Identification and functional analysis of novel protein-encoding sequences related to stress-resistance. - Related Documents




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634001.0000Identification and functional analysis of novel protein-encoding sequences related to stress-resistance. Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process-specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.202337840709
892310.9998The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli. Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms. IMPORTANCE: With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress.201627879333
900220.9998Bacterial strategies to inhabit acidic environments. Bacteria can inhabit a wide range of environmental conditions, including extremes in pH ranging from 1 to 11. The primary strategy employed by bacteria in acidic environments is to maintain a constant cytoplasmic pH value. However, many data demonstrate that bacteria can grow under conditions in which pH values are out of the range in which cytoplasmic pH is kept constant. Based on these observations, a novel notion was proposed that bacteria have strategies to survive even if the cytoplasm is acidified by low external pH. Under these conditions, bacteria are obliged to use acid-resistant systems, implying that multiple systems having the same physiological role are operating at different cytoplasmic pH values. If this is true, it is quite likely that bacteria have genes that are induced by environmental stimuli under different pH conditions. In fact, acid-inducible genes often respond to another factor(s) besides pH. Furthermore, distinct genes might be required for growth or survival at acid pH under different environmental conditions because functions of many systems are dependent on external conditions. Systems operating at acid pH have been described to date, but numerous genes remain to be identified that function to protect bacteria from an acid challenge. Identification and analysis of these genes is critical, not only to elucidate bacterial physiology, but also to increase the understanding of bacterial pathogenesis.200012483574
633930.9998Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment. Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizosphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the transcriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowledge about the different strategies used by microorganisms to survive under extremely acid conditions.201323145860
892240.9998Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches. Burkholderia pseudomallei, a soil and water saprophyte, is responsible for the tropical human disease melioidosis. A hundred years since its discovery, there is still much to learn about B. pseudomallei proteins that are essential for the bacterium's survival in and interaction with the infected host, as well as their roles within the bacterium's natural soil habitat. To address this gap, bacteria grown under conditions mimicking the soil environment were subjected to transcriptome sequencing (RNA-seq) analysis. A dual RNA-seq approach was used on total RNA from spleens isolated from a B. pseudomallei mouse infection model at 5 days postinfection. Under these conditions, a total of 1,434 bacterial genes were induced, with 959 induced in the soil environment and 475 induced in bacteria residing within the host. Genes encoding metabolism and transporter proteins were induced when the bacteria were present in soil, while virulence factors, metabolism, and bacterial defense mechanisms were upregulated during active infection of mice. On the other hand, capsular polysaccharide and quorum-sensing pathways were inhibited during infection. In addition to virulence factors, reactive oxygen species, heat shock proteins, siderophores, and secondary metabolites were also induced to assist bacterial adaptation and survival in the host. Overall, this study provides crucial insights into the transcriptome-level adaptations which facilitate infection by soil-dwelling B. pseudomallei. Targeting novel therapeutics toward B. pseudomallei proteins required for adaptation provides an alternative treatment strategy given its intrinsic antimicrobial resistance and the absence of a vaccine. IMPORTANCE Burkholderia pseudomallei, a soil-dwelling bacterium, is the causative agent of melioidosis, a fatal infectious disease of humans and animals. The bacterium has a large genome consisting of two chromosomes carrying genes that encode proteins with important roles for survival in diverse environments as well as in the infected host. While a general mechanism of pathogenesis has been proposed, it is not clear which proteins have major roles when the bacteria are in the soil and whether the same proteins are key to successful infection and spread. To address this question, we grew the bacteria in soil medium and then in infected mice. At 5 days postinfection, bacteria were recovered from infected mouse organs and their gene expression was compared against that of bacteria grown in soil medium. The analysis revealed a list of genes expressed under soil growth conditions and a different set of genes encoding proteins which may be important for survival, replication, and dissemination in an infected host. These proteins are a potential resource for understanding the full adaptation mechanism of this pathogen. In the absence of a vaccine for melioidosis and with treatment being reliant on combinatorial antibiotic therapy, these proteins may be ideal targets for designing antimicrobials to treat melioidosis.202336856434
900450.9998Shedding 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
928960.9998Artificial 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
838470.9998In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors. Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.201425408687
900180.9998Bacterial Methionine Metabolism Genes Influence Drosophila melanogaster Starvation Resistance. Animal-associated microorganisms (microbiota) dramatically influence the nutritional and physiological traits of their hosts. To expand our understanding of such influences, we predicted bacterial genes that influence a quantitative animal trait by a comparative genomic approach, and we extended these predictions via mutant analysis. We focused on Drosophila melanogaster starvation resistance (SR). We first confirmed that D. melanogaster SR responds to the microbiota by demonstrating that bacterium-free flies have greater SR than flies bearing a standard 5-species microbial community, and we extended this analysis by revealing the species-specific influences of 38 genome-sequenced bacterial species on D. melanogaster SR. A subsequent metagenome-wide association analysis predicted bacterial genes with potential influence on D. melanogaster SR, among which were significant enrichments in bacterial genes for the metabolism of sulfur-containing amino acids and B vitamins. Dietary supplementation experiments established that the addition of methionine, but not B vitamins, to the diets significantly lowered D. melanogaster SR in a way that was additive, but not interactive, with the microbiota. A direct role for bacterial methionine metabolism genes in D. melanogaster SR was subsequently confirmed by analysis of flies that were reared individually with distinct methionine cycle Escherichia coli mutants. The correlated responses of D. melanogaster SR to bacterial methionine metabolism mutants and dietary modification are consistent with the established finding that bacteria can influence fly phenotypes through dietary modification, although we do not provide explicit evidence of this conclusion. Taken together, this work reveals that D. melanogaster SR is a microbiota-responsive trait, and specific bacterial genes underlie these influences.IMPORTANCE Extending descriptive studies of animal-associated microorganisms (microbiota) to define causal mechanistic bases for their influence on animal traits is an emerging imperative. In this study, we reveal that D. melanogaster starvation resistance (SR), a model quantitative trait in animal genetics, responds to the presence and identity of the microbiota. Using a predictive analysis, we reveal that the amino acid methionine has a key influence on D. melanogaster SR and show that bacterial methionine metabolism mutants alter normal patterns of SR in flies bearing the bacteria. Our data further suggest that these effects are additive, and we propose the untested hypothesis that, similar to bacterial effects on fruit fly triacylglyceride deposition, the bacterial influence may be through dietary modification. Together, these findings expand our understanding of the bacterial genetic basis for influence on a nutritionally relevant trait of a model animal host.201829934334
634290.9998Determinants of Extreme β-Lactam Tolerance in the Burkholderia pseudomallei Complex. Slow-growing bacteria are insensitive to killing by antibiotics, a trait known as antibiotic tolerance. In this study, we characterized the genetic basis of an unusually robust β-lactam (meropenem) tolerance seen in Burkholderia species. We identified tolerance genes under three different slow-growth conditions by extensive transposon mutant sequencing (Tn-seq), followed by single mutant validation. There were three principal findings. First, mutations in a small number of genes reduced tolerance under multiple conditions. Most of the functions appeared to be specific to peptidoglycan synthesis and the response to its disruption by meropenem action rather than being associated with more general physiological processes. The top tolerance genes are involved in immunity toward a type VI toxin targeting peptidoglycan (BTH_I0069), peptidoglycan recycling (ldcA), periplasmic regulation by proteolysis (prc), and an envelope stress response (rpoE and degS). Second, most of the tolerance functions did not contribute to growth in the presence of meropenem (intrinsic resistance), indicating that the two traits are largely distinct. Third, orthologues of many of the top Burkholderia thailandensis tolerance genes were also important in Burkholderia pseudomallei Overall, these studies show that the determinants of meropenem tolerance differ considerably depending on cultivation conditions, but that there are a few shared functions with strong mutant phenotypes that are important in multiple Burkholderia species.201829439964
6341100.9998Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression. In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype.202337485524
9349110.9998Gene essentiality analysis based on DEG, a database of essential genes. Essential genes are the genes that are indispensable for the survival of an organism. The genome-scale identification of essential genes has been performed in various organisms, and we consequently constructed DEG, a Database that contains currently available essential genes. Here we analyzed functional distributions of essential genes in DEG, and found that some essential-gene functions are even conserved between the prokaryote (bacteria) and the eukaryote (yeast), e.g., genes involved in information storage and processing are overrepresented, whereas those involved in metabolism are underrepresented in essential genes compared with non-essential ones. In bacteria, species specificity in functional distribution of essential genes is mainly due to those involved in cellular processes. Furthermore, within the category of information storage and processing, function of translation, ribosomal structure, and biogenesis are predominant in essential genes. Finally, some potential pitfalls for analyzing gene essentiality based on DEG are discussed.200818392983
8711120.9998Novel soil bacteria possess diverse genes for secondary metabolite biosynthesis. In soil ecosystems, microorganisms produce diverse secondary metabolites such as antibiotics, antifungals and siderophores that mediate communication, competition and interactions with other organisms and the environment(1,2). Most known antibiotics are derived from a few culturable microbial taxa (3) , and the biosynthetic potential of the vast majority of bacteria in soil has rarely been investigated (4) . Here we reconstruct hundreds of near-complete genomes from grassland soil metagenomes and identify microorganisms from previously understudied phyla that encode diverse polyketide and nonribosomal peptide biosynthetic gene clusters that are divergent from well-studied clusters. These biosynthetic loci are encoded by newly identified members of the Acidobacteria, Verrucomicobia and Gemmatimonadetes, and the candidate phylum Rokubacteria. Bacteria from these groups are highly abundant in soils(5-7), but have not previously been genomically linked to secondary metabolite production with confidence. In particular, large numbers of biosynthetic genes were characterized in newly identified members of the Acidobacteria, which is the most abundant bacterial phylum across soil biomes (5) . We identify two acidobacterial genomes from divergent lineages, each of which encodes an unusually large repertoire of biosynthetic genes with up to fifteen large polyketide and nonribosomal peptide biosynthetic loci per genome. To track gene expression of genes encoding polyketide synthases and nonribosomal peptide synthetases in the soil ecosystem that we studied, we sampled 120 time points in a microcosm manipulation experiment and, using metatranscriptomics, found that gene clusters were differentially co-expressed in response to environmental perturbations. Transcriptional co-expression networks for specific organisms associated biosynthetic genes with two-component systems, transcriptional activation, putative antimicrobial resistance and iron regulation, linking metabolite biosynthesis to processes of environmental sensing and ecological competition. We conclude that the biosynthetic potential of abundant and phylogenetically diverse soil microorganisms has previously been underestimated. These organisms may represent a source of natural products that can address needs for new antibiotics and other pharmaceutical compounds.201829899444
8920130.9998A systems biology approach to drug targets in Pseudomonas aeruginosa biofilm. Antibiotic resistance is an increasing problem in the health care system and we are in a constant race with evolving bacteria. Biofilm-associated growth is thought to play a key role in bacterial adaptability and antibiotic resistance. We employed a systems biology approach to identify candidate drug targets for biofilm-associated bacteria by imitating specific microenvironments found in microbial communities associated with biofilm formation. A previously reconstructed metabolic model of Pseudomonas aeruginosa (PA) was used to study the effect of gene deletion on bacterial growth in planktonic and biofilm-like environmental conditions. A set of 26 genes essential in both conditions was identified. Moreover, these genes have no homology with any human gene. While none of these genes were essential in only one of the conditions, we found condition-dependent genes, which could be used to slow growth specifically in biofilm-associated PA. Furthermore, we performed a double gene deletion study and obtained 17 combinations consisting of 21 different genes, which were conditionally essential. While most of the difference in double essential gene sets could be explained by different medium composition found in biofilm-like and planktonic conditions, we observed a clear effect of changes in oxygen availability on the growth performance. Eight gene pairs were found to be synthetic lethal in oxygen-limited conditions. These gene sets may serve as novel metabolic drug targets to combat particularly biofilm-associated PA. Taken together, this study demonstrates that metabolic modeling of human pathogens can be used to identify oxygen-sensitive drug targets and thus, that this systems biology approach represents a powerful tool to identify novel candidate antibiotic targets.201222523548
8957140.9998Transcriptome Profiling Reveals Interplay of Multifaceted Stress Response in Escherichia coli on Exposure to Glutathione and Ciprofloxacin. We have previously reported that supplementation of exogenous glutathione (GSH) promotes ciprofloxacin resistance in Escherichia coli by neutralizing antibiotic-induced oxidative stress and by enhancing the efflux of antibiotic. In the present study, we used a whole-genome microarray as a tool to analyze the system-level transcriptomic changes of E. coli on exposure to GSH and/or ciprofloxacin. The microarray data revealed that GSH supplementation affects redox function, transport, acid shock, and virulence genes of E. coli. The data further highlighted the interplay of multiple underlying stress response pathways (including those associated with the genes mentioned above and DNA damage repair genes) at the core of GSH, offsetting the effect of ciprofloxacin in E. coli. The results of a large-scale validation of the transcriptomic data using reverse transcription-quantitative PCR (RT-qPCR) analysis for 40 different genes were mostly in agreement with the microarray results. The altered growth profiles of 12 different E. coli strains carrying deletions in the specific genes mentioned above with GSH and/or ciprofloxacin supplementation implicate these genes in the GSH-mediated phenotype not only at the molecular level but also at the functional level. We further associated GSH supplementation with increased acid shock survival of E. coli on the basis of our transcriptomic data. Taking the data together, it can be concluded that GSH supplementation influences the expression of genes of multiple stress response pathways apart from its effect(s) at the physiological level to counter the action of ciprofloxacin in E. coli. IMPORTANCE The emergence and spread of multidrug-resistant bacterial strains have serious medical and clinical consequences. In addition, the rate of discovery of new therapeutic antibiotics has been inadequate in last few decades. Fluoroquinolone antibiotics such as ciprofloxacin represent a precious therapeutic resource in the fight against bacterial pathogens. However, these antibiotics have been gradually losing their appeal due to the emergence and buildup of resistance to them. In this report, we shed light on the genome-level expression changes in bacteria with respect to glutathione (GSH) exposure which act as a trigger for fluoroquinolone antibiotic resistance. The knowledge about different bacterial stress response pathways under conditions of exposure to the conditions described above and potential points of cross talk between them could help us in understanding and formulating the conditions under which buildup and spread of antibiotic resistance could be minimized. Our findings are also relevant because GSH-induced genome-level expression changes have not been reported previously for E. coli.201829468195
8921150.9998Multivariate approach to comparing whole-cell proteomes of Bacillus cereus indicates a biofilm-specific proteome. Biofilm bacteria are widely held to exhibit a unique phenotype, typified by their increased resistance to antimicrobial agents. Numerous studies have been devoted to the identification of biofilm-specific genes, but surprisingly few have been reported to date. We compared the whole cell proteomes of 24 h old Bacillus cereus biofilms and the associated suspended population to exponential, transient and stationary phase planktonic cultures using the unbiased approach of principal component analysis, comparing the quantity variations of the 823 detected spots. The analyses support the hypothesis that biofilms of Gram positive bacteria have a unique pattern of gene expression. The data provides proteomic evidence for a new biofilm and surface influenced planktonic population which is distinct to both planktonic and biofilm cells.200616889414
8387160.9998Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.201728189581
6311170.9998Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces. BACKGROUND: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. RESULTS: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. CONCLUSIONS: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.201728950904
8304180.9997A Shift to Human Body Temperature (37°C) Rapidly Reprograms Multiple Adaptive Responses in Escherichia coli That Would Facilitate Niche Survival and Colonization. One of the first environmental cues sensed by a microbe as it enters a human host is an upshift in temperature to 37°C. In this dynamic time point analysis, we demonstrate that this environmental transition rapidly signals a multitude of gene expression changes in Escherichia coli. Bacteria grown at 23°C under aerobic conditions were shifted to 37°C, and mRNA expression was measured at time points after the shift to 37°C (t = 0.5, 1, and 4 h). The first hour is characterized by a transient shift to anaerobic respiration strategies and stress responses, particularly acid resistance, indicating that temperature serves as a sentinel cue to predict and prepare for various niches within the host. The temperature effects on a subset of stress response genes were shown to be mediated by RpoS and directly correlated with RpoS, DsrA, and RprA levels, and increased acid resistance was observed that was dependent on 23°C growth and RpoS. By 4 h, gene expression shifted to aerobic respiration pathways and decreased stress responses, coupled with increases in genes associated with biosynthesis (amino acid and nucleotides), iron uptake, and host defense. ompT, a gene that confers resistance to antimicrobial peptides, was highly thermoregulated, with a pattern conserved in enteropathogenic and uropathogenic E. coli strains. An immediate decrease in curli gene expression concomitant with an increase in flagellar gene expression implicates temperature in this developmental decision. Together, our studies demonstrate that temperature signals a reprogramming of gene expression immediately upon an upshift that may predict, prepare, and benefit the survival of the bacterium within the host. IMPORTANCE As one of the first cues sensed by the microbe upon entry into a human host, understanding how bacteria like E. coli modulate gene expression in response to temperature improves our understanding of how bacteria immediately initiate responses beneficial for survival and colonization. For pathogens, understanding the various pathways of thermal regulation could yield valuable targets for anti-infective chemotherapeutic drugs or disinfection measures. In addition, our data provide a dynamic examination of the RpoS stress response, providing genome-wide support for how temperature impacts RpoS through changes in RpoS stability and modulation by small regulatory RNAs.202134516284
6338190.9997Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism. The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.201628033422