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.

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.

The bacterial nucleoid is a highly organized and dynamic structure for simultaneous transcription, replication, and segregation of the bacterial genome. Compaction of the nucleoid and disturbance of DNA segregation and cell division by artificially high levels of transcription regulators, as described here, reveals that an excess of DNA-binding protein disturbs nucleoid structuring.

In enteric bacteria organization of the circular chromosomal DNA into a highly dynamic and toroidal shaped nucleoid involves various factors such as DNA supercoiling, nucleoid-associated proteins (NAPs), the structural maintenance of chromatin (SMC) complex, and macro-domain organizing proteins. Here we show that ectopic expression of transcription regulators at high levels leads to nucleoid compaction. This serendipitous result was obtained by fluorescence microscopy upon ectopic expression of the transcription regulator and phosphodiesterase PdeL of Escherichia coli of a strain expressing the mCherry-tagged HU-α subunit (HupA) for nucleoid staining. Nucleoid compaction by PdeL depends on DNA-binding, but not on its enzymatic phosphodiesterase activity. Nucleoid compaction was also observed upon high-level ectopic expression of the transcription regulators LacI, RutR, RcsB, LeuO and Cra, which range from single target gene regulators to global regulators. In case of LacI its high-level expression in presence of the gratuitous inducer IPTG also led to nucleoid compaction indicating that compaction is caused by unspecific DNA-binding. In all cases nucleoid compaction correlated with misplacement of the FtsZ ring and loss of MukB foci, a subunit of the SMC complex. Thus, high levels of several transcription regulators cause nucleoid compaction with consequences on transcription, replication and cell division.