RESUMO
The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.
Assuntos
Bradyrhizobium , Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Glycine max , Fenômenos Fisiológicos Celulares , Fenótipo , SimbioseRESUMO
The guanine/cytosine (GC) content of prokaryotic genomes is species-specific, taking values from 16% to 77%. This diversity of selection for GC content remains contentious. We analyse the correlations between GC content and a range of phenotypic and genotypic data in thousands of prokaryotes. GC content integrates well with these traits into r/K selection theory when phenotypic plasticity is considered. High GC-content prokaryotes are r-strategists with cheaper descendants thanks to a lower average amino acid metabolic cost, colonize unstable environments thanks to flagella and a bacillus form and are generalists in terms of resource opportunism and their defence mechanisms. Low GC content prokaryotes are K-strategists specialized for stable environments that maintain homeostasis via a high-cost outer cell membrane and endospore formation as a response to nutrient deprivation, and attain a higher nutrient-to-biomass yield. The lower proteome cost of high GC content prokaryotes is driven by the association between GC-rich codons and cheaper amino acids in the genetic code, while the correlation between GC content and genome size may be partly due to functional diversity driven by r/K selection. In all, molecular diversity in the GC content of prokaryotes may be a consequence of ecological r/K selection.
Assuntos
Aminoácidos , Células Procarióticas , Composição de Bases , Aminoácidos/análise , Códon , Proteoma/genéticaRESUMO
Microbes are often discussed in terms of dichotomies such as copiotrophic/oligotrophic and fast/slow-growing microbes, defined using the characterisation of microbial growth in isolated cultures. The dichotomies are usually qualitative and/or study-specific, sometimes precluding clear-cut results interpretation. We can unravel microbial dichotomies as life history strategies by combining ecology theory with Monod curves, a laboratory mathematical tool of bacterial physiology that relates the specific growth rate of a microbe with the concentration of a limiting nutrient. Fitting of Monod curves provides quantities that directly correspond to key parameters in ecological theories addressing species coexistence and diversity, such as r/K selection theory, resource competition and community structure theory and the CSR triangle of life strategies. The resulting model allows us to reconcile the copiotrophic/oligotrophic and fast/slow-growing dichotomies as different subsamples of a life history strategy triangle that also includes r/K strategists. We also used the number of known carbon sources together with community structure theory to partially explain the diversity of heterotrophic microbes observed in metagenomics experiments. In sum, we propose a theoretical framework for the study of natural microbial communities that unifies several existing proposals. Its application would require the integration of metagenomics, metametabolomics, Monod curves and carbon source data.
Assuntos
Bactérias , Microbiota , Bactérias/genética , Processos Heterotróficos , Metagenômica , CarbonoRESUMO
Nearly 100 years ago, Winogradsky published a classic communication in which he described two groups of microbes, zymogenic and autochthonous. When organic matter penetrates the soil, zymogenic microbes quickly multiply and degrade it, then giving way to the slow combustion of autochthonous microbes. Although the text was originally written in French, it is often cited by English-speaking authors. We undertook a complete translation of the 1924 publication, which we provide as Supporting information. Here, we introduce the translation and describe how the zymogenic/autochthonous dichotomy shaped research questions in the study of microbial diversity and physiology. We also identify in the literature three additional and closely related dichotomies, which we propose to call exclusive copiotrophs/oligotrophs, coexisting copiotrophs/oligotrophs and fast-growing/slow-growing microbes. While Winogradsky focussed on a successional view of microbial populations over time, the current discussion is focussed on the differences in the specific growth rate of microbes as a function of the concentration of a given limiting substrate. In the future, it will be relevant to keep in mind both nutrient-focussed and time-focussed microbial dichotomies and to design experiments with both isolated laboratory cultures and multi-species communities in the spirit of Winogradsky's direct method.
Assuntos
Bactérias , Microbiologia do Solo , Biodiversidade , Bactérias/classificação , Bactérias/citologia , Bactérias/metabolismo , Solo/química , EcossistemaRESUMO
It is unclear how gene order within the chromosome influences genome evolution. Bacteria cluster transcription and translation genes close to the replication origin (oriC). In Vibrio cholerae, relocation of s10-spc-α locus (S10), the major locus of ribosomal protein genes, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction in growth rate, fitness, and infectivity. To test the long-term impact of this trait, we evolved 12 populations of V. cholerae strains bearing S10 at an oriC-proximal or an oriC-distal location for 1,000 generations. During the first 250 generations, positive selection was the main force driving mutation. After 1,000 generations, we observed more nonadaptative mutations and hypermutator genotypes. Populations fixed inactivating mutations at many genes linked to virulence: flagellum, chemotaxis, biofilm, and quorum sensing. Throughout the experiment, all populations increased their growth rates. However, those bearing S10 close to oriC remained the fittest, indicating that suppressor mutations cannot compensate for the genomic position of the main ribosomal protein locus. Selection and sequencing of the fastest-growing clones allowed us to characterize mutations inactivating, among other sites, flagellum master regulators. Reintroduction of these mutations into the wild-type context led to a ≈10% growth improvement. In conclusion, the genomic location of ribosomal protein genes conditions the evolutionary trajectory of V. cholerae. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and evolution. A lack of suppression enables artificial gene relocation as a tool for genetic circuit reprogramming. IMPORTANCE The bacterial chromosome harbors several entangled processes such as replication, transcription, DNA repair, and segregation. Replication begins bidirectionally at the replication origin (oriC) until the terminal region (ter) organizing the genome along the ori-ter axis gene order along this axis could link genome structure to cell physiology. Fast-growing bacteria cluster translation genes near oriC. In Vibrio cholerae, moving them away was feasible but at the cost of losing fitness and infectivity. Here, we evolved strains harboring ribosomal genes close or far from oriC. Growth rate differences persisted after 1,000 generations. No mutation was able to compensate for the growth defect, showing that ribosomal gene location conditions their evolutionary trajectory. Despite the high plasticity of bacterial genomes, evolution has sculpted gene order to optimize the ecological strategy of the microorganism. We observed growth rate improvement throughout the evolution experiment that occurred at expense of energetically costly processes such the flagellum biosynthesis and virulence-related functions. From the biotechnological point of view, manipulation of gene order enables altering bacterial growth with no escape events.
Assuntos
Vibrio cholerae , Vibrio cholerae/genética , Proteínas Ribossômicas/genética , Genoma Bacteriano , Mutação , Cromossomos , Proteínas de Bactérias/genéticaRESUMO
Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2'-O,4'-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Oligonucleotídeos/química , Etilenos/químicaRESUMO
The human pathogen Acinetobacter baumannii possesses high genetic plasticity and frequently acquires antimicrobial resistance genes. Here we investigated the role of natural transformation in these processes. Genomic DNA from different sources, including from carbapenem-resistant Klebsiella pneumoniae strains, was mixed with A. baumannii A118 cells. Selected transformants were analysed by whole-genome sequencing. In addition, bioinformatics analyses and in silico gene flow prediction were also performed to support the experimental results. Transformant strains included some that became resistant to carbapenems or changed their antimicrobial susceptibility profile. Foreign DNA acquisition was confirmed by whole-genome analysis. The acquired DNA most frequently identified corresponded to mobile genetic elements, antimicrobial resistance genes and operons involved in metabolism. Bioinformatics analyses and in silico gene flow prediction showed continued exchange of genetic material between A. baumannii and K. pneumoniae when they share the same habitat. Natural transformation plays an important role in the plasticity of A. baumannii and concomitantly in the emergence of multidrug-resistant strains.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Klebsiella pneumoniae/genética , Transformação Bacteriana/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , DNA Bacteriano/genética , Genoma Bacteriano/genética , Humanos , Sequências Repetitivas Dispersas/genética , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Sequenciamento Completo do GenomaRESUMO
No-tillage crop production has revolutionized the agriculture worldwide. In our country more than 30 Mha are currently cultivated under no-till schemes, stressing the importance of this management system for crop production. It is widely recognized that soil microbiota is altered under different soil managements. In this regard the structure of Burkholderia populations is affected by soils management practices such as tillage, fertilization, or crop rotation. The stability of these structures, however, has not been evaluated under sustainable schemes where the impact of land practices could be less deleterious to physicochemical soils characteristics. In order to assess the structure of Burkholderia spp. populations in no-till schemes, culturable Burkholderia spp. strains were quantified and their biodiversity evaluated. Results showed that Burkholderia spp. biodiversity, but not their abundance, clearly displayed a dependence on agricultural managements. We also showed that biodiversity was mainly influenced by two soil factors: Total Organic Carbon and Total Nitrogen. Results showed that no-till schemes are not per se sufficient to maintain a richer Burkholderia spp. soil microbiota, and additional traits should be considered when sustainability of productive soils is a goal to fulfil productive agricultural schemes.
Assuntos
Biodiversidade , Burkholderia , Produção Agrícola , Microbiologia do Solo , Solo , Argentina , Burkholderia/classificação , Burkholderia/crescimento & desenvolvimento , Burkholderia/isolamento & purificaçãoRESUMO
The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca(2+)or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Cálcio/farmacologia , Infecção Hospitalar/microbiologia , Competência de Transformação por DNA/efeitos dos fármacos , Albumina Sérica/farmacologia , DNA/genética , Competência de Transformação por DNA/genética , Farmacorresistência Bacteriana/genética , Transferência Genética Horizontal/efeitos dos fármacos , Transferência Genética Horizontal/genética , Genes Bacterianos/genética , HumanosRESUMO
RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.
Assuntos
Oligorribonucleotídeos Antissenso/metabolismo , RNA Bacteriano/metabolismo , RNA Catalítico/metabolismo , Ribonuclease P/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Humanos , Modelos Genéticos , Conformação de Ácido Nucleico , Oligorribonucleotídeos Antissenso/genética , RNA Bacteriano/química , RNA Bacteriano/genéticaRESUMO
We studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxA- and oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6')-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusion-based method to improve the phenotypic detection of aac(6')-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6')-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum ß-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6')-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum ß-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Plasmídeos/genética , Quinolonas/farmacologia , Argentina , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Humanos , Integrons/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNARESUMO
EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Desenho de Fármacos , Oligorribonucleotídeos Antissenso/farmacologia , Clivagem do RNA/efeitos dos fármacos , Ribonuclease P/metabolismo , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli , Microscopia Confocal , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Regiões Terminadoras Genéticas/genéticaRESUMO
Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site.
Assuntos
Escherichia coli/genética , Plasmídeos/genética , Salmonella/genética , Argentina , Sequência de Bases , Evolução Biológica , DNA Bacteriano/genética , Infecções por Escherichia coli/microbiologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Recombinação Genética , Infecções por Salmonella/microbiologiaRESUMO
Delivery inside the cells is essential for practical application of antisense technologies. The hybrid locked nucleic acid (LNA)/DNA CAAGTACTGTTCCACCA (LNA residues are underlined) was labeled by conjugation to Alexa Fluor 488 (fLNA/DNA) and tested to determine its ability to penetrate Escherichia coli cells and reach the cytoplasm. Flow cytometry analysis showed that the fLNA/DNA was associated with 14% of cells from a stationary phase culture, while association with a labeled isosequential oligodeoxynucleotide was negligible. Laser scanning confocal microscopy confirmed that the fLNA/DNA was located inside the cytoplasm.
RESUMO
Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.
Assuntos
Acetiltransferases/antagonistas & inibidores , Amicacina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Ribonuclease P/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Bases , DNA/metabolismo , Endocitose/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6')-Ib-cr within the variable region of integrons. The three arrays differed from bla(CTX-M-2)-bearing integrons, which are broadly distributed in Argentina.