Biology subjects

Blokesch, M.

Publications and source records attributed to Blokesch, M..

18 recordsLinked to original sources

bioRxiv2026

Large-scale recovery of integron cassettes for gene discovery screens

Integrons capture and stockpile adaptive genes encoded in modular mobile genetic elements called integron cassettes (ICs). Present in 17% of bacterial genomes, integrons can harbor hundreds of cassettes, representing a hotspot of genetic variability. Some ICs encode antimicrobial or phage resistance genes, but most remain functionally uncharacterized, representing an untapped source of genes of biotechnological interest. Here, we present two tools, the cassette gatherer and hunter, that allow the swift establishment of libraries of genes either from genetically tractable strains or directly from DNA. By re-engineering a class 1 integron, these platforms capture single cassettes in a sequence and function-independent manner. When applied to Vibrio genomes, they recovered hundreds of single cassettes per assay with >99% specificity. To validate the usability of our tools for the discovery of new genes, we subjected these libraries to screens against phages ICP2 and T4, and identified nine phage-defense systems, including five previously undescribed. Hence, these tools provide a fast and simple method to recover thousands of ICs, in a way amenable to gene-discovery screens.

microbiology↗

bioRxiv2025

Synthetically designed anti-defense proteins overcome barriers to bacterial transformation and phage infection

Bacterial defense systems present considerable barriers to both phage infection and plasmid transformation. These systems target mobile genetic elements, limiting the efficacy of bacteriophage-based therapies and restricting genetic engineering applications. Here, we employ a de-novo protein design approach to generate proteins that bind and inhibit bacterial defense systems. We show that our synthetically designed proteins block defense, and that phages engineered to encode the synthetic proteins can replicate in cells that express the respective defense system. We further demonstrate that a single phage could be engineered with multiple anti-defense proteins, yielding improved infectivity in bacterial strains carrying multiple defense systems. Finally, we show that plasmids that express synthetic anti-defense proteins can be introduced into bacteria that naturally restrict plasmid transformation. Our approach can broaden host ranges of therapeutic phages and can improve genetic engineering efficiency in strains that are typically difficult to transform.

bioengineering↗

bioRxiv2025

Structure and Activation Mechanism of a Lamassu Phage Defence System

Lamassu is a diverse family of defence systems that protect bacteria, including pandemic strains of Vibrio cholerae, against phage infection. They target essential cellular processes, aborting infection and preventing phage propagation by terminating the infected host. The mechanisms by which Lamassu efectors are activated when needed and otherwise suppressed are unknown. Here, we present structures of a Lamassu defence system from Salmonella enterica. We show that an oligomerization domain of the nuclease efector, LmuA, is sequestered by two tightly-folded SMC-like LmuB protomers and LmuC. Upon activation, liberated LmuA proteins assemble into a cyclic homo-tetramer, in which two of four nuclease domains are brought into proximity to create an active site capable of cleaving DNA. We propose tetramer formation is likely a one-way switch that establishes a threshold to limit potential spontaneous activation and cell death. Our findings reveal a mechanism of cellular defence, involving liberation and oligomerization of immune efectors, and shed light on how Lamassu systems balance potent immune responses with self-preservation.

microbiology↗

bioRxiv2024

Diverse phage defence systems define West African South American pandemic Vibrio cholerae

Our understanding of the factors underlying the evolutionary success of different lineages of pandemic Vibrio cholerae remains incomplete. Interestingly, two unique genetic signatures define the West African South American (WASA) lineage of V. cholerae responsible for the 1991-2001 Latin American cholera epidemic. Here we show these signatures encode diverse anti-phage defence systems. Firstly, the WASA-1 prophage encodes a 2-gene abortive-infection system WonAB that renders the lineage resistant to the major predatory vibriophage ICP1, which alongside other phages, is thought to restrict cholera epidemics and has potential for use in prophylaxis. Secondly, a unique set of genes on the Vibrio seventh pandemic island II encodes an unusual modification-dependent restriction system targeting phages with modified genomes, and a new member of the Shedu defence family that defends against vibriophage X29. Taken together, we propose that these anti-phage defence systems have likely contributed to the success of a major epidemic lineage of the ongoing seventh cholera pandemic.

microbiology↗

bioRxiv2024

Exploring Mobile Genetic Elements in Vibrio cholerae

Members of the bacterial species Vibrio cholerae are known both as prominent constituents of marine environments and as the causative agents of cholera, a severe diarrheal disease. While strains responsible for cholera have been extensively studied over the past century, less is known about their environmental counterparts, despite their contributions to the species pangenome. This study analyzed the genome compositions of 46 V. cholerae strains, encompassing both pandemic and non-pandemic, toxigenic, and environmental variants, to explore the diversity of mobile genetic elements (MGEs) and bacterial defense systems. Our findings include both conserved and novel MGEs across the strains, pointing to shared evolutionary pathways and ecological niches. The defensome analysis revealed a wide array of antiviral mechanisms, extending well beyond the traditional CRISPR-Cas and restriction-modification systems. This underscores the dynamic arms race between V. cholerae and MGEs and/or bacteriophages and suggests that non-pandemic strains may act as reservoirs for emerging defense strategies. Moreover, the study showed that MGEs are integrated into genomic hotspots, which may serve as critical platforms for the exchange of defense systems, thereby enhancing V. choleraes adaptive capabilities against phage attacks and invading MGEs. Overall, this research offers foundational insights into V. choleraes genetic complexity and adaptive strategies, with implications for understanding the differences between environmental strains and their pandemic relatives, and the potential evolutionary paths that led to the emergence of pandemic strains.

microbiology↗

bioRxiv2024

Molecular mechanism of plasmid elimination by the DdmDE defense system

Seventh pandemic Vibrio cholerae strains contain two hallmark pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryo-EM to reveal the mechanistic basis for plasmid defense by DdmDE. A cryo-EM structure of the DdmD helicase-nuclease reveals that it adopts an auto-inhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. A structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the non-target DNA strand. Finally, in vitro studies reveal that DdmD translocates in the 5 to 3 direction, while partially degrading the plasmid DNA. These findings provide critical insights into the architecture and mechanism of DdmDE systems in plasmid elimination.

microbiology↗

bioRxiv2024

Capsular Polysaccharide Restrains Type VI Secretion in Acinetobacter baumannii

The type VI secretion system (T6SS) is a sophisticated, contact-dependent nanomachine involved in interbacterial competition. To function effectively, the T6SS must penetrate the membranes of both attacker and target bacteria. Structures associated with the cell envelope, like polysaccharides chains, can therefore introduce spatial separation and steric hindrance, potentially affecting the efficacy of the T6SS. In this study, we examined how the capsular polysaccharide (CPS) of Acinetobacter baumannii affects T6SSs antibacterial function. Our findings show that the CPS confers resistance against T6SS-mediated assaults from rival bacteria. Notably, under typical growth conditions, the presence of the surface-bound capsule also reduces the efficacy of the bacteriums own T6SS. This T6SS impairment is further enhanced when CPS is overproduced due to genetic modifications or antibiotic treatment. Furthermore, we demonstrate that the bacterium adjusts the level of the T6SS inner tube protein Hcp according to its secretion capacity, by initiating a degradation process involving the ClpXP protease. Collectively, our findings contribute to a better understanding of the dynamic relationship between T6SS and CPS and how they respond swiftly to environmental challenges.

microbiology↗

bioRxiv2024

Vibrio cholerae pathogenicity island 2 encodes two distinct types of restriction systems

In response to predation by bacteriophages and invasion by other mobile genetic elements such as plasmids, bacteria have evolved specialised defence systems that are often clustered together on genomic islands. The O1 El Tor strains of Vibrio cholerae responsible for the ongoing seventh cholera pandemic (7PET) contain a characteristic set of genomic islands involved in host colonisation and disease, many of which contain defence systems. Notably, Vibrio pathogenicity island 2 contains several characterised defence systems as well as a putative Type I restriction-modification system (T1RM), which, interestingly, is interrupted by two genes of unknown function. Here, we demonstrate that the T1RM system is active, methylates the host genomes of a representative set of 7PET strains, and identify a specific recognition sequence that targets non-methylated plasmids for restriction. We go on to show that the two genes embedded within the T1RM system encode a novel two-protein modification-dependent restriction system related to the GmrSD family of Type IV restriction enzymes. Indeed, we show that this system has potent anti-phage activity against diverse members of the Tevenvirinae, a subfamily of bacteriophages with hypermodified genomes. Taken together these results expand our understanding of how this highly conserved genomic island contributes to the defence of pandemic V. cholerae against foreign DNA.

microbiology↗

bioRxiv2023

Interactions between pili affect the outcome of bacterial competition driven by the type VI secretion system

The bacterial type VI secretion system (T6SS) is a widespread, kin-discriminatory weapon capable of shaping microbial communities. Due to the systems dependency on contact, cellular interactions can lead to either competition or kin protection. Cell-to-cell contact is often accomplished via surface-exposed type IV pili (T4P). In Vibrio cholerae, these T4P facilitate specific interactions when the bacteria colonize natural chitinous surfaces. However, it has remained unclear whether and, if so, how these interactions affect the bacteriums T6SS-mediated killing. In this study, we demonstrate that pilus-mediated interactions can be harnessed to reduce the population of V. cholerae under liquid growth conditions in a T6SS-dependent manner. We also show that the naturally occurring diversity of pili determines the likelihood of cell-to-cell contact and, consequently, the extent of T6SS-mediated competition. To determine the factors that enable or hinder the T6SSs targeted reduction of competitors carrying pili, we developed a physics-grounded computational model for autoaggregation. Collectively, our research demonstrates that T4P involved in cell-to-cell contact can impose a selective burden when V. cholerae encounters non-kin cells that possess an active T6SS. Additionally, our study underscores the significance of T4P diversity in protecting closely related individuals from T6SS attacks through autoaggregation and spatial segregation.

microbiology↗

bioRxiv2023

DNA modifications impact natural transformation of Acinetobacter baumannii

Acinetobacter baumannii is a dangerous nosocomial pathogen, especially due to its ability to rapidly acquire new genetic traits, including antibiotic resistance genes (ARG). In A. baumannii, natural competence for transformation, one of the primary modes of horizontal gene transfer (HGT), is thought to contribute to ARG acquisition and has therefore been intensively studied. However, knowledge regarding the potential role of epigenetic DNA modification(s) on this process remains lacking. Here, we demonstrate that the methylome pattern of diverse A. baumannii strains differs substantially and that these epigenetic marks influence the fate of transforming DNA. Specifically, we describe a methylome-dependent phenomenon that impacts intra- and inter-species DNA exchange by the competent A. baumannii strain A118. We go on to identify and characterize an A118-specific restrictionmodification (RM) system that impairs transformation when the incoming DNA lacks a specific methylation signature. Collectively, our work contributes towards a more holistic understanding of HGT in this organism and may also aid future endeavors towards tackling the spread of novel ARGs. In particular, our results suggest that DNA exchanges between bacteria that share similar epigenomes are favored and could therefore guide future research into identifying the reservoir(s) of dangerous genetic traits for this multi-drug resistant pathogen.

microbiology↗

bioRxiv2022

A single nucleotide polymorphism determines constitutive versus inducible type VI secretion in Vibrio cholerae

Vibrio cholerae is a well-studied human pathogen that is also a common inhabitant of marine habitats. In both environments, the bacterium is subject to interbacterial competition. A molecular nanomachine that is often involved in such competitive behavior is the type VI secretion system (T6SS). Interestingly and in contrast to non-pandemic or environmental isolates, the T6SS of the O1 El Tor clade of V. cholerae, which is responsible for the ongoing 7th pandemic, is largely silent under standard conditions. Instead, these strains induce their full T6SS capacity only under specific conditions such as growth on chitinous surfaces (signaled through TfoX and QstR) or when the cells encounter low intracellular c-di-GMP levels (TfoY-driven). In this study, we identified a single nucleotide polymorphism (SNP) within an intergenic region of the major T6SS gene cluster of V. cholerae that determines the T6SS status of the cell. We show that SNP conversion is sufficient to induce T6SS production in numerous pandemic strains, while the converse approach renders non-pandemic/environmental V. cholerae strains T6SS-silent. We further demonstrate that SNP-dependent T6SS production occurs independently of the known T6SS regulators TfoX, QstR, and TfoY. Finally, we identify a putative promoter region adjacent to the identified SNP that is required for all forms of T6SS regulation.

microbiology↗

bioRxiv2021

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment. Significance StatementResponsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes.

microbiology↗

bioRxiv2020

Interbacterial competition and anti-predatory behavior of environmental Vibrio cholerae strains

Vibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the hemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti-grazing defense relies on the strains T6SS and its actin-cross-linking domain (ACD)-containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS-dependent interbacterial killing. We explored the latter phenotype through whole-genome sequencing of fourteen isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but nonidentical immunity proteins were insufficient to provide cross-immunity among those wild strains. Originality-Significance StatementThis work contributes to the understanding of phenotypic consequences that differentiate diverse Vibrio cholerae strains. We focused on the type VI secretion system (T6SS) and the pore forming toxin hemolysin, which are tightly regulated in pandemic strains but remain constitutively active in non-pandemic isolates. We unveiled diverse arrays of T6SS effector/immunity modules in a set of environmental strains by long-read whole genome sequencing and de novo assembly. These modules determine whether the strains are able to evade amoebal predation and dictate their level of compatibility or competitiveness with one another.

microbiology↗

bioRxiv2019

Neighbor predation linked to natural competence fosters the transfer of large genomic regions in Vibrio cholerae

Natural competence for transformation is a primary mode of horizontal gene transfer (HGT). Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. And while it is known that transformation contributes to evolution and pathogen emergence in bacteria, there are still questions regarding the general prevalence of non-degraded DNA with sufficient coding capacity. In this context, we previously showed that the naturally competent bacterium Vibrio cholerae uses its type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors under chitin-colonizing conditions. We therefore sought to further explore the role of the T6SS in acquiring DNA, the condition of the DNA released through T6SS-mediated killing versus passive cell lysis, and the extent of the transfers that occur due to these conditions. To do this, we herein measured the frequency and the extent of genetic exchanges in bacterial co-cultures on competence-inducing chitin under various DNA-acquisition conditions. We show that competent V. cholerae strains acquire DNA fragments with an average and maximum length exceeding 50 kbp and 150 kbp, respectively, and that the T6SS is of prime importance for such HGT events. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters HGT and that the coding capacity of the exchanged genetic material is sufficient to significantly accelerate bacterial evolution.\n\nSignificance StatementDNA shuffled from one organism to another in an inheritable manner is a common feature of prokaryotes. It is a significant mechanism by which bacteria acquire new phenotypes, for example by first absorbing foreign DNA and then recombining it into their genome. In this study, we show the remarkable extent of the exchanged genetic material, frequently exceeding 150 genes in a seemingly single transfer event, in Vibrio cholerae. We also show that to best preserve its length and quality, bacteria mainly acquire this DNA by killing adjacent, healthy neighbors then immediately absorbing the released DNA before it can be degraded. These new insights into this prey-killing DNA acquisition process shed light on how bacterial species evolve in the wild.

microbiology↗

bioRxiv2018

Conserved type VI secretion regulation in diverse Vibrio species by the regulatory proteins TfoX and TfoY

Bacteria of the genus Vibrio are common members of aquatic environments where they compete with other prokaryotes and defend themselves against grazing predators. A macromolecular protein complex called the type VI secretion system (T6SS) is used for both purposes. Previous research showed that the sole T6SS of the human pathogen V. cholerae is induced by extracellular (chitin) or intracellular (low c-di-GMP levels) cues and that these cues lead to distinctive signalling pathways for which the proteins TfoX and TfoY serve as master regulators. In this study, we tested whether the TfoX- and TfoY-mediated regulation of T6SS was conserved in non-cholera species, and if so, how these regulators affected the production of individual T6SSs in double-armed vibrios. We show that, alongside representative competence genes, TfoX regulates at least one T6SS in all tested Vibrio species. TfoY, on the other hand, fostered motility in all vibrios but had a more versatile T6SS response in that it did not foster T6SS-mediated killing in V. fischeri while it induced both systems in V. alginolyticus. Collectively, our data provide evidence that the TfoX- and TfoY-mediated signalling pathways are mostly conserved in diverse Vibrio species and important for signal-specific T6SS induction.\n\nOriginality-Significance StatementThis work provides new insight into the regulatory circuits involved in type VI secretion in diverse Vibrio species. Specifically, it is the first study to compare the effects of the two regulatory proteins TfoX and TfoY on the primary or secondary type VI secretion systems of non-cholera vibrios. Importantly, this work also shows that decreased c-di-GMP levels in V. parahaemolyticus lead to TfoY production without changing tfoY transcript levels, thereby indirectly linking TfoY production to surface sensing.

microbiology↗

bioRxiv2018

DNA-uptake pilus of Vibrio cholerae capable of kin-discriminated auto-aggregation

Natural competence for transformation is a widely used and key mode of horizontal gene transfer that can foster rapid bacterial evolution. Competent bacteria take-up DNA from their environment using Type IV pili, a widespread and multi-purpose class of cell surface polymers. However, how pili facilitate DNA-uptake has remained unclear. Here, using direct labelling, we show that in the Gram-negative pathogen Vibrio cholerae DNA-uptake pili are highly dynamic and that they retract prior to DNA-uptake. Unexpectedly, these pili can self-interact to mediate auto-aggregation of cells into macroscopic structures. This phenotype is conserved in disease causing pandemic strains. However, extensive strain-to-strain variability in the major pilin subunit PilA, present in environmental isolates, controls the ability of pili to interact without affecting transformation. We go on to show that interactions between pili are highly specific, enabling cells producing pili composed of different PilA subunits to discriminate between one another. On chitin surfaces, a natural habitat of V. cholerae, pili connect cells within dense networks, suggesting a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonisation. Moreover, our results provide evidence that type IV pili could provide a simple and potentially widespread mechanism for bacterial kin recognition.

microbiology↗

bioRxiv2018

QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae

During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is likely a dual transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

microbiology↗

bioRxiv2017

Molecular insights into Vibrio cholerae’s intra-amoebal host-pathogen interactions

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the hosts osmoregulatory organelle. In this study, we deciphered the molecular mechanisms involved in the maintenance of V. choleraes intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrated that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, play a key role role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

microbiology↗