Estimating mutation rates under heterogeneous stress responses.

Exposure to environmental stressors, including certain antibiotics, induces stress responses in bacteria. Some of these responses increase mutagenesis and thus potentially accelerate resistance evolution. Many studies report increased mutation rates under stress, often using the standard experimental approach of fluctuation assays. However, single-cell studies have revealed that many stress responses are heterogeneously expressed in bacterial populations, which existing estimation methods have not yet addressed. We develop a population dynamic model that considers heterogeneous stress responses (subpopulations of cells with the response off or on) that impact both mutation rate and cell division rate, inspired by the DNA-damage response in Escherichia coli (SOS response). We derive the mutant count distribution arising in fluctuation assays under this model and then implement maximum likelihood estimation of the mutation-rate increase specifically associated with the expression of the stress response. Using simulated mutant count data, we show that our inference method allows for accurate and precise estimation of the mutation-rate increase, provided that this increase is sufficiently large and the induction of the response also reduces the division rate. Moreover, we find that in many cases, either heterogeneity in stress responses or mutant fitness costs could explain similar patterns in fluctuation assay data, suggesting that separate experiments would be required to identify the true underlying process. In cases where stress responses and mutation rates are heterogeneous, current methods still correctly infer the effective increase in population mean mutation rate, but we provide a novel method to infer distinct stress-induced mutation rates, which could be important for parameterising evolutionary models.

Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance.

Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated Epe1 (tEpe1) which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non-cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell integrity MAP kinase stress signaling pathway, mutations in which alter resistance. Thus, environmental changes elicit a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop-plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered.

Growth-dependent heterogeneity in the DNA damage response in Escherichia coli.

In natural environments, bacteria are frequently exposed to sub-lethal levels of DNA damage, which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria, which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub-lethal DNA damage in E. coli. We found heterogeneous expression of the SOS regulon at the single-cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS-induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. The result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrate how cell division and physiology come together to produce growth-dependent heterogeneity in the DNA damage response.

Future Trends in Synthetic Biology-A Report.

Leading researchers working on synthetic biology and its applications gathered at the University of Edinburgh in May 2018 to discuss the latest challenges and opportunities in the field. In addition to the potential socio-economic benefits of synthetic biology, they also examined the ethics and security risks arising from the development of these technologies. Speakers from industry, academia and not-for-profit organizations presented their vision for the future of the field and provided guidance to funding and regulatory bodies to ensure that synthetic biology research is carried out responsibly and can realize its full potential. This report aims to capture the collective views and recommendations that emerged from the discussions that took place. The meeting was held under the Chatham House Rule (i.e., a private invite-only meeting where comments can be freely used but not attributed) to promote open discussion; the findings and quotes included in the report are therefore not attributed to individuals. The goal of the meeting was to identify research priorities and bottlenecks. It also provided the opportunity to discuss how best to manage risk and earn public acceptance of this emerging and disruptive technology.

Rediscovering Bacteria through Single-Molecule Imaging in Living Cells.

Bacteria are microorganisms central to health and disease, serving as important model systems for our understanding of molecular mechanisms and for developing new methodologies and vehicles for biotechnology. In the past few years, our understanding of bacterial cell functions has been enhanced substantially by powerful single-molecule imaging techniques. Using single fluorescent molecules as a means of breaking the optical microscopy limit, we can now reach resolutions of ∼20 nm inside single living cells, a spatial domain previously accessible only by electron microscopy. One can follow a single bacterial protein complex as it performs its functions and directly observe intricate cellular structures as they move and reorganize during the cell cycle. This toolbox enables the use of in vivo quantitative biology by counting molecules, characterizing their intracellular location and mobility, and identifying functionally distinct molecular distributions. Crucially, this can all be achieved while imaging large populations of cells, thus offering detailed views of the heterogeneity in bacterial communities. Here, we examine how this new scientific domain was born and discuss examples of applications to bacterial cellular mechanisms as well as emerging trends and applications.

The processive kinetics of gene conversion in bacteria.

Gene conversion, non-reciprocal transfer from one homologous sequence to another, is a major force in evolutionary dynamics, promoting co-evolution in gene families and maintaining similarities between repeated genes. However, the properties of the transfer - where it initiates, how far it proceeds and how the resulting conversion tracts are affected by mismatch repair - are not well understood. Here, we use the duplicate tuf genes in Salmonella as a quantitatively tractable model system for gene conversion. We selected for conversion in multiple different positions of tuf, and examined the resulting distributions of conversion tracts in mismatch repair-deficient and mismatch repair-proficient strains. A simple stochastic model accounting for the essential steps of conversion showed excellent agreement with the data for all selection points using the same value of the conversion processivity, which is the only kinetic parameter of the model. The analysis suggests that gene conversion effectively initiates uniformly at any position within a tuf gene, and proceeds with an effectively uniform conversion processivity in either direction limited by the bounds of the gene.

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo.

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.

Co-evolution of segregation guide DNA motifs and the FtsK translocase in bacteria: identification of the atypical Lactococcus lactis KOPS motif.

Bacteria use the global bipolarization of their chromosomes into replichores to control the dynamics and segregation of their genome during the cell cycle. This involves the control of protein activities by recognition of specific short DNA motifs whose orientation along the chromosome is highly skewed. The KOPS motifs act in chromosome segregation by orienting the activity of the FtsK DNA translocase towards the terminal replichore junction. KOPS motifs have been identified in γ-Proteobacteria and in Bacillus subtilis as closely related G-rich octamers. We have identified the KOPS motif of Lactococcus lactis, a model bacteria of the Streptococcaceae family harbouring a compact and low GC% genome. This motif, 5'-GAAGAAG-3, was predicted in silico using the occurrence and skew characteristics of known KOPS motifs. We show that it is specifically recognized by L. lactis FtsK in vitro and controls its activity in vivo. L. lactis KOPS is thus an A-rich heptamer motif. Our results show that KOPS-controlled chromosome segregation is conserved in Streptococcaceae but that KOPS may show important variation in sequence and length between bacterial families. This suggests that FtsK adapts to its host genome by selecting motifs with convenient occurrence frequencies and orientation skews to orient its activity.

DNA motifs that sculpt the bacterial chromosome.

During the bacterial cell cycle, the processes of chromosome replication, DNA segregation, DNA repair and cell division are coordinated by precisely defined events. Tremendous progress has been made in recent years in identifying the mechanisms that underlie these processes. A striking feature common to these processes is that non-coding DNA motifs play a central part, thus 'sculpting' the bacterial chromosome. Here, we review the roles of these motifs in the mechanisms that ensure faithful transmission of genetic information to daughter cells. We show how their chromosomal distribution is crucial for their function and how it can be analysed quantitatively. Finally, the potential roles of these motifs in bacterial chromosome evolution are discussed.

Small variable segments constitute a major type of diversity of bacterial genomes at the species level.

BACKGROUND: Analysis of large scale diversity in bacterial genomes has mainly focused on elements such as pathogenicity islands, or more generally, genomic islands. These comprise numerous genes and confer important phenotypes, which are present or absent depending on strains. We report that despite this widely accepted notion, most diversity at the species level is composed of much smaller DNA segments, 20 to 500 bp in size, which we call microdiversity. RESULTS: We performed a systematic analysis of the variable segments detected by multiple whole genome alignments at the DNA level on three species for which the greatest number of genomes have been sequenced: Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes. Among the numerous sites of variability, 62 to 73% were loci of microdiversity, many of which were located within genes. They contribute to phenotypic variations, as 3 to 6% of all genes harbor microdiversity, and 1 to 9% of total genes are located downstream from a microdiversity locus. Microdiversity loci are particularly abundant in genes encoding membrane proteins. In-depth analysis of the E. coli alignments shows that most of the diversity does not correspond to known mobile or repeated elements, and it is likely that they were generated by illegitimate recombination. An intriguing class of microdiversity includes small blocks of highly diverged sequences, whose origin is discussed. CONCLUSIONS: This analysis uncovers the importance of this small-sized genome diversity, which we expect to be present in a wide range of bacteria, and possibly also in many eukaryotic genomes.

Inactivation of the Lactococcus lactis high-affinity phosphate transporter confers oxygen and thiol resistance and alters metal homeostasis.

Numerous strategies allowing bacteria to detect and respond to oxidative conditions depend on the cell redox state. Here we examined the ability of Lactococcus lactis to survive aerobically in the presence of the reducing agent dithiothreitol (DTT), which would be expected to modify the cell redox state and disable the oxidative stress response. DTT inhibited L. lactis growth at 37 degrees C in aerobic conditions, but not in anaerobiosis. Mutants selected as DTT resistant all mapped to the pstFEDCBA locus, encoding a high-affinity phosphate transporter. Transcription of pstFEDCBA and a downstream putative regulator of stress response, phoU, was deregulated in a pstA strain, but amounts of major oxidative stress proteins were unchanged. As metals participate in oxygen radical formation, we compared metal sensitivity of wild-type and pstA strains. The pstA mutant showed approximately 100-fold increased resistance to copper and zinc. Furthermore, copper or zinc addition exacerbated the sensitivity of a wild-type L. lactis strain to DTT. Inactivation of pstA conferred a more general resistance to oxidative stress, alleviating the oxygen- and thermo-sensitivity of a clpP mutant. This study establishes a role for the pst locus in metal homeostasis, suggesting that pst inactivation lowers intracellular reactivity of copper and zinc, which would limit bacterial sensitivity to oxygen.

A genomic distance based on MUM indicates discontinuity between most bacterial species and genera.

The fundamental unit of biological diversity is the species. However, a remarkable extent of intraspecies diversity in bacteria was discovered by genome sequencing, and it reveals the need to develop clear criteria to group strains within a species. Two main types of analyses used to quantify intraspecies variation at the genome level are the average nucleotide identity (ANI), which detects the DNA conservation of the core genome, and the DNA content, which calculates the proportion of DNA shared by two genomes. Both estimates are based on BLAST alignments for the definition of DNA sequences common to the genome pair. Interestingly, however, results using these methods on intraspecies pairs are not well correlated. This prompted us to develop a genomic-distance index taking into account both criteria of diversity, which are based on DNA maximal unique matches (MUM) shared by two genomes. The values, called MUMi, for MUM index, correlate better with the ANI than with the DNA content. Moreover, the MUMi groups strains in a way that is congruent with routinely used multilocus sequence-typing trees, as well as with ANI-based trees. We used the MUMi to determine the relatedness of all available genome pairs at the species and genus levels. Our analysis reveals a certain consistency in the current notion of bacterial species, in that the bulk of intraspecies and intragenus values are clearly separable. It also confirms that some species are much more diverse than most. As the MUMi is fast to calculate, it offers the possibility of measuring genome distances on the whole database of available genomes.

MOSAIC: an online database dedicated to the comparative genomics of bacterial strains at the intra-species level.

BACKGROUND: The recent availability of complete sequences for numerous closely related bacterial genomes opens up new challenges in comparative genomics. Several methods have been developed to align complete genomes at the nucleotide level but their use and the biological interpretation of results are not straightforward. It is therefore necessary to develop new resources to access, analyze, and visualize genome comparisons. DESCRIPTION: Here we present recent developments on MOSAIC, a generalist comparative bacterial genome database. This database provides the bacteriologist community with easy access to comparisons of complete bacterial genomes at the intra-species level. The strategy we developed for comparison allows us to define two types of regions in bacterial genomes: backbone segments (i.e., regions conserved in all compared strains) and variable segments (i.e., regions that are either specific to or variable in one of the aligned genomes). Definition of these segments at the nucleotide level allows precise comparative and evolutionary analyses of both coding and non-coding regions of bacterial genomes. Such work is easily performed using the MOSAIC Web interface, which allows browsing and graphical visualization of genome comparisons. CONCLUSION: The MOSAIC database now includes 493 pairwise comparisons and 35 multiple maximal comparisons representing 78 bacterial species. Genome conserved regions (backbones) and variable segments are presented in various formats for further analysis. A graphical interface allows visualization of aligned genomes and functional annotations. The MOSAIC database is available online at http://genome.jouy.inra.fr/mosaic.

The MatP/matS site-specific system organizes the terminus region of the E. coli chromosome into a macrodomain.

The organization of the Escherichia coli chromosome into insulated macrodomains influences the segregation of sister chromatids and the mobility of chromosomal DNA. Here, we report that organization of the Terminus region (Ter) into a macrodomain relies on the presence of a 13 bp motif called matS repeated 23 times in the 800-kb-long domain. matS sites are the main targets in the E. coli chromosome of a newly identified protein designated MatP. MatP accumulates in the cell as a discrete focus that colocalizes with the Ter macrodomain. The effects of MatP inactivation reveal its role as main organizer of the Ter macrodomain: in the absence of MatP, DNA is less compacted, the mobility of markers is increased, and segregation of Ter macrodomain occurs early in the cell cycle. Our results indicate that a specific organizational system is required in the Terminus region for bacterial chromosome management during the cell cycle.

FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae.

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

Identification of DNA motifs implicated in maintenance of bacterial core genomes by predictive modeling.

Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the "crossover hotspot instigator," or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions.

KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase.

Bacterial chromosomes are organized in replichores of opposite sequence polarity. This conserved feature suggests a role in chromosome dynamics. Indeed, sequence polarity controls resolution of chromosome dimers in Escherichia coli. Chromosome dimers form by homologous recombination between sister chromosomes. They are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific chromosomal site, dif, and a DNA translocase, FtsK, which is anchored at the division septum and sorts chromosomal DNA to daughter cells. Evidences suggest that DNA motifs oriented from the replication origin towards dif provide FtsK with the necessary information to faithfully distribute chromosomal DNA to either side of the septum, thereby bringing the dif sites together at the end of this process. However, the nature of the DNA motifs acting as FtsK orienting polar sequences (KOPS) was unknown. Using genetics, bioinformatics and biochemistry, we have identified a family of DNA motifs in the E. coli chromosome with KOPS activity.