, (iii) metabolism of amino acids, carbohydrates, nucleotides and lipids, (iv) energy production and conversion, (v) utilization of toxic organic compounds (e.g., naphthalene), and (vi) resistance to heavy metals, metalloids and antibiotics. Some of the plasmids also contain type II restriction-modification systems, which are involved in both plasmid stabilization and protection against foreign DNA. Moreover, approx. 50% of the analyzed plasmids carry genetic modules responsible for conjugal transfer or mobilization for transfer, which may facilitate the spread of these replicons among various bacteria, including across species boundaries.201425426110
437210.9999Plasmidome of Listeria spp.-The repA-Family Business. Bacteria of the genus Listeria (phylum Firmicutes) include both human and animal pathogens, as well as saprophytic strains. A common component of Listeria spp. genomes are plasmids, i.e., extrachromosomal replicons that contribute to gene flux in bacteria. This study provides an in-depth insight into the structure, diversity and evolution of plasmids occurring in Listeria strains inhabiting various environments under different anthropogenic pressures. Apart from the components of the conserved plasmid backbone (providing replication, stable maintenance and conjugational transfer functions), these replicons contain numerous adaptive genes possibly involved in: (i) resistance to antibiotics, heavy metals, metalloids and sanitizers, and (ii) responses to heat, oxidative, acid and high salinity stressors. Their genomes are also enriched by numerous transposable elements, which have influenced the plasmid architecture. The plasmidome of Listeria is dominated by a group of related replicons encoding the RepA replication initiation protein. Detailed comparative analyses provide valuable data on the level of conservation of these replicons and their role in shaping the structure of the Listeria pangenome, as well as their relationship to plasmids of other genera of Firmicutes, which demonstrates the range and direction of flow of genetic information in this important group of bacteria.202134638661
437120.9999Independent origins and evolution of the secondary replicons of the class Gammaproteobacteria. Multipartite genomes, consisting of more than one replicon, have been found in approximately 10 % of bacteria, many of which belong to the phylum Proteobacteria. Many aspects of their origin and evolution, and the possible advantages related to this type of genome structure, remain to be elucidated. Here, we performed a systematic analysis of the presence and distribution of multipartite genomes in the class Gammaproteobacteria, which includes several genera with diverse lifestyles. Within this class, multipartite genomes are mainly found in the order Alteromonadales (mostly in the genus Pseudoalteromonas) and in the family Vibrionaceae. Our data suggest that the emergence of secondary replicons in Gammaproteobacteria is rare and that they derive from plasmids. Despite their multiple origins, we highlighted the presence of evolutionary trends such as the inverse proportionality of the genome to chromosome size ratio, which appears to be a general feature of bacteria with multipartite genomes irrespective of taxonomic group. We also highlighted some functional trends. The core gene set of the secondary replicons is extremely small, probably limited to essential genes or genes that favour their maintenance in the genome, while the other genes are less conserved. This hypothesis agrees with the idea that the primary advantage of secondary replicons could be to facilitate gene acquisition through horizontal gene transfer, resulting in replicons enriched in genes associated with adaptation to different ecological niches. Indeed, secondary replicons are enriched both in genes that could promote adaptation to harsh environments, such as those involved in antibiotic, biocide and metal resistance, and in functional categories related to the exploitation of environmental resources (e.g. carbohydrates), which can complement chromosomal functions.202337185344
928930.9999Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity. When organisms are subjected to environmental challenges, including growth inhibitors and toxins, evolution often selects for the duplication of endogenous genes, whose overexpression can provide a selective advantage. Such events occur both in natural environments and in clinical settings. Microbial cells-with their large populations and short generation times-frequently evolve resistance to a range of antimicrobials. While microbial resistance to antibiotic drugs is well documented, less attention has been given to the genetic elements responsible for resistance to metal toxicity. To assess which overexpressed genes can endow gram-negative bacteria with resistance to metal toxicity, we transformed a collection of plasmids overexpressing all E. coli open reading frames (ORFs) into naive cells, and selected for survival in toxic concentrations of six transition metals: Cd, Co, Cu, Ni, Ag, Zn. These selections identified 48 hits. In each of these hits, the overexpression of an endogenous E. coli gene provided a selective advantage in the presence of at least one of the toxic metals. Surprisingly, the majority of these cases (28/48) were not previously known to function in metal resistance or homeostasis. These findings highlight the diverse mechanisms that biological systems can deploy to adapt to environments containing toxic concentrations of metals.201829356848
436740.9998Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistance to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.19979167257
416850.9998Various pathways leading to the acquisition of antibiotic resistance by natural transformation. Natural transformation can lead to exchange of DNA between taxonomically diverse bacteria. In the case of chromosomal DNA, homology-based recombination with the recipient genome is usually necessary for heritable stability. In our recent study, we have shown that natural transformation can promote the transfer of transposons, IS elements, and integrons and gene cassettes, largely independent of the genetic relationship between the donor and recipient bacteria. Additional results from our study suggest that natural transformation with species-foreign DNA might result in the uptake of a wide range of DNA fragments; leading to changes in the antimicrobial susceptibility profile and contributing to the generation of antimicrobial resistance in bacteria.201223482877
437460.9998Core genes can have higher recombination rates than accessory genes within global microbial populations. Recombination is essential to microbial evolution, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche. However, assessing the impact of homologous recombination on accessory genes which are only present in a subset of strains of a given species remains challenging due to their complex phylogenetic relationships. Quantifying homologous recombination for accessory genes (which are important for niche-specific adaptations) in comparison to core genes (which are present in all strains and have essential functions) is critical to understanding how selection acts on variation to shape species diversity and genome structures of bacteria. Here, we apply a computationally efficient, non-phylogenetic approach to measure homologous recombination rates in the core and accessory genome using >100,000 whole genome sequences from Streptococcus pneumoniae and several additional species. By analyzing diverse sets of sequence clusters, we show that core genes often have higher recombination rates than accessory genes, and for some bacterial species the associated effect sizes for these differences are pronounced. In a subset of species, we find that gene frequency and homologous recombination rate are positively correlated. For S. pneumoniae and several additional species, we find that while the recombination rate is higher for the core genome, the mutational divergence is lower, indicating that divergence-based homologous recombination barriers could contribute to differences in recombination rates between the core and accessory genome. Homologous recombination may therefore play a key role in increasing the efficiency of selection in the most conserved parts of the genome.202235801696
930870.9998Integrons: natural tools for bacterial genome evolution. Integrons were first identified as the primary mechanism for antibiotic resistance gene capture and dissemination among Gram-negative bacteria. More recently, their role in genome evolution has been extended with the discovery of larger integron structures, the super-integrons, as genuine components of the genomes of many species throughout the gamma-proteobacterial radiation. The functional platforms of these integrons appear to be sedentary, whereas their gene cassette contents are highly variable. Nevertheless, the gene cassettes for which an activity has been experimentally demonstrated encode proteins related to simple adaptive functions and their recruitment is seen as providing the bacterial host with a selective advantage. The widespread occurrence of the integron system among Gram-negative bacteria is discussed, with special focus on the super-integrons. Some of the adaptive functions encoded by these genes are also reviewed, and implications of integron-mediated genome evolution in the emergence of novel bacterial species are highlighted.200111587934
437680.9998Genetic exchanges are more frequent in bacteria encoding capsules. Capsules allow bacteria to colonize novel environments, to withstand numerous stresses, and to resist antibiotics. Yet, even though genetic exchanges with other cells should be adaptive under such circumstances, it has been suggested that capsules lower the rates of homologous recombination and horizontal gene transfer. We analysed over one hundred pan-genomes and thousands of bacterial genomes for the evidence of an association between genetic exchanges (or lack thereof) and the presence of a capsule system. We found that bacteria encoding capsules have larger pan-genomes, higher rates of horizontal gene transfer, and higher rates of homologous recombination in their core genomes. Accordingly, genomes encoding capsules have more plasmids, conjugative elements, transposases, prophages, and integrons. Furthermore, capsular loci are frequent in plasmids, and can be found in prophages. These results are valid for Bacteria, independently of their ability to be naturally transformable. Since we have shown previously that capsules are commonly present in nosocomial pathogens, we analysed their co-occurrence with antibiotic resistance genes. Genomes encoding capsules have more antibiotic resistance genes, especially those encoding efflux pumps, and they constitute the majority of the most worrisome nosocomial bacteria. We conclude that bacteria with capsule systems are more genetically diverse and have fast-evolving gene repertoires, which may further contribute to their success in colonizing novel niches such as humans under antibiotic therapy.201830576310
931190.9998Various plasmid strategies limit the effect of bacterial restriction-modification systems against conjugation. In bacteria, genes conferring antibiotic resistance are mostly carried on conjugative plasmids, mobile genetic elements that spread horizontally between bacterial hosts. Bacteria carry defence systems that defend them against genetic parasites, but how effective these are against plasmid conjugation is poorly understood. Here, we study to what extent restriction-modification (RM) systems-by far the most prevalent bacterial defence systems-act as a barrier against plasmids. Using 10 different RM systems and 13 natural plasmids conferring antibiotic resistance in Escherichia coli, we uncovered variation in defence efficiency ranging from none to 105-fold protection. Further analysis revealed genetic features of plasmids that explain the observed variation in defence levels. First, the number of RM recognition sites present on the plasmids generally correlates with defence levels, with higher numbers of sites being associated with stronger defence. Second, some plasmids encode methylases that protect against restriction activity. Finally, we show that a high number of plasmids in our collection encode anti-restriction genes that provide protection against several types of RM systems. Overall, our results show that it is common for plasmids to encode anti-RM strategies, and that, as a consequence, RM systems form only a weak barrier for plasmid transfer by conjugation.202439413206
9312100.9998Why There Are No Essential Genes on Plasmids. Mobile genetic elements such as plasmids are important for the evolution of prokaryotes. It has been suggested that there are differences between functions coded for by mobile genes and those in the "core" genome and that these differences can be seen between plasmids and chromosomes. In particular, it has been suggested that essential genes, such as those involved in the formation of structural proteins or in basic metabolic functions, are rarely located on plasmids. We model competition between genotypically varying bacteria within a single population to investigate whether selection favors a chromosomal location for essential genes. We find that in general, chromosomal locations for essential genes are indeed favored. This is because the inheritance of chromosomes is more stable than that for plasmids. We define the "degradation" rate as the rate at which chance genetic processes, for example, mutation, deletion, or translocation, render essential genes nonfunctioning. The only way in which plasmids can be a location for functioning essential genes is if chromosomal genes degrade faster than plasmid genes. If the two degradation rates are equal, or if plasmid genes degrade faster than chromosomal genes, functioning essential genes will be found only on chromosomes.201525540453
4164110.9998Broad-host-range IncP-1 plasmids and their resistance potential. The plasmids of the incompatibility (Inc) group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals, and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance, and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad-host-range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.201323471189
6335120.9998Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli. The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.202134756069
9004130.9998Shedding light on the bacterial resistance to toxic UV filters: a comparative genomic study. UV filters are toxic to marine bacteria that dominate the marine biomass. Ecotoxicology often studies the organism response but rarely integrates the toxicity mechanisms at the molecular level. In this study, in silico comparative genomics between UV filters sensitive and resistant bacteria were conducted in order to unravel the genes responsible for a resistance phenotype. The genomes of two environmentally relevant Bacteroidetes and three Firmicutes species were compared through pairwise comparison. Larger genomes were carried by bacteria exhibiting a resistant phenotype, favoring their ability to adapt to environmental stresses. While the antitoxin and CRISPR systems were the only distinctive features in resistant Bacteroidetes, Firmicutes displayed multiple unique genes that could support the difference between sensitive and resistant phenotypes. Several genes involved in ROS response, vitamin biosynthesis, xenobiotic degradation, multidrug resistance, and lipophilic compound permeability were shown to be exclusive to resistant species. Our investigation contributes to a better understanding of UV filters resistance phenotypes, by identifying pivotal genes involved in key pathways.202134760358
3837140.9998Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance. The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.201626668183
4375150.9998Evidence of a large novel gene pool associated with prokaryotic genomic islands. Microbial genes that are "novel" (no detectable homologs in other species) have become of increasing interest as environmental sampling suggests that there are many more such novel genes in yet-to-be-cultured microorganisms. By analyzing known microbial genomic islands and prophages, we developed criteria for systematic identification of putative genomic islands (clusters of genes of probable horizontal origin in a prokaryotic genome) in 63 prokaryotic genomes, and then characterized the distribution of novel genes and other features. All but a few of the genomes examined contained significantly higher proportions of novel genes in their predicted genomic islands compared with the rest of their genome (Paired t test = 4.43E-14 to 1.27E-18, depending on method). Moreover, the reverse observation (i.e., higher proportions of novel genes outside of islands) never reached statistical significance in any organism examined. We show that this higher proportion of novel genes in predicted genomic islands is not due to less accurate gene prediction in genomic island regions, but likely reflects a genuine increase in novel genes in these regions for both bacteria and archaea. This represents the first comprehensive analysis of novel genes in prokaryotic genomic islands and provides clues regarding the origin of novel genes. Our collective results imply that there are different gene pools associated with recently horizontally transmitted genomic regions versus regions that are primarily vertically inherited. Moreover, there are more novel genes within the gene pool associated with genomic islands. Since genomic islands are frequently associated with a particular microbial adaptation, such as antibiotic resistance, pathogen virulence, or metal resistance, this suggests that microbes may have access to a larger "arsenal" of novel genes for adaptation than previously thought.200516299586
3796160.9998The presence of plasmids in bacterial hosts alters phage isolation and infectivity. Antibiotic resistance genes are often carried by plasmids, which spread intra- and inter genera bacterial populations, and also play a critical role in bacteria conferring phage resistance. However, it remains unknown about the influence of plasmids present in bacterial hosts on phage isolation and subsequent infectivity. In this study, using both Escherichia coli and Pseudomonas putida bacteria containing different plasmids, eight phages were isolated and characterized in terms of phage morphology and host range analysis, in conjunction with DNA and protein sequencing. We found that plasmids can influence both the phage isolation process and phage infectivity. In particular, the isolated phages exhibited different phage plaquing infectivity towards the same bacterial species containing different plasmids. Furthermore, the presence of plasmids was found to alter the expression of bacteria membrane protein, which correlates with bacterial cell surface receptors recognized by phages, thus affecting phage isolation and infectivity. Given the diverse and ubiquitous nature of plasmids, our findings highlight the need to consider plasmids as factors that can influence both phage isolation and infectivity.202237938681
9287170.9998Use of DNA probes and plasmid capture in a search for new interesting environmental genes. Adaptation to a stressed environment leads to organisms bearing DNA, encoding defense mechanisms. These mechanisms can be heavy metal resistance, catabolism of organic xenobiotics or stress reactions. Genes responsible for these mechanisms can be used for monitoring changing environments and therefore it can be important to store such bacteria in a bank. DNA-probing will be presented by the use of DNA fragments (of Alcaligenes eutrophus) coding for heavy metal resistance or xenobiotic degradation. Some strains do not grow on petri dishes and accordingly cannot be isolated from soils. In order to isolate plasmids from such strains, coding for heavy metal resistances or xenobiotic degradations, an exogenous plasmid isolation method was developed. In this method, the endogenous population is conjugated with Pseudomonas or Alcaligenes strains bearing a retrotransfer plasmid like RP4. In that way new plasmids from various sources including non-culturable strains could be obtained. With these methods, a large number of specimens adapted to stressed situations can be isolated or constructed (in the case of the exogenous plasmid isolation method). They form a source of interesting genetic material that can be used to restore polluted areas in natural areas, if necessary with the aid of genetic engineering (in vitro or in vivo techniques). Full knowledge of such bacteria and their resistance mechanisms or degradation pathways, can lead to new constructions able to attack recalcitrant mixtures of different organics and to resist heavy metals.19938272850
4383180.9998Importance of Core Genome Functions for an Extreme Antibiotic Resistance Trait. Extreme antibiotic resistance in bacteria is associated with the expression of powerful inactivating enzymes and other functions encoded in accessory genomic elements. The contribution of core genome processes to high-level resistance in such bacteria has been unclear. In the work reported here, we evaluated the relative importance of core and accessory functions for high-level resistance to the aminoglycoside tobramycin in the nosocomial pathogen Acinetobacter baumannii Three lines of evidence establish the primacy of core functions in this resistance. First, in a genome scale mutant analysis using transposon sequencing and validation with 594 individual mutants, nearly all mutations reducing tobramycin resistance inactivated core genes, some with stronger phenotypes than those caused by the elimination of aminoglycoside-inactivating enzymes. Second, the core functions mediating resistance were nearly identical in the wild type and a deletion mutant lacking a genome resistance island that encodes the inactivating enzymes. Thus, most or all of the core resistance determinants important in the absence of the enzymes are also important in their presence. Third, reductions in tobramycin resistance caused by different core mutations were additive, and highly sensitive double and triple mutants (with 250-fold reductions in the MIC) that retained accessory resistance genes could be constructed. Core processes that contribute most strongly to intrinsic tobramycin resistance include phospholipid biosynthesis, phosphate regulation, and envelope homeostasis.IMPORTANCE The inexorable increase in bacterial antibiotic resistance threatens to undermine many of the procedures that transformed medicine in the last century. One strategy to meet the challenge antibiotic resistance poses is the development of drugs that undermine resistance. To identify potential targets for such adjuvants, we identified the functions underlying resistance to an important class of antibiotics for one of the most highly resistant pathogens known.201729233894
9405190.9998Functional Metagenomic Screening for Antimicrobial Resistance in the Oral Microbiome. A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance.202134410638