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1.
Microbiology (Reading) ; 168(4)2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35411846

RESUMO

Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in Escherichia coli and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNAFRT, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless E. coli strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNAFRT/Cas9 may be generally applicable to cure FRT scar-sites of E. coli strains constructed by classical λ-Red recombineering.


Assuntos
Sistemas CRISPR-Cas , Escherichia coli , DNA de Cadeia Simples , Escherichia coli/genética , Engenharia Genética/métodos
2.
Microbiology (Reading) ; 168(4)2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35471195

RESUMO

Complex carbohydrates shape the gut microbiota, and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analysed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (∆sus Bt) can grow within the halo. Furthermore, when these ∆sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.


Assuntos
Bacteroides thetaiotaomicron , Amilopectina , Bactérias/metabolismo , Bacteroides thetaiotaomicron/metabolismo , Glucose , Amido Resistente , Ruminococcus , Amido/metabolismo
3.
J Bacteriol ; 2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318048

RESUMO

PdeL is a transcription regulator and catalytically active c-di-GMP phosphodiesterases (PDE) in Escherichia coli PdeL has been shown to be a transcription autoregulator, while no other target genes have been identified so far. Here, we show that PdeL represses transcription of the flagella class II operon, fliFGHIJK, and activates sslE encoding an extracellular anchored metalloprotease, among additional loci. DNA-binding studies and expression analyses using plasmidic reporters suggest that regulation of the fliF and sslE promoters by PdeL is direct. Transcription repression of the fliFGHIJK operon, encoding protein required for assembly of the flagellar basal body, results in inhibition of motility on soft agar plates and reduction of flagella assembly, as shown by fluorescence staining of the flagella hook protein FlgE. PdeL-mediated repression of motility is independent of its phosphodiesterase activity. Thus, in motility control the transcription regulator function of PdeL reducing the number of assembled flagella is apparently epistatic to its phosphodiesterase function, which can indirectly promote the activity of the flagellar motor by lowering the c-di-GMP concentration.Bacteria adopt different lifestyles depending on their environment and physiological condition. In Escherichia coli and other enteric bacteria the transition between the motile and the sessile state is controlled at multiple levels from the regulation of gene expression to the modulation of various processes by the second messenger c-di-GMP as signaling molecule. The significance of our research is in identifying PdeL, a protein of dual function that hydrolyzes c-di-GMP and that regulates transcription of genes, as a repressor of Flagella gene expression and an inhibitor of motility, which adds an additional regulatory switch to the control of motility.

4.
Microbiology (Reading) ; 166(7): 624-628, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32416743

RESUMO

Unique morphologies can enable bacteria to survive in their native environment. Furthermore, many bacteria change their cell shape to adapt to different environmental conditions. For instance, some bacteria increase their surface area under carbon or nitrogen starvation. Bacteriodes thetaiotaomicron is an abundant human gut species; it efficiently degrades a number of carbohydrates and also supports the growth of other bacteria by breaking down complex polysaccharides. The gut provides a variable environment as nutrient availability is subject to the diet and health of the host, yet how gut bacteria adapt and change their morphologies under different nutrient conditions has not been studied. Here, for the first time, we report an elongated B. thetaiotaomicron morphology under sugar-limited conditions using live-cell imaging; this elongated morphology is enhanced in the presence of sodium bicarbonate. Similarly, we also observed that sodium bicarbonate produces an elongated-length phenotype in another Gram-negative gut bacterium, Escherichia coli. The increase in cell length might provide an adaptive advantage for cells to survive under nutrient-limited conditions.


Assuntos
Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Estresse Fisiológico , Açúcares/metabolismo , Bacteroides thetaiotaomicron/metabolismo , Escherichia coli/metabolismo , Trato Gastrointestinal/microbiologia , Humanos , Morfogênese , Fenótipo , Bicarbonato de Sódio/metabolismo
6.
Mol Microbiol ; 108(3): 226-239, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29424946

RESUMO

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/biossíntese , Sítios de Ligação/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/biossíntese , RNA Polimerases Dirigidas por DNA/metabolismo , Repressão Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Transcrição Gênica/fisiologia
7.
Methods Mol Biol ; 2819: 225-240, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028509

RESUMO

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.


Assuntos
Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli , Transcrição Gênica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Genes Reporter , Plasmídeos/genética , DNA Bacteriano/genética
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