Opening Session

The flagellar type 3 secretion-specificity switch

• Kelly Hughes (University of Utah)

Type 3 secretion (T3S) is the only secretion system in Biology known to switch from a given set of secretion substrates to a completely different set of protein secretion substrates. We devised a simple genetic selection that revealed the presence of a protein lock, a gatekeeper, that holds the apparatus into "early" secretion mode. Upon completion of an intermediate flagellum assembly structure, the hook-basal body (HBB), a secreted molecular ruler measures that the HBB has reached its terminal length of 80 nm. The C-terminus of the final secreting ruler performs protein intercourse with the gatekeeper that is holding the flagellar T3S apparatus in early substrate specific secretion mode. The result of this protein intercourse is the opening of the gatekeeper's hydrophobic core that allows it to escape from its held position, which results in the flipping the flagellar T3S-specificity switch.

Polar accumulation of pyoverdin and exit from stationary phase

• Clara Moreno-Fenoll (ESPCI Paris, Max Planck Institute for Evolutionary Biology)

Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus Pseudomonas and used for the acquisition of insoluble ferric iron. Although freely diffusible in aqueous environments, preferential dissemination of pyoverdin among adjacent cells, fine-tuning of intracellular siderophore concentrations, and fitness advantages to pyoverdin-producing versus nonproducing cells, indicate control of location and release. Here, using time-lapse fluorescence microscopy to track single cells in growing microcolonies of Pseudomonas fluorescens SBW25, we show accumulation of pyoverdin at cell poles. Accumulation occurs on cessation of cell growth, is achieved by cross-feeding in pyoverdin-nonproducing mutants and is reversible. Moreover, accumulation coincides with localization of a fluorescent periplasmic reporter, suggesting that pyoverdin accumulation at cell poles is part of the general cellular response to starvation. Compatible with this conclusion is absence of non-accumulating phenotypes in a range of pyoverdin mutants. Analysis of the performance of pyoverdin-producing and nonproducing cells under conditions promoting polar accumulation shows an advantage to accumulation on resumption of growth after stress. Examination of pyoverdin polar accumulation in a multispecies community and in a range of laboratory and natural species of Pseudomonas, including P. aeruginosa PAO1 and P. putida KT2440, confirms that the phenotype is characteristic of Pseudomonas.

Metabolic differentiation during endospore formation

• Javier Lopez-Garrido (MPI for Evolutionary Biology)

Bacillus subtilis sporulation entails a dramatic transformation of the two cells required to assemble a dormant spore, with the larger mother cell engulfing the smaller forespore to produce the cell-within-a-cell structure that is a hallmark of endospore formation. Sporulation also entails metabolic differentiation, whereby key metabolic enzymes are depleted from the forespore but maintained in the mother cell. This reduces the metabolic potential of the forespore, which becomes dependent on mother-cell metabolism and the SpoIIQ-SpoIIIA channel to obtain metabolic building blocks necessary for development. We demonstrate that metabolic differentiation depends on the ClpCP protease and a forespore-produced protein encoded by the yjbA gene, which we have renamed MdfA (metabolic differentiation factor A). MdfA is conserved in aerobic endospore-formers and required for spore resistance to hypochlorite. Using mass spectrometry and quantitative fluorescence microscopy, we show that MdfA mediates the depletion of dozens of metabolic enzymes and key transcription factors from the forespore. An accompanying study by Massoni, Evans and collaborators demonstrates that MdfA is a ClpC adaptor protein that directly interacts with and stimulates ClpCP activity. Together, these results document a developmentally-regulated proteolytic pathway that reshapes forespore metabolism, reinforces differentiation, and is required to produce spores resistant to the oxidant hypochlorite.

Genetic response of Achromobacter to phage therapy in the lungs of CF patients

• Anca Segall (San Diego State University)

Patients with cystic fibrosis (CF) face chronic antibiotic-resistant bacterial infections in their airways, lungs, other mucosal surfaces, and in some cases, bloodstream. Over the patients’ lifetime, these infections become exceedingly difficult to treat. Despite the profound benefits of corrector and modulator drugs that restore some function to the CFTR on quality of life and lifespan, ~10% patients have mutant channels that are not responsive to these drugs. Bacteriophage therapy (PT) in conjunction with antibiotics holds promise for these patients, despite PT lacking clear parameters such as phage doses, delivery, length of treatment, or interactions with antibiotics. Additionally, there are few if any clear-cut criteria for measuring success. We have safely treated with antibiotics and phages several CF patients with multi-drug resistant Achromobacter xylosoxidans infections; one of these patients was treated twice, ~2 years apart. Metagenomic analyses of sputum and blood samples showed that Achromobacter sequences decreased as much as 80% and remained reduced for months. During each round of therapy, some of the isolates became sensitive to antibiotics. No neutralizing antibodies were detected after the first round of therapy (the results from the second round are still being analyzed). Microbiological, genomic, and metagenomic data of Achromobacter strains isolated before, during, and after PT showed changes in both phage and antibiotic sensitivity. Some of the mutations correspond with those conferring phage resistance in vitro. These highlight the dynamics between bacterial fitness and the three-way interactions between the phages, the target pathogen, and antibiotics within human lungs.

Spore alanine racemases modulate germination in structured environments

• Alejandro Bonive Boscan

Endospores of multiple bacterial species carry alanine racemase on their envelopes, an enzyme that interconverts the germinant L-alanine and the germination inhibitor D-alanine1, 2. However, the role of alanine racemization in sporulation and spore ecology remains unclear. Here, we confirm that Bacillus subtilis spores possess two distinct alanine racemases3: AlrB, a sporulation-specific enzyme4, 5, and AlrA, the essential vegetative racemase that provides D-alanine for cell wall synthesis during growth. Both are recruited to the spore coat in a CotE-dependent manner and contribute to alanine racemization in purified spore preparations. Using cell-specific inducible protein degradation, we depleted both racemases during sporulation, bypassing the requirement for D-alanine supplementation during growth. Spores lacking both racemases developed normally, without premature germination within the mother cell, indicating that alanine racemization is not essential for sporulation in B. subtilis. However, these spores were insensitive to germination inhibition at high spore densities6. In structured environments within a microfluidic chamber, alanine racemization reduced germination in regions of high spore density. Wild-type spores exposed to intermittent L-alanine pulses remained dormant, whereas mutant spores incapable of racemizing alanine germinated under identical conditions. These findings suggest that alanine racemization enables B. subtilis spore populations to regulate germination, preventing it when the presence of L-alanine does not reliably indicate favorable conditions for vegetative growth.

Exploring the co-evolution of germinant selection and metabolism in endospore-forming bacteria

• Iqra Kasu (MPI for Evolutionary Biology)

Recent studies have demonstrated that the catabolism of germinant amino acids, such as alanine and valine, is crucial for preventing the premature germination of spores within their mother cells or shortly after their release into spent sporulation medium (1), (2). In Bacillus subtilis, mutants lacking alanine dehydrogenase (Ald) are unable to catabolize alanine, leading to its accumulation in the medium and triggering the premature germination of newly formed spores. This observation suggests that metabolism and germinant selection may co-evolve in sporulating bacteria—specifically, only molecules that can be efficiently catabolized and cleared from the environment function effectively as germinants. To explore this hypothesis, we implemented an experimental evolution strategy to isolate suppressor mutants that prevent the premature germination phenotype of Ald⁻ strains. The resulting suppressor mutations fall into two main categories: modifications in germinant receptors and alterations in proteins involved in spore coat assembly. These findings suggest that sporulating bacteria can mitigate the uncoupling of germinant catabolism and selection by either adjusting the affinity of germinant receptors for their ligands or modulating spore coat permeability to specific germinants. Ongoing experiments and analyses aim to elucidate these mechanisms further.

The fascinating complexity of synthetic lethality

• Andrei Kuzminov (University of Illinois at Urbana-Champaign)

As genetic analysis increasingly moves from one dimension (genotype-phenotype) to the second dimension (interactions between genotype-phenotype pairs) and even third dimension (additional mutations modifying the two-gene interactions), a better understanding of the complex effects is required to use this powerful tool intelligently. Of the four general types of genetic interactions (epistasis, additivity, synergy and compensation), synergy creates the most excitement — perhaps because it is both rare and unexpected, but also because of its dark, mysterious extreme. Indeed, the ultimate synergy yields synthetic lethality, or colethality — a combination of two otherwise modest-effect mutations that proves completely dead. Colethality is typically attributed to “inactivation of two redundant activities both contributing to the same essential function” — but it is rarely the case in real life. The major explanation of colethal combinations is “potentiation”, when one genetic defect greatly potentiates the problems of the second defect. We call these gene pairs “the avoidance-repair couples” around an essential cellular molecule or structure. And there is also a third explanation, “loss-of-support”, due to the complications coming from colethals in which one mutation is a hypomorph of an essential gene. One way to distinguish between the three explanation is via suppressors, which takes our genetic analysis to the third dimension.

Experimental evidence of loss of autoregulation in release factor

• Sungbin Lim (MPI for Evolutionary Biology)

Translation termination involves the recognition of stop codons by release factors (RFs). In bacteria, RF2 recognizes UGA and UAA stop codons and is encoded by the prfB gene. The translation of prfB is interrupted by an internal UGA stop codon on its coding sequence, resulting in negative autoregulation controlled by RF2 concentration. Complete translation can only occur when the internal stop codon is bypassed by a +1 programmed ribosomal frameshifting (PRF) event, which is more likely to occur when cellular RF2 level are low. In addition to RF2 concentration, frameshifting rate depends on conserved sequences near the internal stop codon, which interact with the ribosomal complex and facilitate frameshifting, thereby allowing sufficient expression of RF2. Although the PRF system is widely conserved across bacteria (PRF⁺ species), some bacterial species lack the internal stop codon in their prfB genes (PRF⁻ species) so that no frameshifting required while translation. It has been proposed that PRF loss may be beneficial in species with high UGA or UAA stop codon usage, but conclusive evidence is missing. In this study, we combine phylogenetic correlation analysis and experimental evolution to investigate the evolutionary pressures that lead to PRF loss. From phylogenetic analysis, loss of PRF did not correlate with increased usage of UGA or UAA stop codons, indicating that it is not driven by the genomic context. To further explore potential mechanisms of PRF loss, we constructed set of mutations in the sequences surrounding the internal stop codon of the prfB gene in Pseudomonas fluorescens SBW25 to disrupt its interaction with the ribosomal complex, leading to a reduced frameshifting rate. Using experimental evolution, we identified two compensatory strategies to overcome reduced frameshifting rate: (i) secondary mutations in ribosomal genes that can increase the frameshifting rate, and (ii) a single base pair deletion upstream of the internal stop codon, which shifts the reading frame and thereby bypasses the stop codon without recognition. These results demonstrate that the loss of PRF can occur as a consequence of compensatory evolution in response to deleterious mutations that reduce the frameshifting rate. This evolutionary trajectory aligns with the nearly neutral theory of molecular evolution, rather than with direct adaptive selection driven by stop codon usage.

Unravelling the hidden genetics of the air-liquid Interface colonization

• Gisela Rodríguez-Sánchez (MPI for Evolutionary Biology)

Natural selection is the non-random differential reproduction of individuals in a population. Selection occurs upon a pool of already exiting variation. Thus, the outcome of selection depends on available phenotypic variation. Although mutations in the genome are random, generation of variant types is not random because of constraints and redundancy. Therefore, from all possible genotypes, only a fraction of phenotypes is likely to be realized. The bacterium Pseudomonas fluorescens SBW25 is a good model to investigate the biases in the generation of variation and its effects on selection. In microcosms, bacterial growth in unshaken conditions results in depletion of oxygen in the liquid. Mutants that arise that are able to grow at the air-liquid interface (ALI), where Oxygen is available, are selected for. There are multiple ways by which SBW25 mutants can grow at the ALI. Overproduction of polymers is one, overproduction of proteinaceous fibers is another. We always observe mutants taking the first solution and, despite the enormous efforts on understanding the genetics of adaptation of SBW25 ALI colonizer mutants, we have never observed mutants with the second solution. In this work we used a combination of transcriptomics, functional genetics and experimental evolution to show how the proteinaceous fiber Fap, although playing a subtle role in the ALI colonization by the canonical mutants using the polymer solution, are themselves sufficient for the ALI colonization but never realized. We later revealed that fap regulation is tightly regulated and shared with polymers regulation, because its activation depends on the same second messenger that regulates polymers, c-di-GMP. The initial mutation required for the Fap phenotype is one that elevates c-di-GMP. This mutation also results in polymer activation, which allows mutants to grow in the ALI. Consequently, selection is reduced, and the other Fap-activating mutations become detrimental. Therefore, the Fap-phenotype is not independently (from the polymers) accessible by mutation and therefore never realized. These results underscore the complexity of adaptive evolution, which involves several layers of explanation, including available genome potential, gene regulation, fitness effects, and the likelihoods of alternative pathways. While some phenotypes are fit and possible solutions to the same problem, some will never be seen by selection.

Bacteriophage host range/Mapping the Microverse

• Bas E. Dutilh (Friedrich Schiller Universität Jena)

Experimental capture of genomic islands defines a widespread class of genetic element capable of non-autonomous transfer

• Yansong Zhao (MPI for Evolutionary Biology)

Bacteria acquire new genes by horizontal gene transfer (HGT). Acquisition is typically mediated by mobile genetic elements (MGEs), however, beyond plasmids, bacteriophages and certain integrative conjugative elements (ICEs), the nature and diversity of MGEs is poorly understood. The bacterium Pseudomonas fluorescens SBW25 was propagated by serial transfer in the presence of filtrate obtained from garden compost communities. Genome sequencing of derived colonies revealed acquisition of three different mobile elements, each integrated immediately downstream of tmRNA. All are flanked by direct repeats and harbour a tyrosine integrase (intY), followed by a cargo of accessory genes including putative phage defence systems. Although characteristic of genomic islands, MGE-classifiers showed no matches to mobile elements. Interrogation of DNA sequence databases showed that similar elements are widespread in the genus Pseudomonas and beyond, with Vibrio Pathogenicity Island-1 (VPI-1) from V. cholerae being a notable example. Bioinformatic analyses demonstrate frequent transfer among diverse hosts. With focus on a single 55 kb element (I55) we show that intY is necessary for excision and circularisation, that the element is incapable of autonomous horizontal transfer, but is mobilizable – in the absence of direct cell-cell contact – upon addition of community filtrate. Recent evidence from filtrate sequencing suggests that the transfer is mediated by a jumbo phage. Further analyses demonstrate that I55 enhances host fitness in the presence of community filtrate, which stems in part from its ability, equipped by a type II restriction-modification system encoded by I55, to defend against another phage in the filtrate. Together, this work demonstrates the potential for real-time evolution experiments in capturing and characterising mobile genetic elements with phage defence activity.

The role of mobile genetic elements in community dynamics

• Catriona Thompson (John Innes Centre)

Successful recruitment, colonisation and stability of plant-associated microbial communities depends on the microbes’ ability to successfully interpret and appropriately respond to environmental signals. We have made considerable progress towards understanding soil community signalling and plant colonisation. However, the central role of mobile genetic elements (MGEs) – bacteriophages, plasmids, and integrative conjugative elements- within the rhizosphere community, and how they interact as part of the wider mobilome remains poorly understood. MGEs disseminate genetic information, including ecologically important traits such as virulence and metabolic competence across large phylogenetic distances, and disrupt both regulatory networks and niche preferences. Therefore, understanding how they influence rhizosphere microbiome assembly and maintenance is paramount for advancing plant health. My work aims to understand how the mobilome shapes microbial interactions within the rhizosphere, the role of the plant in shaping the mobilome, and how different MGEs influence each other. I have recently shown that MGEs alter bacterial behaviour and rhizosphere colonisation at a systemic level. Accessory genes, frequently encoded on plasmids, are able to alter bacterial behaviour by hijacking key regulatory pathways that are important for rhizosphere success. These genes encode diverse signalling proteins that can cause widespread regulatory impacts, that ultimately lead to changes in bacterial social behaviours in keystone species. In an environment where community instability and niche collapse can have devastating consequences, understanding the role of MGEs, and the regulators which they encode is paramount. In this talk I will discuss how I am using model wheat-P. fluorescens-pQBR103 system, in combination with synthetic, plant specific communities, to unpick the role of plasmids and their encoded regulators on the dynamics and community ecology of the rhizosphere.

The Microbial Proteome Allocation Concept

• Alfred Spormann (Stanford University)

Microbial proteome allocation has been introduced as a useful concept to rationalize metabolic switching and ribosome biology in fast microorganisms such as E. coli. When utilizing aerobically glucose at higher growth rates, E. coli initiates an overflow metabolism resulting in inefficient glucose utilization to accommodate the increased demand for ribosomes in the cellular proteome. Our work in CO2-reducing methanogens and acetogens, however, reveals a fundamentally different picture: Proteomic resource allocation plays only a minor role when growth rates change two orders of magnitude. In contrast, posttranslational regulation of metabolism is turning out to be a key aspect of microbial acclimation to different growth rates. Ribosomes operate over a much larger ranges of translation rates than in E. coli, and at low growth rates are limited by rRNA and not ribosomal protein. A refined model for proteome allocation is presented for chemolithoautotrophs.

Special Talk - The bacterial nucleoid

• Andrei Kuzminov

rRNA operon redundancy as a bacterial genome stability insurance policy

• Ivan Matic (Institut Cochin, Paris)

The ability to modulate translation capacity, which resides greatly on a number of ribosomes, provides robustness in fluctuating environments. Because translation is energetically the most expensive process in cells, cells must constantly adapt the rate of ribosome production to resource availability. This is primarily achieved by regulating ribosomal RNA (rRNA) synthesis, to which ribosomal proteins synthesis is adjusted. The multiplicity of rRNA encoding operons per bacterial genome exceeds requirements for the maximal growth rates in non-stress conditions. We will provide evidence that a major function of rRNA operon multiplicity is to ensure that individual operons are not saturated by RNA polymerases during adaptation to environmental fluctuations, which can result in catastrophic chromosome replication failure and cell death.

Investigating the genome dynamics of Saccharomyces interspecific hybridization

• Danielle Adekunle (IRCAN – Institute for Research on Cancer and Aging, Nice, University of Nice)

Hybridization has shaped the evolutionary history of every kingdom of life. Hybridization inherently forces genomes that are not optimized to function together into the same cell. Remarkably, despite in some cases extreme sequence divergence upwards of 30%, cells can survive this challenge and even exploit it to rapidly evolve to new environments and create novelty. However, much of what we know of hybrids derives from extant hybrids, thus how populations resolve the challenges of hybridization and the genome dynamics of this process are still not fully understood. Taking advantage of Saccharomyces yeast as a model we generated hybrids ranging from 0.5 to approximately 40% parental sequence divergence. We used two approaches to do so, one being a single-cell mating approach which reduces competition and selection and has the advantage of allowing for lineage tracking, the other being a standard mass mating approach which is characterized by high selection, to generate all crosses. We found, using a heat shock protein reporter, that hybridization initially creates an acute stress response that is later attenuated. We subjected our panel of hybrids to genome sequencing and found that hybrids across a range of sequence divergences rapidly fixed aneuploidies and loss of heterozygosity. Interestingly, genome instability was not restricted to nuclear genomes but extrachromosomal sequences and mitochondrial genomes both exhibited copy number variations across hybrid lineages. Through both Hi-C and genome sequencing we strikingly observed convergent evolution around spontaneous polyploidization events in various hybrid lineages across a range of sequence divergences of parental founders when natural selection was minimized and maximized. A number of these polyploidization events perfectly recapitulated polyploidization events that have been identified by similar extant hybrids in the wild. Ongoing investigation is being conducted to track the timing, the frequency, the level of convergence across lineages, and the persistence of these genomic changes over evolutionary time in various environments. Thus far, our results provide unique empirical insights into the evolutionary dynamics and impacts of hybridization and identify a multitude of genomic responses and when they arise in hybrid evolutionary trajectories.

How plasmid-borne regulatory proteins influence bacterial behaviour

• Jacob Malone (John Innes Centre)

Horizontal gene transfer (HGT) is a fundamental process in bacterial evolution and is typically driven by mobile genetic elements, with conjugative plasmids one of the most significant sources of bacterial HGT. In addition to functional traits such as antibiotic resistance, plasmids also frequently encode homologs of bacterial regulators that can dramatically change the expression of chromosomally-encoded bacterial traits, a process known as plasmid-chromosome crosstalk (PCC). We recently characterised two PCC regulators from the megaplasmid pQBR103 that subvert ecologically relevant pathways in the soil bacterium Pseudomonas fluorescens. These plasmid-borne regulators appear similar to host proteins, but act in strikingly different ways, subverting host regulation and behaviour to benefit plasmid transmission. For example, RsmQ is a homolog of the bacterial translational regulator RsmA that disrupts the host Gac/Rsm pathway, influencing chemotaxis, metabolism and plasmid conjugation rate. Meanwhile, the plasmid-borne ParB-mimic ParQ functions as a transcriptional regulator of bacterial motility and chemotaxis. The RsmQ and ParQ regulons interact with one another and with the host cyclic-di-GMP network, with deletions in specific diguanylate cyclase genes recovering motility defects associated with plasmid carriage. pQBR103 apparently contains multiple putative PCC regulatory proteins, whose function is under active investigation in my lab. The intersection of plasmid and bacterial signalling driven by PCC regulators promotes plant colonisation, sessility and biofilm formation, traits that in turn support community transmission of the pQBR103 plasmid. Together, these findings expand our knowledge of the regulatory mechanisms underpinning HGT, and have broad implications for our understanding of how plasmids influence microbial communities.

Genetic Battles on the X chromosome

• Rajalekshmi Narayana Sarma (MPI for Evolutionary Biology)

Reproductive isolation is central to speciation, and hybrid sterility in Mus musculus subspecies provides a powerful model to study its genetic basis. A key player in this process is the HstX2 locus on the X chromosome, which in tandem with the only known hybrid sterility gene in vertebrates PRDM9, intriguingly regulates both meiotic recombination rate variation and hybrid male sterility, two interconnected processes that shape genome evolution and reproductive barriers. This system follows classical models of hybrid sterility: 1. Dobzhansky-Muller Incompatibilities (DMIs): Independent evolution of genetic elements in different subspecies leads to incompatibilities in hybrids, triggering sterility through genetic conflict. 2. Haldane’s Rule & Dominance Theory: Hybrid sterility disproportionately affects the heterogametic sex (XY males in mammals), as recessive incompatibilities on the X chromosome are fully expressed. 3. The Large-X Effect & Faster-X Hypothesis: The X chromosome evolves rapidly, accumulating genetic changes that drive hybrid sterility and recombination divergence. At the core of HstX2 is a cluster of microRNAs, the SpermiRs, which evolve through copy number variation and single nucleotide polymorphisms. These genetic changes modulate hybrid sterility in a dosage-dependent manner and modulate recombination rate by differential interaction with target proteins respectively, suggesting a shared regulatory mechanism between these two evolutionary forces. By dissecting the evolution of this locus across Mus musculus subspecies, we gain insight into how genetic conflict, epigenetic regulation, and recombination dynamics interact to drive postzygotic isolation.

The extreme mutational hotspot in the rpoS promoter: causes and consequences

• Andrew Farr (MPI for Evolutionary Biology)

Mutation rates vary within genomes, leading to biases in the genetic change that fuels evolution. Some regions have extreme rates of mutation and are referred to as mutational ‘hotspots’. Understanding the causes of hotspots are important if evolutionary biology is to become a more predictive science. We have taken advantage of a chance observation to show that genetic regulation can influence mutation rates inside a promoter. While performing experiments with the bacterium Pseudomonas fluorescens, we observed near deterministic levels of parallel evolution caused by an identical point mutation inside the highly conserved promoter of rpoS. We have shown this promoter has a mutational hotspot, with transcriptional regulatory proteins required for maximisation of mutation rate (~5000-fold above expectation). This talk will present evidence for this transcription-dependent hotspot, explore the conditions under which similar hotspots might appear in other promoters and organisms, and discusses the ecological and evolutionary consequences of promoter-located hotspots.

Special Session: In memoriam We will honor the memory of our dear friends and mentors, Pepe Casadesús and Rolf Menzel. Talk: Roberto Balbontin, The legacy of Pepe Casadesús

Prophage warfare in Salmonella

• Lionello Bossi (University Paris-Saclay)

Free-living bacteria often harbor multiple prophages integrated into their genomes, where these elements can cooperate to provide beneficial functions to their host. However, the peaceful coexistence among prophages ends if the host experiences genotoxic stress or other conditions that trigger the phage lytic cycle. Upon induction, prophages enter a competitive state. Phage competition comes in different flavors, but a common denominator is the struggle for cellular resources. The nature of the resource(s) that become rate-limiting for viral reproduction has remained elusive. I will present and discuss evidence that prophages begin competing even before excising from the chromosome, with the primary goal being the highjacking of the host’s DNA replication machinery.

New insights in phage P22-mediated transduction dynamics

• Kevin Broux (KU Leuven)

Generalized transduction has been intensively studied for its driving role in bacterial evolution and the spread of antibiotic resistance. Studying P22 packaging preferences with genetics and sequencing allowed us to genetically engineer a P22-based transduction platform that enables live scrutiny of transduction events at single-cell resolution with time-lapse fluorescence microscopy. This not only allowed us to compare successful and failed/aborted transduction events, but also to study some of the early-discovered transduction-affected P22 mutants. In fact, synthetic reconstruction of such spontaneous mutants lead to new insights into the role of P22 ejection proteins.

Phage Mu Utilizes the Sliding Clamp for Late Gene Transcription

• Rasika Harshey (University of Texas at Austin)

MuC, along with core RNAP and σ70, is required for transcription of late genes of phage Mu. We observed that overexpression of MuC is lethal in E. coli and that host replication was over-initiating under these conditions. Suppressors of MuC lethality occurred in dnaA, diaA, and dnaX, genes involved in initiation of DNA replication. We hypothesized that lethality was likely due to increase in ATP-DnaA levels, which are normally kept in check by Hda-DnaN (β clamp). We noticed that MuC harbors a well-defined motif for interaction with the β clamp. We provide experimental support for this interaction and show that disrupting it through mutations in the clamp-binding motif on MuC, expressed from both plasmid and the phage genome, abrogated transcription from a MuC-dependent promoter as well as production of viable phage, respectively. Furthermore, we show that inactivation of β-clamp specifically inactivates C-dependent transcription but not σ70-dependent transcription. We conclude that MuC requires the  sliding clamp for transcribing late phage genes. To the best of our knowledge, this is the first demonstration of the involvement of the clamp, a processivity factor essential for DNA replication, for transcription by E. coli RNAP.

The extended life cycle of phage λ

• Abram Aertsen (KU Leuven)

The life of phage Lambda (λ) has been extensively studied and has led to the current paradigmatic bifurcation of temperate phage life cycles along either lytic or lysogenic reproduction strategies. However, infection dynamics have long been studied using classical plating techniques that inevitably capture the more stable infection outcomes and tend to neglect the more transient phage-host interactions. Using genetics and live cell biology, we scrutinized λ infection dynamics under host scarcity conditions and observed an unexpected heterogeneity of λ-host-interactions in which episomal states tend to defy stable vertical inheritance. Both experiments and modelling reveal that these states profoundly extend the textbook life cycle of this model phage and complicate its longer-term infection dynamics.

Experimental evolution strategies to modulate bacteriophage life-history traits

• Manuela Reuter (MPI for Evolutionary Biology)

Experimental evolution strategies to modulate bacteriophage life-history traits Manuela Reuter, Michael Sieber1, Octavio Reyes-Matte1, Christina Vasileiou1, Jordan Romeyer-Dherbey2, Christopher Böhmker1, Javier Lopez-Garrido1, Frederic Bertels1 1Max Planck Institute for Evolutionary Biology, Plön, Germany 2University of Cambridge, Cambridge, United Kingdom Bacteriophage’s life-history traits such as the rate of attachment to the host, the duration of the infection cycle, the number of progeny produced per infected cell, and the ability to persist in the environment determine whether a phage infection will be productive. Here, we show that subtle differences in transfer times during serial passaging can select for drastically different phage life-history trait phenotypes. Long transfer times led to highly persistent phages with a high adsorption rate, while short transfers selected for slow adsorbing mutants with low persistence. Simulations of both transfer regimes predicted that slow adsorption alone could explain the evolution of the short-transfer mutant on the observed time scale, while selection on persistence and adsorption was required to predict the emergence of long transfer mutants. Changes in adsorption and persistence were caused by a single-point mutation in the major capsid protein. The proximity of these very different phenotypes in genotypic space may be the result of frequent adaptation to different environments that select for life history traits associated with a specific plaque phenotype. Our results demonstrate that evolution experiments can be used to efficiently switch between these phenotypes, which may also be important for improving the efficacy of therapeutic phages.

Discovering new phage genome variants

• Rob Edwards (Flinders University)

We resolved hundreds of bacteriophage genomes into distinct structural components through high-resolution assembly graph analysis of metagenomic datasets. We uncovered extensive genomic variation and quasispecies dynamics. Our analyses revealed a striking anti-correlation between genome plasticity and dominance, suggesting that phage populations balance genetic variability and selective advantage. We provide new insights into how phage populations diversify and adapt by integrating assembly graphs, gene annotations, and ANI comparisons across clinical (IBD) and environmental (Tara Oceans) samples.

With a little help from a friend: How Rho factor supports H-NS in gene silencing.

• Nara Figueroa-Bossi (University of Paris-Saclay)

The nucleoid-structuring protein H-NS silences segments of the enterobacterial genomes by binding to AT-rich nucleation sites and forming nucleoprotein filaments that block transcription over extended regions. H-NS:DNA filaments assemble on negatively supercoiled DNA and are stabilized by this DNA topology. In Salmonella, H-NS silences all major pathogenicity islands when the bacteria are outside their hosts. Given that H-NS is largely thought to obstruct RNA polymerase (RNAP) access to promoters, we were surprised to find that impairing Rho-dependent transcription termination leads to the constitutive activation of Salmonella pathogenicity islands (SPIs). How could termination play a role in regions that are not supposed to be transcribed? Our work on Salmonella pathogenicity island 1 (SPI-1) revealed that while H-NS efficiently represses bona fide promoters, it does not completely prevent RNAP from binding to intragenic promoter-like sequences that arise at high frequency in AT-rich regions. This is where Rho factor comes into play. Aided (or recruited) by the transcription elongation factor NusG, Rho terminates transcription originating from these spurious promoters. This function is critical for maintaining H-NS-mediated SPI-1 silencing. When Rho or NusG is defective, spurious transcripts elongate, generating positive DNA supercoiling. This supercoiling propagates and invades the negatively supercoiled DNA:H-NS filament. As positive and negative supercoils neutralize each other, the loss of negative superhelicity destabilizes the H-NS:DNA complex, leading to filament disassembly. This disruption grants RNAP access to bona fide promoters, triggering a feedforward cascade that ultimately activates the entire 48-Kb island.

Regulatory adaptation to low-iron growth conditions in Staphylococcus aureus

• Philippe Bouloc

We previously designed a competition assay with a collection of Staphylococcus aureus sRNA mutants to identify growth defects/advantages associated with sRNAs. We applied this assay to various conditions, including growth with a sublethal concentration of antibiotics, resulting in the identification of sRNAs associated with antibiotic tolerance. By subjecting the library to a medium containing sequestered iron, we identified IsrR-sRNA as being required for optimal growth when iron is unavailable. IsrR shows functional similarities to E. coli/Salmonella RyhB sRNA. It down-regulates the translation of numerous mRNAs encoding iron-containing proteins. These include the citB mRNA encoding aconitase and the ccpE mRNA encoding the aconitase transcriptional activator. IsrR is the master regulator of a feedback loop controlling aconitase levels. Moreover, in an iron-depleted environment, aconitase, a moonlighting protein (as reported in other species), becomes an RNA-binding protein, ensuring its autoregulation and controlling other genes. Our results highlight complex regulations for adapting to iron shortage, a condition faced by a pathogen within the host. A part of these results was recently published. I will present the continuation of this work with the characterization of IsrR and aconitase targets, and the contribution of moonlighting proteins to bacterial adaptation to growth conditions.

Bacterial insertion sequences as agents of fitness and evolvability

• Roderich Römhild (IST Austria)

Insertion sequence elements (ISEs) are small integrative genetic elements and abundant in bacterial genomes. ISE integration is a frequent cause of both pseudogenisation and promoter capture. Additionally, ISEs are recombination hotspots that stimulate spontaneous gene duplications amplifications and deletions. To study the hypothesised key role of ISEs in bacterial gene expression flexibility and evolvability, we deleted the entire set of 45 ISEs from the E. coli chromosome. These 45 ISEs belong to different families (e.g. 7x IS1, 11x IS5, 7x IS3, 5x IS2, 3x IS186 etc) with varying genetic mobility, target preference and host control. I will present data from genetic analyses, evolution and competition experiments that indicate a central importance of ISEs as agents of genome plasticity and fitness in bacteria.

Round Table: The future of analytical genetics

Interactions of mucus monosaccharides and the epidermal microbiome in four benthic Elasmobranchs

• Elizabeth Dinsdale (Flinders University)

Interactions between microbes and vertebrate host is hypothesized to be regulated through the mucus, therefore, host associated microbiomes will have genes to utilize the carbohydrates that are produced in the host mucus. Therefore, we quantified the carbohydrate (monosaccharide) composition of the mucus from four sharks and rays (Elasmobranchii) and investigate the genomic machinery of the microbiome. Elasmobranchii had low amounts of mucus and low proportion of carbohydrates (<10 %) compared with other marine organisms. Four key monosaccharides; glucose, glucosamine, galactose, and fucose, were identified in mucus samples. Hosts exhibited distinct, species-specific monosaccharide signatures. We identified key carbohydrate microbial genes from host and water microbiomes. Elasmobranch microbiomes had a higher relative abundance of carbon utilization genes compared to the water column microbiome and contained gene pathways for the utilization specific monosaccharides found in host mucus, suggesting that the host mucus was a regulator of the microbiome. Elasmobranch epidermal microbiomes had the genetic machinery required for detecting, transporting, and metabolizing monosaccharides and other carbohydrates present in the host mucus, demonstrating the selective nature of Elasmobranch epidermal mucus.

Combining Genetics and Pharmacology to understand chronic disease

• Baldomero Olivera (University of Utah)

Chronic disease states are the most challenging to address, in part because they are not understood at a basic mechanistic level. Genetics has been a powerful tool to understanding the most intractable pathologies, but I will discuss why combining genetics with pharmacology is essential, using chronic pain as one specific example.

Taking control of Staphylococcus aureus phospholipids shows how extreme lipid changes affect growth, cell division, and antibiotic resistance.

• Alexandra Gruss (Micalis - Univ Paris-Saclay, INRAE, Jouy en Josas)

Fatty acids determine structural flexibility and thickness of membrane phospholipids, and changes in their composition necessarily affect cell function. While fatty acid composition would expectedly be strictly controlled, it is not: various cells incorporate environmental fatty acids directly into phospholipids, such that they ‘lose control’ of their membrane lipid composition. We are exploring the limits of membrane phospholipid distortion tolerated by a live bacterium, using Staphylococcus aureus as the model. An S. aureus fatty acid auxotroph was constructed in which the phospholipid fatty acid composition is fully controlled by the experimenter. This system allows us to introduce extreme variations in fatty acid composition, and identify how they affect growth, cell division, and antibiotic tolerance. The findings I will present are relevant to S. aureus behavior in the diverse lipid environments encountered during infection.

The evolution of evolvability

• Paul Rainey (MPI for Evolutionary Biology)

I will discuss the hypothesis that life may have evolved to evolve — a provocative idea given that it appears to require foresight on the part of natural selection. The controversy arises because evolvability is a property of lineages, not individuals, and thus implies that selection can operate at levels above single entities. To probe the mechanisms underlying evolvability, I will describe an experiment performed by Michael Barnett who conducted a long-term evolution experiment using bacteria. Over three years, lineages evolved under a selective regime in which success depended on capacity to mutate between two phenotypic states. Lineages capable of generating the necessary phenotype for future environmental conditions displaced those lacking this capacity. After a series of 46 mutations, a type emerged that appeared to 'anticipate' the future via localised hyper-mutation — a mechanism analogous to contingency loci in pathogenic bacteria. I will describe the stepwise emergence of this capacity and describe experiments demonstrating that the locus itself is evolvable. Additionally, I will highlight an unanticipated consequence of enhanced evolvability: its facilitation of secondary adaptive mutations. In closing, I will argue that selection acting on lineages may be more prevalent and consequential than currently recognised.