Phase variable expression of pdcB, a phosphodiesterase influences sporulation in Clostridioides difficile

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to hypervirulence strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovered that PdcB, a phosphodiesterase to influence sporulation in C. difficile UK1 strain positively. Through genetic and biochemical assays, we have shown that phase variable expression of pdcB results in hypo- and hyper-sporulation phenotype. In the ON orientation, the identified promotor is the right orientation to drive the expression of pdcB. Production of PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP concentration, resulting in hyper-sporulation phenotype. The OFF orientation of pdcB switch or mutating pdcB results in increased cyclic-di-GMP and hypo-sporulating phenotype. Additionally, we demonstrated that CodY binds to the upstream region of pdcB to represses its expression, and CodY mediated repression is relieved by the DNA inversion.

The Genome and Transcriptome Analysis of the Vigna mungo Chloroplast

Vigna mungo is cultivated in approximately 5 million hectares worldwide. The chloroplast genome of this species has not been previously reported. In this study, we sequenced the genome and transcriptome of the V. mungo chloroplast. We identified many positively selected genes in the photosynthetic pathway (e.g., rbcL, ndhF, and atpF) and RNA polymerase genes (e.g., rpoC2) from the comparison of the chloroplast genome of V. mungo, temperate legume species, and tropical legume species. Our transcriptome data from PacBio isoform sequencing showed that the 51-kb DNA inversion could affect the transcriptional regulation of accD polycistronic. Using Illumina deep RNA sequencing, we found RNA editing of clpP in the leaf, shoot, flower, fruit, and root tissues of V. mungo. We also found three G-to-A RNA editing events that change guanine to adenine in the transcripts transcribed from the adenine-rich regions of the ycf4 gene. The edited guanine bases were found particularly in the chloroplast genome of the Vigna species. These G-to-A RNA editing events were likely to provide a mechanism for correcting DNA base mutations. The V. mungo chloroplast genome sequence and the analysis results obtained in this study can apply to phylogenetic studies and chloroplast genome engineering.

Crystal structure of the nucleoid-associated protein Fis (PA4853) from Pseudomonas aeruginosa

Factor for inversion stimulation (Fis) is a versatile bacterial nucleoid-associated protein that can directly bind and bend DNA to influence DNA topology. It also plays crucial roles in regulating bacterial virulence factors and in optimizing bacterial adaptation to various environments. Fis from Pseudomonas aeruginosa (PA4853, referred to as PaFis) has recently been found to be required for virulence by regulating the expression of type III secretion system (T3SS) genes. PaFis can specifically bind to the promoter region of exsA, which functions as a T3SS master regulator, to regulate its expression and plays an essential role in transcription elongation from exsB to exsA. Here, the crystal structure of PaFis, which is composed of a four-helix bundle and forms a homodimer, is reported. PaFis shows remarkable structural similarities to the well studied Escherichia coli Fis (EcFis), including an N-terminal flexible loop and a C-terminal helix–turn–helix (HTH) motif. However, the critical residues for Hin-catalyzed DNA inversion in the N-terminal loop of EcFis are not conserved in PaFis and further studies are required to investigate its exact role. A gel-electrophoresis mobility-shift assay showed that PaFis can efficiently bind to the promoter region of exsA. Structure-based mutagenesis revealed that several conserved basic residues in the HTH motif play essential roles in DNA binding. These structural and biochemical studies may help in understanding the role of PaFis in the regulation of T3SS expression and in virulence.

Exploring the design space of recombinase logic circuits.

Logic circuits operating in living cells are generally built by mimicking electronic layouts, and scale-up is accomplished using additional layers of elementary logic gates like NOT and NOR gates. Recombinase-based logic, in which logic is implemented using DNA inversion or excision, allows for highly efficient, compact and single-layer design architectures. However, recombinase logic architectures depart from electronic design principles, and gate design performed empirically is challenging for an increasing number of inputs. Here we used a combinatorial approach to explore the design space of recombinase logic devices. We generated combinations and permutations of recombination sites, genes, and regulatory elements, for a total of ~19 million designs supporting the implementation of all 2- and 3-input logic functions and up to 92% of 4-input logic functions. We estimated the influence of different design constraints on the number of executable functions, and found that the use of DNA inversion and transcriptional terminators were key factors to implement the vast majority of logic functions. We provide a user-friendly interface, called RECOMBINATOR (http://recombinator.lirmm.fr/index.php), that enable users to navigate the design space of recombinase-based logic, find architectures implementing a specific logic function and sort them according to various biological criteria. Finally, we define a set of 16 architectures from which all 256 3-input logic functions can be derived. This work provides a theoretical foundation for the systematic exploration and design of single-layer recombinase logic devices.

Invertible promoters mediate bacterial phase variation, antibiotic resistance, and host adaptation in the gut

Phase variation, the reversible alternation between genetic states, enables infection by pathogens and colonization by commensals. However, the diversity of phase variation remains underexplored. We developed the PhaseFinder algorithm to quantify DNA inversion–mediated phase variation. A systematic search of 54,875 bacterial genomes identified 4686 intergenic invertible DNA regions (invertons), revealing an enrichment in host-associated bacteria. Invertons containing promoters often regulate extracellular products, underscoring the importance of surface diversity for gut colonization. We found invertons containing promoters regulating antibiotic resistance genes that shift to the ON orientation after antibiotic treatment in human metagenomic data and in vitro, thereby mitigating the cost of antibiotic resistance. We observed that the orientations of some invertons diverge after fecal microbiota transplant, potentially as a result of individual-specific selective forces.

Molecular Mechanisms ofhsdSInversions in thecodLocus ofStreptococcus pneumoniae

ABSTRACTStreptococcus pneumoniae(pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferasehsdSgenes (hsdSA,hsdSB, andhsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all threehsdSgenes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression ofhsdSAbut nothsdSBandhsdSC, indicating thathsdSBandhsdSCdo not produce functional proteins and instead act as sources for altering the sequence ofhsdSAby DNA inversions. Consistent with our previous finding that thehsdSinversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion betweenhsdSAandhsdSB. Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in thepsrAmutant. Surprisingly, PsrA-independent inversions were also detected in thehsdSsequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed thatS. pneumoniaevaries the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in thecodlocus.IMPORTANCEStreptococcus pneumoniaeis a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferasehsdSgenes of thecodlocus. This work revealed that only thehsdSgene that is in the same orientation ashsdMis actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of thehsdSinversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed thatS. pneumoniaeswitches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.

Mathematical model of a serine integrase-controlled toggle switch with a single input

Dual-state genetic switches that can change their state in response to input signals can be used in synthetic biology to encode memory and control gene expression. A transcriptional toggle switch (TTS), with two mutually repressing transcription regulators, was previously used for switching between two expression states. In other studies, serine integrases have been used to control DNA inversion switches that can alternate between two different states. Both of these switches use two different inputs to switch ON or OFF. Here, we use mathematical modelling to design a robust one-input binary switch, which combines a TTS with a DNA inversion switch. This combined circuit switches between the two states every time it receives a pulse of a single-input signal. The robustness of the switch is based on the bistability of its TTS, while integrase recombination allows single-input control. Unidirectional integrase-RDF-mediated recombination is provided by a recently developed integrase-RDF fusion protein. We show that the switch is stable against parameter variations and molecular noise, making it a promising candidate for further use as a basic element of binary counting devices.