A microfluidic mechano-chemostat for tissues and organisms reveals that confined growth is accompanied with increased macromolecular crowding.

Conventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellular assemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they often do not allow the control of compressive forces emerging when cells proliferate in a confined environment. Here, we present a generic microfluidic device to control both chemical and mechanical compressive forces. This device relies on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable closed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression.

Atypical low-copy number plasmid segregation systems, all in one?

Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.

DNA Segregation in Enterobacteria.

DNA segregation ensures that cell offspring receive at least one copy of each DNA molecule, or replicon, after their replication. This important cellular process includes different phases leading to the physical separation of the replicons and their movement toward the future daughter cells. Here, we review these phases and processes in enterobacteria with emphasis on the molecular mechanisms at play and their controls.

Characterization of the DNA Binding Domain of StbA, A Key Protein of A New Type of DNA Segregation System.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

TnpAREP and REP sequences dissemination in bacterial genomes: REP recognition determinants.

REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpAREP, the first prokaryotic domesticated transposase associated with REP. TnpAREP, fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpAREP processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpAREP. To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpAREP/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpAREP groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.

Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells.

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.

Detection and Characterization of Transposons in Bacteria.

Bacterial transposons, through their ability to transfer DNA sequences from one position in the genome to another, play a central role in the shape and the evolution of genomes. Extensive studies have been performed during the last five decades to understand the molecular mechanisms involved in the transposition of a variety of elements. Among the methods used, the papillation and the mating out coupled to arbitrary primed PCR assays described in this chapter are widely used as very powerful approaches to detect and characterize transposition events in vivo.

First Biochemical Steps on Bacterial Transposition Pathways.

Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and hence, to the continuous emergence of new multiresistant pathogens. Contrasting with their biological and societal impact, only a few bacterial transposons have been the subject of detailed molecular studies. In this chapter, we propose a set of reliable biochemical methods as a primary route for studying new transposition mechanisms. These methods include (a) a straightforward approach termed "thermal shift induction" to produce the transposase in a soluble and properly folded configuration prior to its purification, (b) an adaptation of classical electrophoretic mobility shift assays (EMSA) combined to fluorescently labeled DNA substrates to determine the DNA content of different complexes assembled by the transposase, and (c) a highly sensitive "in-gel" DNA footprinting assay to further characterize these complexes at the base pair resolution level. A combination of these approaches was recently applied to decipher the molecular organization of key intermediates in the Tn3-family transposition pathway, a mechanism that has long been refractory to biochemical studies.

Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering.

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction network between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions.

Single strand transposition at the host replication fork.

Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the 'peel and paste' pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the ß sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork.

IS200/IS605 family single-strand transposition: mechanism of IS608 strand transfer.

Transposase, TnpA, of the IS200/IS605 family member IS608, catalyses single-strand DNA transposition and is dimeric with hybrid catalytic sites composed of an HUH motif from one monomer and a catalytic Y127 present in an α-helix (αD) from the other (trans configuration). αD is attached to the main body by a flexible loop. Although the reactions leading to excision of a transposition intermediate are well characterized, little is known about the dynamic behaviour of the transpososome that drives this process. We provide evidence strongly supporting a strand transfer model involving rotation of both αD helices from the trans to the cis configuration (HUH and Y residues from the same monomer). Studies with TnpA heterodimers suggest that TnpA cleaves DNA in the trans configuration, and that the catalytic tyrosines linked to the 5'-phosphates exchange positions to allow rejoining of the cleaved strands (strand transfer) in the cis configuration. They further imply that, after excision of the transposon junction, TnpA should be reset to a trans configuration before the cleavage required for integration. Analysis also suggests that this mechanism is conserved among members of the IS200/IS605 family.

The stb operon balances the requirements for vegetative stability and conjugative transfer of plasmid R388.

The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed.

Plasmid segregation without partition.

Low-copy number plasmids need a segregation mechanism to assort one half of the plasmid copies to each daughter cell during cell division. This can be achieved directly by partitioning plasmid copies through a mechanism reminiscent of eukaryotic mitosis. Briefly, plasmid copies are paired around a centromere-like site, and then separated toward the daughter cells at cell division. Partition mechanisms are used by a majority of well-studied plasmids. They involve two proteins, a DNA-binding protein and a motor protein, besides the centromeric site. However, some plasmids do not encode typical partition systems, so alternative segregation mechanisms must be considered. For instance, chromosome segregation could provide the driving force for plasmid movement, through a "pilot-fish"-like mechanism. In support of this assumption, we recently demonstrated that plasmid R388 segregation, which does not involve a plasmid-encoded motor protein, requires a single plasmid-encoded DNA-binding protein. Besides, the new segregation system becomes essential when the plasmid encodes conjugation machinery, providing a new understanding of how plasmids integrate conjugative transfer with segregation.

Single-stranded DNA transposition is coupled to host replication.

DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the "top" IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication.

Resetting the site: redirecting integration of an insertion sequence in a predictable way.

Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Though most transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using single-stranded DNA, always inserts just 3' of a TTAC tetranucleotide. Our results from studies on the IS608 transposition mechanism demonstrated that the transposase recognizes its target site by co-opting an internal segment of transposon DNA and utilizes it for specific recognition of the target sites through base-pairing. This suggested a way to redirect IS608 transposition to novel target sites. As we demonstrate here, we can now direct insertions in a predictable way into a variety of different chosen target sequences, both in vitro and in vivo.

Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection.

The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.

In vitro reconstitution of a single-stranded transposition mechanism of IS608.

Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any other known transposition system, they strictly require single-strand DNA. TnpA, the shortest identified transposase, uses a nucleophilic tyrosine for these reactions. It recognizes and cleaves only the IS608 "top strand." The results support a transposition model involving excision of a single-strand circle with abutted left (LE) and right (RE) IS ends. Insertion occurs site specifically 3' to conserved and essential TTAC tetranucleotide and appears to be driven by LE. This single-strand transposition mode has important implications not only for dispersion of IS608 but also for the other members of this very large IS family.

Control of IS911 target selection: how OrfA may ensure IS dispersion.

IS911 transposition involves a closed circular insertion sequence intermediate (IS-circle) and two IS-encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N-terminal region with OrfA. This includes a helix-turn-helix (HTH) motif and the first three of four heptads of a leucine zipper (LZ). OrfAB binds specifically to IS911 ends via its HTH whereas OrfA does not. We show here: that OrfA binds DNA non-specifically and that this requires the HTH; that OrfA LZ is required for its multimerization; and that both motifs are essential for OrfA activity. We propose that these OrfA properties are required to assemble a nucleoprotein complex committed to random IS911 insertion. This control of IS911 insertion activity by OrfA in this way would assure its dispersion.

Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria.

TonB-dependent receptors (TBDRs) are outer membrane proteins mainly known for the active transport of iron siderophore complexes in Gram-negative bacteria. Analysis of the genome of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc), predicts 72 TBDRs. Such an overrepresentation is common in Xanthomonas species but is limited to only a small number of bacteria. Here, we show that one Xcc TBDR transports sucrose with a very high affinity, suggesting that it might be a sucrose scavenger. This TBDR acts with an inner membrane transporter, an amylosucrase and a regulator to utilize sucrose, thus defining a new type of carbohydrate utilization locus, named CUT locus, involving a TBDR for the transport of substrate across the outer membrane. This sucrose CUT locus is required for full pathogenicity on Arabidopsis, showing its importance for the adaptation to host plants. A systematic analysis of Xcc TBDR genes and a genome context survey suggested that several Xcc TBDRs belong to other CUT loci involved in the utilization of various plant carbohydrates. Interestingly, several Xcc TBDRs and CUT loci are conserved in aquatic bacteria such as Caulobacter crescentus, Colwellia psychrerythraea, Saccharophagus degradans, Shewanella spp., Sphingomonas spp. or Pseudoalteromonas spp., which share the ability to degrade a wide variety of complex carbohydrates and display TBDR overrepresentation. We therefore propose that TBDR overrepresentation and the presence of CUT loci designate the ability to scavenge carbohydrates. Thus CUT loci, which seem to participate to the adaptation of phytopathogenic bacteria to their host plants, might also play a very important role in the biogeochemical cycling of plant-derived nutrients in marine environments. Moreover, the TBDRs and CUT loci identified in this study are clearly different from those characterized in the human gut symbiont Bacteroides thetaiotaomicron, which allow glycan foraging, suggesting a convergent evolution of TBDRs in Proteobacteria and Bacteroidetes.

Active site sharing and subterminal hairpin recognition in a new class of DNA transposases.

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg2+ and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.