RESUMO
Cigars and cigarillos are emerging as popular tobacco alternatives to cigarettes. However, these products may be equally harmful to human health than cigarettes and are associated with similar adverse health effects. We used 16S rRNA gene amplicon sequencing to extensively characterize the microbial diversity and investigate differences in microbial composition across 23 different products representing three different cigar product categories: filtered cigar, cigarillo, and large cigar. High throughput sequencing of the V4 hypervariable region of the 16 s rRNA gene revealed 2124 Operational Taxonomic Units (OTUs). Our findings showed that the three categories of cigars differed significantly in observed richness and Shannon diversity, with filtered cigars exhibiting lower diversity compared to large cigars and cigarillos. We also found a shared and unique microbiota among different product types. Firmicutes was the most abundant phylum in all product categories, followed by Actinobacteria. Among the 16 genera shared across all product types were Bacillus, Staphylococcus, Pseudomonas, and Pantoea. Nine genera were exclusively shared by large cigars and cigarillos and an additional thirteen genera were exclusive to filtered cigars. Analysis of individual cigar products showed consistent microbial composition across replicates for most large cigars and cigarillos while filtered cigars showed more inter-product variability. These findings provide important insights into the microbial diversity of the different cigar product types.
Assuntos
Bactérias , Biodiversidade , Microbiota , RNA Ribossômico 16S , Produtos do Tabaco , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Filogenia , Sequenciamento de Nucleotídeos em Larga Escala , DNA Bacteriano/genéticaRESUMO
IMPORTANCE: Production of ethanol from sugars and yeast is an ancient, ostensibly simple process. The source of sugars varies depending on the desired product and can include fruits, vegetables, molasses, honey, or grains, among other things. The source of yeast can be natural in the case of spontaneous ferments, but dry yeast addition is typical for large-scale fermentations. While the polymicrobial nature of some alcoholic fermentations is appreciated (e.g., for wine), most grain-based ethanol producers view microbes, apart from the added yeast, as "contaminants" meant to be controlled in order to maximize efficiency of ethanol production per unit of sugar. Nonetheless, despite rigorous cleaning-in-place measures and cooking the mash, bacteria are routinely cultured from these fermentations. We now know that bacteria can contribute to fermentation efficiency on an industrial scale, yet nothing is known about the makeup and stability of microbial communities in distilled spirit fermentations. The work here establishes the roles of mash recipes and distillery practices in microbial community assembly and dynamics over the course of fermentation. This represents an important first step in appreciating the myriad roles of bacteria in the production of distilled spirits.
Assuntos
Etanol , Saccharomyces cerevisiae , Fermentação , Bactérias/genética , AçúcaresRESUMO
Our previous studies showed that invertebrate animal serine racemase (SerR) and aspartate racemase (AspR) evolved from a common ancestral gene and are widely distributed. However, the overall molecular evolutionary background of these genes has remained unclear. In the present study, we have cloned, expressed and characterized five SerR and three AspR genes from six invertebrate species. The coexistence of SerR and AspR paralogs has been observed in some species, and the presence of both SerR and AspR is here confirmed in the flatworm Macrostomum lignano, the feather star Anneissia japonica, the ark shell Anadara broughtonii and the sea hare Aplysia californica. Comparison of the gene structures revealed the evolution of SerR and AspR. The ancestral species of metazoans probably had a single SerR gene, and the first gene duplication in the common ancestor species of the eumetazoans occurred after the divergence of porifera and eumetazoans, yielding two SerR genes. Most eumetazoans lost one of the two SerR genes, while the echinoderm A. japonica retained both genes. Furthermore, it is clear that invertebrate AspR genes arose through parallel evolution by duplication of the SerR gene followed by substitution of amino acid residues necessary for substrate recognition in multiple lineages.
Assuntos
Ácido Aspártico , Serina , Animais , Ácido Aspártico/metabolismo , Serina/metabolismo , Invertebrados/genética , Invertebrados/metabolismo , Evolução Molecular , FilogeniaRESUMO
Mammals harbor diverse gut microbiomes (GMs) that perform critical functions for host health and fitness. Identifying factors associated with GM variation can help illuminate the role of microbial symbionts in mediating host ecological interactions and evolutionary processes, including diversification and adaptation. Many mammals demonstrate phylosymbiosis-a pattern in which more closely-related species harbor more similar GMs-while others show overwhelming influences of diet and habitat. Here, we generated 16S rRNA sequence data from fecal samples of 15 species of wild lemurs across southern Madagascar to (1) test a hypothesis of phylosymbiosis, and (2) test trait correlations between dietary guild, habitat, and GM diversity. Our results provide strong evidence of phylosymbiosis, though some closely-related species with substantial ecological niche overlap exhibited greater GM similarity than expected under Brownian motion. Phylogenetic regressions also showed a significant correlation between dietary guild and UniFrac diversity, but not Bray-Curtis or Jaccard. This discrepancy between beta diversity metrics suggests that older microbial clades have stronger associations with diet than younger clades, as UniFrac weights older clades more heavily. We conclude that GM diversity is predominantly shaped by host phylogeny, and that microbes associated with diet were likely acquired before evolutionary radiations within the lemur families examined.
RESUMO
Species distributions are dependent on interactions with abiotic and biotic factors in the environment. Abiotic factors like temperature, moisture, and soil nutrients, along with biotic interactions within and between species, can all have strong influences on spatial distributions of plants and animals. Terrestrial Antarctic habitats are relatively simple and thus good systems to study ecological factors that drive species distributions and abundance. However, these environments are also sensitive to perturbation, and thus understanding the ecological drivers of species distribution is critical for predicting responses to environmental change. The Antarctic midge, Belgica antarctica, is the only endemic insect on the continent and has a patchy distribution along the Antarctic Peninsula. While its life history and physiology are well studied, factors that underlie variation in population density within its range are unknown. Previous work on Antarctic microfauna indicates that distribution over broad scales is primarily regulated by soil moisture, nitrogen content, and the presence of suitable plant life, but whether these patterns are true over smaller spatial scales has not been investigated. Here we sampled midges across five islands on the Antarctic Peninsula and tested a series of hypotheses to determine the relative influences of abiotic and biotic factors on midge abundance. While historical literature suggests that Antarctic organisms are limited by the abiotic environment, our best-supported hypothesis indicated that abundance is predicted by a combination of abiotic and biotic conditions. Our results are consistent with a growing body of literature that biotic interactions are more important in Antarctic ecosystems than historically appreciated.
Assuntos
Ecossistema , Solo , Animais , Regiões Antárticas , Ilhas , PlantasRESUMO
Previous studies have shown that several d-amino acids are widely present in plants, and serine racemase (SerR), which synthesizes d-serine in vivo, has already been identified from three plant species. However, the full picture of the d-amino acid synthesis pathway in plants is not well understood. To clarify the distribution of amino acid racemases in plants, we have cloned, expressed and characterized eight SerR homologous genes from five plant species, including green alga. These SerR homologs exhibited racemase activity towards serine or aspartate and were identified on the basis of their maximum activity as SerR or aspartate racemase (AspR). The plant AspR gene is identified for the first time from Medicago truncatula, Manihot esculenta, Solanum lycopersicum, Sphagnum girgensohnii and Spirogyra pratensis. In addition to the AspR gene, three SerR genes are identified in the former three species. Phylogenetic tree analysis showed that SerR and AspR are widely distributed in plants and form a serine/aspartate racemase family cluster. The catalytic efficiency (kcat/Km) of plant AspRs was more than 100 times higher than that of plant SerRs, suggesting that d-aspartate, as well as d-serine, can be synthesized in vivo by AspR. The amino acid sequence alignment and comparison of the chromosomal gene arrangement have revealed that plant AspR genes independently evolved from SerR in each ancestral lineage of plant species by gene duplication and acquisition of two serine residues at position 150 to 152.
Assuntos
Isomerases de Aminoácido/metabolismo , Racemases e Epimerases/metabolismo , Isomerases de Aminoácido/genética , Biocatálise , Regulação Enzimológica da Expressão Gênica/genética , Solanum lycopersicum/enzimologia , Manihot/enzimologia , Medicago truncatula/enzimologia , Filogenia , Racemases e Epimerases/genética , Sphagnopsida/enzimologia , Spirogyra/enzimologiaRESUMO
Previously, we demonstrated that the animal aspartate racemase (AspR) gene has evolved from the serine racemase (SerR) gene by acquisition of three consecutive serine residues (Ser155-Ser156-Ser157) involved in the strong AspR activity, and this event has occurred independently and frequently during animal evolution. In the present study, we cloned and characterized two mammalian SerR homologous genes from the hemichordate acorn worm (Saccoglossus kowalevskii). The enzymes have been identified as an AspR and an aspartate/glutamate racemase (Asp/GluR) on the basis of their kinetic parameters. The S. kowalevskii Asp/GluR shows comparable substrate affinity and high catalytic efficiency (kcat/Km) for both aspartate and glutamate and is the first reported enzyme from animals that can synthesize d-glutamate. Amino acid sequence alignment analysis and site-directed mutagenesis studies have revealed that the amino acid residue at position 156, which is serine in AspR and alanine in Asp/GluR, is associated with binding and recognition of glutamate and aspartate. Phylogenetic analysis suggests that the S. kowalevskii AspR gene has evolved from the SerR gene after the divergence of hemichordata and vertebrate lineages by acquisition of the three serine residues at position 155 to 157 as in the case of other animal AspR genes. Furthermore, the S. kowalevskii Asp/GluR gene is the result of AspR gene duplication and several amino acid substitutions including that of the 156th serine residue with alanine. The fact that SerR has acquired substrate specificity towards aspartate or glutamate raises the possibility that synthesis of other d-amino acids is carried out by enzymes evolved from SerR.
Assuntos
Isomerases de Aminoácido , Cordados não Vertebrados , Filogenia , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico/economia , Ácido Aspártico/metabolismo , Cordados não Vertebrados/enzimologia , Cordados não Vertebrados/genética , Clonagem MolecularRESUMO
The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of d-Lys in the growth medium. An alr gene knockout strain (Δalr) was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δalr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δalr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δalr strain exhibited a limited capacity for catabolism of l-Lys and l-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of l-Lys by virtue of its ability to convert l-Lys to d-Lys, which is further catabolized through the l-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of l-Arg, although the pathway by which d-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism.
RESUMO
Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150-152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to <0.04% of wild-type after substitution of the triple serine loop region. Conversely, introducing the triple serine loop region into Acropora, Crassostrea, and Penaeus SerR drastically increased the AspR activity. Those mutants showed similar or higher substrate affinity for aspartate than serine and showed 11-683-fold higher k cat and 28-351-fold higher k cat/K m values for aspartate than serine racemization. Furthermore, we introduced serine residues in all combinations at position 150-152 in mouse SerR. These mutants revealed that a change in the enzyme function from SerR to AspR can be caused by introduction of Ser151 and Ser152, and addition of the third serine residue at position 150 further enhances the enzyme specificity for aspartate due to a decrease in the serine racemase and serine dehydratase activity. Here, we provide convincing evidence that the AspR gene has evolved from the SerR gene by acquisition of the triple serine loop region.
Assuntos
Isomerases de Aminoácido , Antozoários , Proteínas de Artrópodes , Crassostrea , Mutação de Sentido Incorreto , Penaeidae , Racemases e Epimerases , Isomerases de Aminoácido/química , Isomerases de Aminoácido/genética , Substituição de Aminoácidos , Animais , Antozoários/enzimologia , Antozoários/genética , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Crassostrea/enzimologia , Crassostrea/genética , Camundongos , Penaeidae/enzimologia , Penaeidae/genética , Estrutura Secundária de Proteína , Racemases e Epimerases/química , Racemases e Epimerases/genéticaRESUMO
UNLABELLED: Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator. IMPORTANCE: Plant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.
Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Glucose 1-Desidrogenase/genética , Cofator PQQ/genética , Pseudomonas putida/genética , Proteínas de Bactérias/metabolismo , Glucose 1-Desidrogenase/metabolismo , Óperon , Cofator PQQ/metabolismo , Pseudomonas putida/metabolismo , RizosferaRESUMO
Soil and rhizosphere environments were examined in order to determine the identity and relative abundance of bacteria that catabolize d- and l-amino acids as the sole source of carbon and nitrogen. All substrates were readily catabolized by bacteria from both environments, with most d-amino acids giving similar CFU counts to their l-amino acid counterparts. CFU count ratios between l- and d-amino acids typically ranged between 2 and 1. Isolates were phylogenetically typed in order to determine the identity of d-amino acid catabolizers. Actinobacteria, specifically the Arthrobacter genus, were abundant along with members of the α- and ß-Proteobacteria classes.
Assuntos
Aminoácidos/metabolismo , Bactérias/metabolismo , Microbiologia do Solo , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Biodiversidade , Carbono/metabolismo , Contagem de Colônia Microbiana , Metabolismo , Nitrogênio/metabolismoRESUMO
Tobacco-specific nitrosamines are carcinogenic N-nitrosamine compounds present at very low levels in freshly harvested tobacco leaves that accumulate during leaf curing. Formation of N-nitrosamine compounds is associated with high nitrate levels in the leaf at harvest, and nitrate is presumed to be the source from which the N-nitrosation species originates. More specifically, nitrite is considered to be a direct precursor, and nitrite is linked with N-nitrosation in many environmental matrices where it occurs via microbial nitrate reduction. Here, we initiate work exploring the role of leaf microbial communities in formation of tobacco-specific nitrosamines. Leaves from burley tobacco line TN90H were air cured under various temperature and relative humidity levels, and 22 cured tobacco samples were analyzed for their microbial communities and leaf chemistry. Analysis of nitrate, nitrite, and total tobacco-specific nitrosamine levels revealed a strong positive correlation between the three variables, as well as a strong positive correlation with increasing relative humidity during cure conditions. 16S rRNA gene amplicon sequencing was used to assess microbial communities in each of the samples. In most samples, Proteobacteria predominated at the phylum level, accounting for >90 % of the OTUs. However, a distinct shift was noted among members of the high tobacco-specific nitrosamine group, with increases in Firmicutes and Actinobacteria. Several OTUs were identified that correlate strongly (positive and negative) with tobacco-specific nitrosamine content. Copy number of bacterial nitrate reductase genes, obtained using quantitative PCR, did not correlate strongly with tobacco-specific nitrosamine content. Incomplete denitrification is potentially implicated in tobacco-specific nitrosamine levels.
Assuntos
Bactérias/classificação , Nicotiana/microbiologia , Nitrosaminas/análise , Folhas de Planta/química , Bactérias/enzimologia , Bactérias/isolamento & purificação , Carcinógenos/análise , DNA Bacteriano/genética , Nitrato Redutase/genética , Nitratos/análise , Nitritos/análise , Folhas de Planta/microbiologia , RNA Ribossômico 16S/genética , Temperatura , Nicotiana/químicaRESUMO
Our goal was to investigate how root exudate flavonoids influence the soil bacterial community structure and to identify members of the community that change their relative abundance in response to flavonoid exudation. Using a model system that approximates flavonoid exudation of Medicago sativa roots, we treated a soil with 7,4'-dihydroxyflavone and naringenin in two separate experiments using three different rates: medium (equivalent to the exudation rate of 7,4'-dihydroxyflavone from M. sativa seedlings), high (10× the medium rate), and low (0.1× the medium rate). Controls received no flavonoid. Soil samples were subjected to ATP assays and 16S rRNA gene amplicon sequencing. The flavonoid treatments caused no significant change in the soil ATP content. With the high 7,4'-dihydroxyflavone treatment rate, operational taxonomic units (OTUs) classified as Acidobacteria subdivision 4 increased in relative abundance compared with the control samples, whereas OTUs classified as Gaiellales, Nocardioidaceae, and Thermomonosporaceae were more prevalent in the control. The naringenin treatments did not cause significant changes in the soil bacterial community structure. Our results suggest that the root exudate flavonoid 7,4'-dihydroxyflavone can interact with a diverse range of soil bacteria and may have other functions in the rhizosphere in addition to nod gene induction in legume-rhizobia symbiosis.
Assuntos
Bactérias/efeitos dos fármacos , Flavanonas/farmacologia , Flavonoides/farmacologia , Medicago sativa/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Microbiologia do Solo , Bactérias/genética , RNA Ribossômico 16S/genéticaRESUMO
Root-associated microbes have a profound impact on plant health, yet little is known about the distribution of root-associated microbes among different root morphologies or between rhizosphere and root environments. We explore these issues here with two commercial varieties of burley tobacco (Nicotiana tabacum) using 16S rRNA gene amplicon sequencing from rhizosphere soil, as well as from primary, secondary, and fine roots. While rhizosphere soils exhibited a fairly rich and even distribution, root samples were dominated by Proteobacteria. A comparison of abundant operational taxonomic units (OTUs) between rhizosphere and root samples indicated that Nicotiana roots select for rare taxa (predominantly Proteobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, and Acidobacteria) from their corresponding rhizosphere environments. The majority of root-inhabiting OTUs (~80 %) exhibited habitat generalism across the different root morphological habitats, although habitat specialists were noted. These results suggest a specific process whereby roots select rare taxa from a larger community.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Nicotiana/microbiologia , Raízes de Plantas/microbiologia , Rizosfera , Microbiologia do Solo , Bactérias/genética , FilogeniaRESUMO
Through livestock manure fertilization, antibiotics, antibiotic-resistant bacteria and genes are transferred to agricultural soils, resulting in a high prevalence of antibiotic-resistant bacteria in the soil. It is not clear, however, whether a correlation exists between resistant bacterial populations in manure and manure-amended soil. In this work, we demonstrate that the prevalence of cephalexin-, amoxicillin-, kanamycin- and gentamicin-resistant bacteria as well as bacteria simultaneously resistant to all four antibiotics was much higher in manure-amended soils than in manure-free soil. 454-pyrosequencing indicated that the ARB and multiple antibiotic-resistant bacteria (MARB) in swine or chicken manure and manure-amended soil were mainly distributed among Sphingobacterium, Myroides, Enterococcus, Comamonas and unclassified Flavobacteriaceae. The genus Sphingobacterium was highly prevalent among ARB from swine manure and manure-amended soil, and was also the most dominant genus among MARB from chicken manure and manure-amended soil. Other dominant genera among ARB or MARB populations in manure samples, including Myroides, Enterococcus and Comamonas, could not be detected or were detected at very low relative abundance in manure-amended soil. The present study suggests the possibility of transfer of ARBs from livestock manures to soils and persistence of ARB in these environments.
Assuntos
Bactérias/isolamento & purificação , Farmacorresistência Bacteriana Múltipla , Esterco/microbiologia , Microbiologia do Solo , Animais , Bactérias/genética , Galinhas/microbiologia , China , Contagem de Colônia Microbiana , Reação em Cadeia da Polimerase , Sus scrofa/microbiologiaRESUMO
Free D-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150-152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR.
Assuntos
Isomerases de Aminoácido/genética , Ácido Aspártico/metabolismo , Invertebrados/enzimologia , Racemases e Epimerases/genética , Serina/metabolismo , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Invertebrados/genética , Filogenia , Fosfato de Piridoxal/metabolismo , Racemases e Epimerases/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Corn-based fuel ethanol facilities mix enzymatically treated, gelatinized corn starch with water to generate a "mash" that is used as the substrate in large-scale (â¼500,000 gallon) yeast-based fermentations. In contrast to other food and beverage fermentations (e.g., cheese, wine), bioethanol production is presumed to be optimal when bacteria are absent from the fermentation-thus maximizing conversion of glucose to ethanol-yet the facilities are not sterilized. Culture-based analysis has suggested that lactic acid bacteria occupy this niche and, under certain circumstances, can outcompete the dedicated fermentation yeast for nutrients. Here, we use 16S rRNA gene amplicon sequencing to probe bacterial community structure during bioethanol fermentation. Nineteen total batches from five corn-based fuel ethanol fermentation facilities were analyzed. From each batch, five samples were taken. This includes the contents of the yeast propagation tank at inoculation, three samples taken at intervals during the fermentation, and a sample taken at the end of fermentation. Bacterial community structure was compared with time, between facility, between fermentor, between batches from the same fermentor, and against environmental variables within each fermentation. Communities were dominated by members of the Firmicutes and Proteobacteria phyla, with lactic acid bacteria dominating the communities in two of the five facilities. In the other facilities, Proteobacteria (largely members of the Pseudomonas and Escherichia-Shigella genera) outcompete the lactic acid bacteria. In most cases, the yeast propagation tank inoculum imparted a rich bacterial community, but the batches vary regarding whether this inoculum was the primary driver of the fermentation community structure.
Assuntos
Bactérias/metabolismo , Etanol/metabolismo , Zea mays/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , Reatores Biológicos/microbiologia , Fermentação , Filogenia , Zea mays/metabolismoRESUMO
Functional metagenomic analysis of soil metagenomes is a method for uncovering as-yet unidentified mechanisms for antibiotic resistance. Here we report an unconventional mode by which a response regulator derived from a soil metagenome confers resistance to the ß-lactam antibiotic carbenicillin in Escherichia coli. A recombinant clone (ßlr16) harboring a 5,169 bp DNA insert was selected from a metagenomic library previously constructed from a remote Alaskan soil. The ßlr16 clone conferred specific resistance to carbenicillin, with limited increases in resistance to other tested antibiotics, including other ß-lactams (penicillins and cephalosporins), rifampin, ciprofloxacin, erythromycin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin. Resistance was more pronounced at 24°C than at 37°C. Zone-of-inhibition assays suggested that the mechanism of carbenicillin resistance was not due to antibiotic inactivation. The DNA insert did not encode any genes known to confer antibiotic resistance, but did have two putative open reading frames (ORFs) that were annotated as a metallopeptidase and a two-component response regulator. Transposon mutagenesis and subcloning of the two ORFs followed by phenotypic assays showed that the response regulator gene was necessary and sufficient to confer the resistance phenotype. Quantitative reverse transcriptase PCR showed that the response regulator suppressed expression of the ompF porin gene, independently of the small RNA regulator micF, and enhanced expression of the acrD, mdtA, and mdtB efflux pump genes. This work demonstrates that antibiotic resistance can be achieved by the modulation of gene regulation by heterologous DNA. Functional analyses such as these can be important for making discoveries in antibiotic resistance gene biology and ecology.
Assuntos
Proteínas de Bactérias/genética , Carbenicilina , Escherichia coli/genética , Metagenoma , Microbiologia do Solo , Resistência beta-Lactâmica/genética , Proteínas de Bactérias/biossíntese , DNA Bacteriano/genética , Escherichia coli/metabolismo , Fases de Leitura AbertaRESUMO
Multitrophic level microbial loop interactions mediated by protist predators, bacteria, and viruses drive eco- and agro-biotechnological processes such as bioremediation, wastewater treatment, plant growth promotion, and ecosystem functioning. To what extent these microbial interactions are context-dependent in performing biotechnological and ecosystem processes remains largely unstudied. Theory-driven research may advance the understanding of eco-evolutionary processes underlying the patterns and functioning of microbial interactions for successful development of microbe-based biotechnologies for real world applications. This could also be a great avenue to test the validity or limitations of ecology theory for managing diverse microbial resources in an era of altering microbial niches, multitrophic interactions, and microbial diversity loss caused by climate and land use changes.
Assuntos
Agricultura , Biodegradação Ambiental , Biotecnologia , Microbiologia Industrial , Interações MicrobianasRESUMO
An Enterobacter cloacae strain (E. cloacae F3S3) that was collected as part of a project to assess antibiotic resistance among bacteria isolated from bioethanol fermentation facilities demonstrated high levels of resistance to antibiotics added prophylactically to bioethanol fermentors. PCR assays revealed the presence of canonical genes encoding resistance to penicillin (ampC) and erythromycin (ermG). Assays measuring biofilm formation under antibiotic stress indicated that erythromycin induced biofilm formation in E. cloacae F3S3. Planktonic growth and biofilm formation were observed at a high ethanol content, indicating E. cloacae F3S3 can persist in a bioethanol fermentor under the highly variable environmental conditions found in fermentors.