Analysis tools for the interplay between genome layout and regulation

Genome layout and gene regulation appear to be interdependent. Understanding this interdependence is key to exploring the dynamic nature of chromosome conformation and to engineering functional genomes. Evidence for non-random genome layout, defined as the relative positioning of either co-functional or co-regulated genes, stems from two main approaches. Firstly, the analysis of contiguous genome segments across species, has highlighted the conservation of gene arrangement (synteny) along chromosomal regions. Secondly, the study of long-range interactions along a chromosome has emphasised regularities in the positioning of microbial genes that are co-regulated, co-expressed or evolutionarily correlated. While one-dimensional pattern analysis is a mature field, it is often powerless on biological datasets which tend to be incomplete, and partly incorrect. Moreover, there is a lack of comprehensive, user-friendly tools to systematically analyse, visualise, integrate and exploit regularities along genomes.Here we present the Genome REgulatory and Architecture Tools SCAN (GREAT:SCAN) software for the systematic study of the interplay between genome layout and gene expression regulation.SCAN is a collection of related and interconnected applications currently able to perform systematic analyses of genome regularities as well as to improve transcription factor binding sites (TFBS) and gene regulatory network predictions based on gene positional information.We demonstrate the capabilities of these tools by studying on one hand the regular patterns of genome layout in the major regulons of the bacterium Escherichia coli. On the other hand, we demonstrate the capabilities to improve TFBS prediction in microbes. Finally, we highlight, by visualisation of multivariate techniques, the interplay between position and sequence information for effective transcription regulation

Bacterial riboproteogenomics : the era of N-terminal proteoform existence revealed

With the rapid increase in the number of sequenced prokaryotic genomes, relying on automated gene annotation became a necessity. Multiple lines of evidence, however, suggest that current bacterial genome annotations may contain inconsistencies and are incomplete, even for so-called well-annotated genomes. We here discuss underexplored sources of protein diversity and new methodologies for high-throughput genome re-annotation. The expression of multiple molecular forms of proteins (proteoforms) from a single gene, particularly driven by alternative translation initiation, is gaining interest as a prominent contributor to bacterial protein diversity. In consequence, riboproteogenomic pipelines were proposed to comprehensively capture proteoform expression in prokaryotes by the complementary use of (positional) proteomics and the direct readout of translated genomic regions using ribosome profiling. To complement these discoveries, tailored strategies are required for the functional characterization of newly discovered bacterial proteoforms

Synthetic biology—putting engineering into biology

Synthetic biology is interpreted as the engineering-driven building of increasingly complex biological entities for novel applications. Encouraged by progress in the design of artificial gene networks, de novo DNA synthesis and protein engineering, we review the case for this emerging discipline. Key aspects of an engineering approach are purpose-orientation, deep insight into the underlying scientific principles, a hierarchy of abstraction including suitable interfaces between and within the levels of the hierarchy, standardization and the separation of design and fabrication. Synthetic biology investigates possibilities to implement these requirements into the process of engineering biological systems. This is illustrated on the DNA level by the implementation of engineering-inspired artificial operations such as toggle switching, oscillating or production of spatial patterns. On the protein level, the functionally self-contained domain structure of a number of proteins suggests possibilities for essentially Lego-like recombination which can be exploited for reprogramming DNA binding domain specificities or signaling pathways. Alternatively, computational design emerges to rationally reprogram enzyme function. Finally, the increasing facility of de novo DNA synthesis—synthetic biology’s system fabrication process—supplies the possibility to implement novel designs for ever more complex systems. Some of these elements have merged to realize the first tangible synthetic biology applications in the area of manufacturing of pharmaceutical compounds.

Implications of Rewiring Bacterial Quorum Sensing

Bacteria employ quorum sensing, a form of cell-cell communication, to sense changes in population density and regulate gene expression accordingly. This work investigated the rewiring of one quorum-sensing module, the lux circuit from the marine bacterium Vibrio fischeri. Steady-state experiments demonstrate that rewiring the network architecture of this module can yield graded, threshold, and bistable gene expression as predicted by a mathematical model. The experiments also show that the native lux operon is most consistent with a threshold, as opposed to a bistable, response. Each of the rewired networks yielded functional population sensors at biologically relevant conditions, suggesting that this operon is particularly robust. These findings (i) permit prediction of the behaviors of quorum-sensing operons in bacterial pathogens and (ii) facilitate forward engineering of synthetic gene circuits

Coexistence of different base periodicities in prokaryotic genomes as related to DNA curvature, supercoiling, and transcription

We analyzed the periodic patterns in E. coli promoters and compared the distributions of the corresponding patterns in promoters and in the complete genome to elucidate their function. Except the three-base periodicity, coincident with that in the coding regions and growing stronger in the region downstream from the transcriptions start (TS), all other salient periodicities are peaked upstream of TS. We found that helical periodicities with the lengths about B-helix pitch ~10.2-10.5 bp and A-helix pitch ~10.8-11.1 bp coexist in the genomic sequences. We mapped the distributions of stretches with A-, B-, and Z- like DNA periodicities onto E.coli genome. All three periodicities tend to concentrate within non-coding regions when their intensity becomes stronger and prevail in the promoter sequences. The comparison with available experimental data indicates that promoters with the most pronounced periodicities may be related to the supercoiling-sensitive genes.Comment: 23 pages, 6 figures, 2 table

Predicting Transcription Factor Specificity with All-Atom Models

The binding of a transcription factor (TF) to a DNA operator site can initiate or repress the expression of a gene. Computational prediction of sites recognized by a TF has traditionally relied upon knowledge of several cognate sites, rather than an ab initio approach. Here, we examine the possibility of using structure-based energy calculations that require no knowledge of bound sites but rather start with the structure of a protein-DNA complex. We study the PurR E. coli TF, and explore to which extent atomistic models of protein-DNA complexes can be used to distinguish between cognate and non-cognate DNA sites. Particular emphasis is placed on systematic evaluation of this approach by comparing its performance with bioinformatic methods, by testing it against random decoys and sites of homologous TFs. We also examine a set of experimental mutations in both DNA and the protein. Using our explicit estimates of energy, we show that the specificity for PurR is dominated by direct protein-DNA interactions, and weakly influenced by bending of DNA.Comment: 26 pages, 3 figure

The Integrity of the Cell Wall and Its Remodeling during Heterocyst Differentiation Are Regulated by Phylogenetically Conserved Small RNA Yfr1 in Nostoc sp. Strain PCC 7120

Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation.IMPORTANCE Bacterial small RNAs (sRNAs) are important players affecting the regulation of essentially every aspect of bacterial physiology. The cell wall is a highly dynamic structure that protects bacteria from their fluctuating environment. Cell envelope remodeling is particularly critical for bacteria that undergo differentiation processes, such as spore formation or differentiation of heterocysts. Heterocyst development involves the deposition of additional layers of glycolipids and polysaccharides outside the outer membrane. Here, we show that a cyanobacterial phylogenetically conserved small regulatory RNA, Yfr1, coordinates the expression of proteins involved in cell wall-related processes, including peptidoglycan metabolism and transport of different molecules, as well as expression of several proteins involved in heterocyst differentiation.España Ministerio de Educación, Cultura y Deporte (FPU014/05123 and EST16-00088)España Ministerio de Economía y Competitividad BFU2013-48282-C2-1España Agencia Estatal de Investigación (AEI), Ministerio de Economía, Industria y Competitividad, both cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER) BFU2016-74943-C2-1-