Deletion of FRT-sites by no-SCAR recombineering in Escherichia coli.

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.

Ruminococcus bromii enables the growth of proximal Bacteroides thetaiotaomicron by releasing glucose during starch degradation.

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.

The transcription regulator and c-di-GMP phosphodiesterase PdeL represses motility in Escherichia coli.

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.

Nutrient-dependent morphological variability of Bacteroides thetaiotaomicron.

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.

Interference of transcription across H-NS binding sites and repression by H-NS.

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.

Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia coli.

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.

Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae.

The signaling molecule cyclic di-GMP (cdG) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of cdG have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of cdG are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize cdG to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyse cdG to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing cdG levels, we followed a timecourse of overexpression of either the V. harveyi diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases cdG levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on cdG levels plateaus at 30 minutes, indicating tight adaptive control of cdG levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to cdG, leads to higher baseline levels of cdG and continuously increasing cdG through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on cdG levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC, is activated by VpsR at high cdG concentrations, mediating this negative feedback on cdG levels. We further identify a transcript encoded within, and antisense to, the cdgC open reading frame which we name sRNA negative regulator of CdgC (SnrC). RNA polymerase ChIP-seq and RNA-seq demonstrate SnrC to be expressed specifically under conditions of high cdG in the absence of VpsR. Ectopic SnrC expression increases cdG levels in a manner dependent on CdgC, demonstrating that its effect on cdG levels is likely through interference with CdgC production. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of cdG to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.