RESUMO
The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3'-5' exoribonuclease involved in mRNA decay in Escherichia coli The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.
Assuntos
Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , RNA Interferente Pequeno/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Homeostase , Óperon , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.
Assuntos
Clostridioides difficile/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Mensageiro/genética , Clostridioides difficile/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , Ligação Proteica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , beta-Galactosidase/metabolismoRESUMO
Bacteria suffer various stresses in their unpredictable environment. In response, clonal populations may exhibit cell-to-cell variation, hypothetically to maximize their survival. The origins, propagation, and consequences of this variability remain poorly understood. Variability persists through cell division events, yet detailed lineage information for individual stress-response phenotypes is scarce. This work combines time-lapse microscopy and microfluidics to uniformly manipulate the environmental changes experienced by clonal bacteria. We quantify the growth rates and RpoH-driven heat-shock responses of individual Escherichia coli within their lineage context, stressed by low streptomycin concentrations. We observe an increased variation in phenotypes, as different as survival from death, that can be traced to asymmetric division events occurring prior to stress induction. Epigenetic inheritance contributes to the propagation of the observed phenotypic variation, resulting in three-fold increase of the RpoH-driven expression autocorrelation time following stress induction. We propose that the increased permeability of streptomycin-stressed cells serves as a positive feedback loop underlying this epigenetic effect. Our results suggest that stochasticity, pre-disposition, and epigenetic effects are at the source of stress-induced variability. Unlike in a bet-hedging strategy, we observe that cells with a higher investment in maintenance, measured as the basal RpoH transcriptional activity prior to antibiotic treatment, are more likely to give rise to stressed, frail progeny.
Assuntos
Epigênese Genética , Escherichia coli , Predisposição Genética para Doença , Resposta ao Choque Térmico , Divisão Celular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico , Análise de SobrevidaRESUMO
In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base-pairing with mRNA targets to affect protein translation or mRNA stability. In Escherichia coli, the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR, encoding a transcription regulator and ydaL, encoding a small MutS-related protein. We show that McaS is a â¼95nt transcript whose expression increases over growth, peaking in early-to-mid stationary phase, or when glucose is limiting. McaS uses three discrete single-stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly ß-1,6-N-acetyl-d-glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.
Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Flagelos/metabolismo , RNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , Transativadores/metabolismoRESUMO
Undernutrition affects about one out of five children worldwide. It is associated with impaired growth, neurodevelopment deficits, and increased infectious morbidity and mortality. Undernutrition, however, cannot be solely attributed to a lack of food or nutrient deficiency but rather results from a complex mix of biological and environmental factors. Recent research has shown that the gut microbiome is intimately involved in the metabolism of dietary components, in growth, in the training of the immune system, and in healthy development. In this review, we look at these features in the first three years of life, which is a critical window for both microbiome establishment and maturation and child development. We also discuss the potential of the microbiome in undernutrition interventions, which could increase efficacy and improve child health outcomes.
Assuntos
Microbioma Gastrointestinal , Desnutrição , Microbiota , Criança , Humanos , Desenvolvimento Infantil , DietaRESUMO
Escherichia coli synthesize over 60 poorly understood small proteins of less than 50 amino acids. A striking feature of these proteins is that 65% contain a predicted α-helical transmembrane (TM) domain. This prompted us to examine the localization, topology, and membrane insertion of the small proteins. Biochemical fractionation showed that, consistent with the predicted TM helix, the small proteins generally are most abundant in the inner membrane fraction. Examples of both N(in)-C(out) and N(out)-C(in) orientations were found in assays of topology-reporter fusions to representative small TM proteins. Interestingly, however, three of nine tested proteins display dual topology. Positive residues close to the transmembrane domains are conserved, and mutational analysis of one small protein, YohP, showed that the positive inside rule applies for single transmembrane domain proteins as has been observed for larger proteins. Finally, fractionation analysis of small protein localization in strains depleted of the Sec or YidC membrane insertion pathways uncovered differential requirements. Some small proteins appear to be affected by both Sec and YidC depletion, others showed more dependence on one or the other insertion pathway, whereas one protein was not affected by depletion of either Sec or YidC. Thus, despite their diminutive size, small proteins display considerable diversity in topology, biochemical features, and insertion pathways.
Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologiaRESUMO
The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18- to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.
Assuntos
Escherichia coli/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/genética , Sequência de Bases , Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Potenciais da Membrana , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Transformação BacterianaRESUMO
Historically, small proteins (sproteins) of less than 50 amino acids, in their final processed forms or genetically encoded as such, have been understudied. However, both serendipity and more recent focused efforts have led to the identification of a number of new sproteins in both Gram-negative and Gram-positive bacteria. Increasing evidence demonstrates that sproteins participate in a wide array of cellular processes and exhibit great diversity in their mechanisms of action, yet general principles of sprotein function are emerging. This review highlights examples of sproteins that participate in cell signaling, act as antibiotics and toxins, and serve as structural proteins. We also describe roles for sproteins in detecting and altering membrane features, acting as chaperones, and regulating the functions of larger proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Transdução de SinaisRESUMO
There has been considerable investigation into the survival of bacterial cells under stress conditions, but little is known about the causes of mortality in the absence of exogenous stress. That there is a basal frequency of cell death in such populations may reflect that it is either impossible to avoid all lethal events, or alternatively, that it is too costly. Here, through a genetic screen in the model organism Escherichia coli, we identify two mutants with lower frequencies of mortality: rssB and fliA. Intriguingly, these two genes both affect the levels of different sigma factors within the cell. The rssB mutant displays enhanced resistance to multiple external stresses, possibly indicating that the cell gains its increased vitality through elevated resistance to spontaneous, endogenous stresses. The loss of fliA does not result in elevated stress resistance; rather, its survival is apparently due to a decreased physical stress linked to the insertion of the flagellum through the membrane and energy saved through the loss of the motor proteins. The identification of these two mutants implies that reducing mortality is not impossible; rather, due to its cost, it is subject to trade-offs with other traits that contribute to the competitive success of the organism.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Viabilidade Microbiana , Mutagênese Insercional , Fator sigma/genética , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Pressão Osmótica , Fenótipo , Fator sigma/metabolismo , Fator sigma/fisiologia , Fatores de Transcrição/fisiologia , Transposases/genéticaRESUMO
In the frame of a survey of potentially endophytic N2-fixing Burkholderia associated with maize in Mexico, its country of origin, the soil of an indigenous maize field near Oaxaca was studied. Under laboratory conditions, plant seedlings of two ancient maize varieties were used as a trap to select endophyte candidates from the soil sample. Among the N2 fixers isolated from inside plant tissues and able to grow on PCAT medium, the most abundant isolates belonged to genus Burkholderia (API 20NE, rrs sequences). Representative isolates obtained from roots and shoots of different plants appeared identical (rrs and nifH RFLP), showing that they were closely related. In addition, their 16S rDNA sequences differed from described Burkholderia species and, phylogenetically, they constituted a separate deep-branching new lineage in genus Burkholderia. This indicated that these isolates probably constituted a new species. An inoculation experiment confirmed that these N2-fixing Burkholderia isolates could densely colonize the plant tissues of maize. More isolates of this group were subsequently obtained from field-grown maize and teosinte plants. It was hypothesized that strains of this species had developed a sort of primitive symbiosis with one of their host plants, teosinte, which persisted during the domestication of teosinte into maize.