DNA Recombination Research Articles

What do we gain when tolerating loss? The information bottleneck wrings out recombination.

Most microbes have the capacity to acquire genetic material from their environment. Recombination of foreign DNA yields genomes that are, at least in part, incongruent with the vertical history of their species. Dominant approaches for detecting these transfers are phylogenetic, requiring a painstaking series of analyses including alignment and tree reconstruction. But these methods do not scale. Here we propose an unsupervised, alignment-free and tree-free technique based on the sequential information bottleneck (SIB), an optimization procedure designed to extract some portion of relevant information from one random variable conditioned on another. In our case, this joint probability distribution tabulates occurrence counts of k-mers against their genomes of origin with the expectation that recombination will create a strong signal that unifies certain sets of co-occuring k-mers. We conceptualize the technique as a rate-distortion problem, measuring distortion in the relevance information as k-mers are compressed into clusters based on their co-occurrence in the source genomes. The result is fast, model-free, lossy compression of k-mers into learned groups of shared genome sequence, differentiating recombined elements from the vertically inherited core. We show that the technique yields a new recombination measure based purely on information, divorced from any biases and limitations inherent to alignment and phylogeny.

Advancements in understanding the role and mechanism of sirtuin family (SIRT1-7) in breast cancer management.

Breast cancer (BC) is the most prevalent type of cancer in women worldwide and it is classified into a few distinct molecular subtypes based on the expression of growth factor and hormone receptors. Though significant progress has been achieved in the search for novel medications through traditional and advanced approaches, still we need more efficacious and reliable treatment options to treat different types and stages of BC. Sirtuins (SIRT1-7) a class III histone deacetylase play a major role in combating various cancers including BC. Studies reveal thateach sirtuin has a unique and well-balanced biology, indicating that it regulates a variety of biological processes that result in the initiation, progression,and metastasis of BC. SIRT also plays a major role in numerous vital biological functions, including apoptosis, axonal protection, transcriptional silencing, DNA recombination and repair, fat mobilization, and aging. As per the current demand, we wish to outline the structural insights into sirtuin's catalytic site, substantial variations among all SIRT types, and their mechanism in BC management. Additionally, this review will focus on the application of SIRT modulators along with their clinical significance, hurdles, and future perspective to develop successful SIRT-based drug candidates to conquer the BC problem.

Pain in rheumatoid arthritis: Emerging role of high mobility group box 1 protein-HMGB1.

Rheumatoid arthritis (RA) is a chronic inflammatory disease where pain, driven by both inflammatory and non-inflammatory processes, is a major concern for patients. This pain can persist even after joint inflammation subsides. High mobility group box-1 (HMGB1) is a non-histone-DNA binding protein located in the nucleus that plays a key role in processes such as DNA transcription, recombination, and replication. HMGB1 can be released into the extracellular space through both passive and active mechanisms. Extracellular HMGB1 contributes to synovial inflammation, bone degradation, and the production of cytokines in RA by binding to toll-like receptors (TLRs) and receptors for advanced glycation end products (RAGE). It also forms complexes with molecules like lipopolysaccharide (LPS) and IL-1β, amplifying inflammatory responses. Due to its central role in these processes, HMGB1 is considered a promising therapeutic target in RA. It also acts as a nociceptive molecule in mediating pain in diseases such as diabetes and bone cancer. In this review, we explore how HMGB1 contributes to chronic pain in RA, supported by both in vitro and in vivo models. We begin by providing an overview of the mechanisms of pain in RA, the structure of HMGB1, its release mechanisms, and the therapeutic potential of targeting HMGB1 in RA. Following this, we highlight its role in peripheral and central pain sensitization through direct activation of the TLR4/MAPK/NF-κB pathway, as well as indirectly through downstream mediators, underscoring its potential as a target for managing RA pain.

Lake salinization on the Qinghai–Tibetan Plateau alters viral community composition and lifestyles

Global warming has accelerated lake salinization, driving changes in microbial community structure and function. However, the dynamics of viral communities in response to salinity remain unclear. Here, we apply metagenomic sequencing to a lake on the Qinghai–Tibet Plateau, spanning a broad salinity gradient, to investigate viral community dynamics. Our findings reveal that salinity strongly influences viral composition and modulates viral lifestyles. Temperate viruses increase in relative abundance along the salinity gradient, whereas virulent viruses show a corresponding decline. These shifts are mirrored in the prokaryotic communities, with Alphaproteobacteria and their infecting temperate viruses, notably Casadabanvirus, becoming more prevalent in higher salinity zones. Viral genomes encode genes associated with osmotic stress adaptation, DNA recombination, and nutrient transport, which may facilitate host adaptation to saline stress. This study provides valuable insights into the interplay between viral and prokaryotic communities in response to lake salinization.

Site-specific free energy surface parameters from single-molecule fluorescence measurements of exciton-coupled (iCy3)2 dimer probes positioned at DNA replication fork junctions.

Single-stranded-double-stranded DNA (ss-dsDNA) replication forks and primer-template junctions are important recognition sites for the assembly and function of proteins involved in DNA replication, recombination, and repair. DNA 'breathing' - i.e. thermally induced local fluctuations of the sugar-phosphate backbones and bases - can populate metastable conformational macrostates at positions near such junctions and likely play key roles in the functional interactions of the regulatory proteins that bind at these sites. Recently, Maurer etal. [1] performed polarization-sweep single-molecule fluorescence (PS-SMF) studies on exciton-coupled (iCy3)2 dimer-labeled ss-dsDNA fork constructs, which revealed that the nucleobases and backbones immediately adjacent to the dimer probes undergo conformational fluctuations on time scales ranging from hundreds of microseconds to hundreds of milliseconds. The local conformations sensed by the dimer probes consist of four quasi-stable macrostates whose populations and dynamics depend on dimer probe position relative to these junctions. Here we present theoretical analyses of these PS-SMF data that quantify the relative stabilities and activation barriers of the free energy surfaces of site-specific DNA 'breathing' events at key positions within these junctions. Our results suggest a detailed molecular picture for DNA 'breathing' at these positions, thus providing insights into understanding the molecular mechanisms of the proteins that operate at these sites.

Local structural dynamics of Rad51 protomers revealed by cryo-electron microscopy of Rad51-ssDNA filaments.

Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) to ascertain the density map of the wild-type budding yeast Rad51-ssDNA filament bound to ADP-AlF3, achieving a resolution of 2.35 Å without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF3. It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains. Notably, R293 in Loop 1 adopts multiple conformations to facilitate L296 and V331 in separating and twisting the DNA triplets. We also analyzed the crystal structure of Rad51-I345T and the predicted structure of yeast Rad51-K342E using the Rad51-ssDNA structure from this study as a reference.

Identifying a non-conserved site for achieving allosteric covalent inhibition of CECR2.

The bromodomain (BRD) represents a highly conserved structural module that provides BRD proteins with fundamental functionality in modulating protein-protein interactions involved in diverse biological processes such as chromatin-mediated gene transcription, DNA recombination, replication and repair. Consequently, dysregulation of BRD proteins has been implicated in the pathogenesis of numerous human diseases. In recent years, considerable scientific endeavors have focused on unraveling the molecular mechanisms underlying BRDs and developing inhibitors that target these domains. While these inhibitors compete for binding with the acetylated lysine binding site of BRDs, achieving inhibition of BRD proteins via competitive pocket binding has proven challenging due to the conserved nature of these pockets. To address this limitation, the present study employed dynamic simulations for a comprehensive analysis, leading to the identification of a non-conserved pocket in CECR2 for achieving BRD family inhibition through allosteric modulation. Subsequently, the compound BAY 11-7085 was proven capable of covalently binding to C494 of this pocket after covalent docking and biological verification in vitro. The allosteric inhibition strategy of CECR2 was further verified by the structurally optimized compound LC-CE-7, which is an allosteric covalent CECR2 inhibitor with anti-cancer effects in MDA-MB-231 cells.

Structural basis of directionality control in large serine integrases.

Large serine integrases (LSIs) catalyze unidirectional site-specific DNA recombination reactions, yet those reactions are reversed by the presence of a cognate recombination directionality factor (RDF). Mechanistic understanding of directionality control has been hampered by a lack of structural information. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of six SPbeta integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways, at 4.16-7.18Å resolution. Our findings reveal how RDF-mediated repositioning of an integrase subdomain (1) dictates which pairs of DNA sites can be assembled into a synaptic complex to initiate recombination and (2) dictates which product complexes will be conformationally locked, preventing the back reaction. These mechanistic insights provide a conceptual framework for engineering efficient and versatile genome editing tools.

Nuclear Intron Sequence Variation of the Bulinus globosus Complex (Mollusca: Planorbidae): Implications for Molecular Systematic Analyses.

Urinary schistosomiasis is caused by the blood fluke Schistosoma haematobium, which is predominantly found in Africa. The freshwater snail Bulinus globosus is its main intermediate host. The species that make up the B. globosus group are genetically complex, and their taxonomic status remains controversial. Genetic variation, heterozygosity, and DNA recombination in B. globosus were examined using the mitochondrial cytochrome c oxidase subunit 1 (COI) and the intron 3 region of the arginine kinase gene (AkInt3). A total of 81 B. globosus snails were collected from three different localities in Kwale County, Kenya. Genomic diversity, heterozygosity, DNA recombination, and haplotype network were calculated using AkInt3 sequences. Low polymorphism in the COI sequence divided B. globosus into six haplotypes (C1-C6). However, AkInt3 sequencing studies showed high polymorphisms, classifying 81 B. globosus snails into 44 haplotypes (H1-H44). These haplotypes were separated into three haplogroups (I-III). AkInt3 sequence heterozygosity was also found. DNA recombination haplotypes between haplogroups were commonly found in heterozygous samples. AkInt3 sequence studies showed high levels of genetic polymorphism and heterozygosity, supporting its use as a genetic marker for elucidating the population genetics of B. globosus. Furthermore, our study showed that B. globosus populations in Kenya form a "species complex".

Genome Mining and Characterization of Two Novel Lacticaseibacillus rhamnosus Probiotic Candidates with Bile Salt Hydrolase Activity.

Bile salt hydrolase (BSH; EC 3.5.1.24) is the microbial enzyme that catalyzes the conversion of primary bile acids (BAs) into secondary ones, promoting microbial adaptation and modulating several host's biological functions. Probiotics with BSH activity are supposed to survive harsh intestinal conditions and exert a cholesterol-lowering effect. Here, Lacticaseibacillus rhamnosus strains (VB4 and VB1), isolated from the vaginal ecosystem, were submitted to a genomic survey, in vitro BSH activity, and BAs tolerance assay to unravel their probiotic potential as BAs modulators. The draft genomes of Lcb. rhamnosus VB4 and VB1 strains comprised 2769 and 2704 CDSs, respectively. Gene annotation revealed numerous strain-specific genes involved in metabolism and transport, as well as in DNA recombination. Each strain harbors a single bsh gene, encoding a C-N amide hydrolase, which conserved the essential residues required in the BSH core site. According to the results, compared to VB1, the VB4 strain tolerated better BAs stress and was more active in deconjugating BAs. However, BAs stress increased the bsh gene transcription in the VB1 strain but not in the VB4 strain, suggesting a partially nonlinear relationship between BSH activity and gene expression. In conclusion, despite the complexity of the BSH transcriptional system, the results support the VB4 strain as a promising BAs-deconjugating probiotic candidate.

Inactivation of GSK3β by Ser389 phosphorylation prevents thymocyte necroptosis and impacts Tcr repertoire diversity.

The assembly of Tcrb and Tcra genes require double negative (DN) thymocytes to undergo multiple rounds of programmed DNA double-strand breaks (DSBs), followed by their efficient repair. However, mechanisms governing cell cycle checkpoints and specific survival pathways during the repair process remain unclear. Here, we report high-resolution scRNA-seq analyses of individually sorted mouse DN3 and DN4 thymocytes, which reveals a G2M cell cycle checkpoint, in addition to the known G1 checkpoint, during Tcrb and Tcra recombination. We also show that inactivation of GSK3β by phosphorylation on Ser389 is essential for DN3/DN4 thymocytes to survive while being stalled at the G1 and G2/M checkpoints. GSK3β promotes death by necroptosis, but not by apoptosis, of DN3/DN4 thymocytes during V(D)J recombination. Failure to inactivate GSK3β in DN3 thymocytes alters the Tcrb gene repertoire primarily through Trbv segment utilization. In addition, preferential recombination of proximal V segments in Tcra depends on GSK3β inactivation. Our study identifies a unique thymocyte survival pathway, enabling them to undergo cell cycle checkpoints for DNA repair during V(D)J recombination of Tcrb and Tcra genes. Thymocyte survival during cell cycle checkpoints for V(D)J recombination DNA repair determines TCRα/β repertoire.

Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair.

FHIT is a fragile site tumor suppressor that is primarily inactivated upon tobacco smoking. FHIT loss is frequently observed in lung cancer, making it an important biomarker for the development of targeted therapy for lung cancer. Here, we report that inhibitors of glycogen synthase kinase 3 beta (GSK3β) and the homologous recombination DNA repair (HRR) pathway are synthetic lethal with FHIT loss in lung cancer. Pharmacological inhibition or siRNA depletion of GSK3β selectively suppressed the growth of FHIT-deficient lung cancer tumors in vitro and in animal models. We further showed that FHIT inactivation leads to the activation of DNA damage repair pathways, including the HRR and NHEJ pathways, in lung cancer cells. Conversely, FHIT-deficient cells are highly dependent on HRR for survival under DNA damage stress. The inhibition of GSK3β in FHIT-deficient cells suppressed the ATR/BRCA1/RAD51 axis in HRR signaling via two distinct pathways and suppressed DNA double-strand break repair, leading to the accumulation of DNA damage and apoptosis. Small molecule inhibitors of HRR, but not NHEJ or PARP, induced synthetic lethality in FHIT-deficient lung cancer cells. The findings of this study suggest that the GSK3β and HRR pathways are potential drug targets in lung cancer patients with FHIT loss.

Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons.

Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110-family transposases, which catalyse RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-O-methylation using C/D-box snoRNAs. On the basis of conservation of protein sequence, domain architecture, three-dimensional structure and non-coding RNA features, alongside phylogenetic analyses, we propose that programmable RNA modification emerged through the exaptation of components derived from IS110-like transposons. These findings underscore how recurrent domestication events of transposable elements have driven the evolution of RNA-guided mechanisms.

The Shu complex is an ATPase that regulates Rad51 filaments during homologous recombination in the DNA damage response.

Rad51 filaments are Rad51-coated single-stranded DNA and essential in homologous recombination (HR). The yeast Shu complex (Shu) is a conserved regulator of homologous recombination, working through its modulation on Rad51 filaments to direct HR-associated DNA damage response. However, the biochemical properties of Shu remain unclear, which hinders molecular insights into Shu's role in HR and the DNA damage response. In this work, we biochemically characterized Shu and analyzed its molecular actions on single-stranded DNA and Rad51 filaments. First, we revealed that Shu preferentially binds fork-shaped DNA with 20nt ssDNA components. Then, we identified and validated, through site-specific mutagenesis, that Shu is an ATPase and hydrolyzes ATP in a DNA-dependent manner. Furthermore, we showed that Shu interacts with ssDNA and Rad51 filaments and alters the properties of ssDNA and the filaments with a 5'-3' polarity. The alterations depend on the ATP hydrolysis of Shu, suggesting that the ATPase activity of Shu is important in regulating its functions. The preference of Shu for acting on the 5' end of Rad51 filaments aligns with the observation that Shu promotes lesion bypass at the lagging strand of a replication fork. Our work on Shu, a prototype modulator of Rad51 filaments in eukaryotes, provides a general molecular mechanism for Rad51-mediated error-free DNA lesion bypass.

Solubilization Inclusion Bodies from Synthetic Recombinant PGA Gene Expressed in E. coli BL21(DE3) by Denaturing and Non-denaturing Agents

Background: With the rise in green chemistry, the synthesis of antibiotic compounds through enzymatic processes is a preferred option. Penicillin-G acylase (PGA) is an important enzyme for producing important antibiotics, such as penicillin and its derivatives. Therefore, studies on PGA have been conducted worldwide. In the penicillin biosynthetic pathway, PGA catalyzes the conversion of penicillin G into 6-amino penicillanic acid (6-APA), a precursor for the enzymatic synthesis of penicillin derivatives. Unfortunately, bacteria naturally produce PGA in small quantities. Objective: One strategy for producing this enzyme in large quantities is DNA recombination, which is expressed in Escherichia coli. The formation of inclusion bodies (IBs) is a common obstacle to protein overexpression in Escherichia coli. In this study, we discuss IBs solubilization methods for recombinant PGA derived from E. coli (rPGAEc) expressed in E. coli BL21 (DE3). Recombinant E. coli BL21 (DE3) cells harboring rPGAEc were induced with IPTG for enzyme expression. Induction was performed at 16 °C for 4 h and 24 h. The PGA enzyme expressed in the IBs form was then incubated in two solutions containing 8 M urea and 0.2% sarcosine to obtain a soluble enzyme. Results: Based on protein analysis by SDS-PAGE, a solution containing 8 M urea solubilized PGA more abundantly than 0,2% sarcosine. Conclusion: The solubilization technique of PGA expressed by E. coli proposed in this study is an alternative solution that can be considered for this purpose.

RecA deletion disrupts protein homeostasis, leading to deamidation, oxidation, and impaired glycolysis in Cronobacter sakazakii.

Cronobacter sakazakii is a foodborne pathogen linked to severe infections in infants and often associated with contaminated powdered infant formula. The RecA protein, a key player in DNA repair and recombination, also influences bacterial resilience and virulence. This study investigated the impact of recA deletion on the pathogenicity and environmental stress tolerance of C. sakazakii BAA-894. A recA knockout mutant displayed impaired growth, desiccation tolerance, and biofilm formation. In a rat model, the mutant demonstrated significantly reduced virulence evidenced by higher host survival rates and lower bacterial loads in blood and tissues compared to the wild-type strain. Proteomic analysis revealed extensive disruptions in protein expression, particularly downregulation of carbohydrate metabolism and respiration-related proteins, alongside increased protein deamidation and oxidation. Functional assays identified fructose-bisphosphate aldolase as a target of oxidative and deamidative damage, resulting in reduced enzymatic activity and glycolytic disruption. These findings highlight the critical role of RecA in maintaining protein homeostasis, environmental resilience, and pathogenicity in C. sakazakii, providing valuable insights for developing targeted interventions against this pathogen.IMPORTANCECronobacter sakazakii poses significant risks due to its ability to persist in low-moisture environments and cause severe neonatal infections. This study identifies RecA as a key factor in environmental resilience and virulence, making it a promising target for mitigating infections and contamination. Inhibiting RecA function could sensitize C. sakazakii to stress during production and sterilization processes, reducing its persistence in powdered infant formula. Future research on RecA-specific inhibitors may lead to innovative strategies for enhancing food safety and preventing infections caused by this pathogen.

Genomic characteristics of PD-L1-Induced resistance to EGFR-TKIs in lung adenocarcinoma.

The co-occurrence of PD-L1 positivity and EGFR mutations in advanced NSCLC often limits EGFR-TKIs effectiveness, with unclear mechanisms. We analyzed 103 treatment-naive EGFR-mutant LUAD patients from three centers, assessing PD-L1 expression and performing NGS analysis. SMO mutations and MET amplification were significantly higher in the PD-L1 ≥ 1% group versus PD-L1 < 1% group (SMO: 8% vs. 0%, p = 0.048; MET: 18% vs. 7%, p = 0.023). The DNA Damage Response and Repair (DDR) pathogenic deficiency mutations, along with biological processes and signaling pathways related to DNA recombination, cell cycle transition and abnormal phosphorylation, were more prevalent in the PD-L1 ≥ 1% group. PIK3CA and RARA clonal alterations were more common in PD-L1 < 1% group, while TP53 clonal mutations predominated in PD-L1 ≥ 1% group. Retrospective analysis showed EGFR-TKIs plus chemotherapy extended median PFS by 9.8 months, potentially overcoming EGFR-TKI monotherapy resistance. This study elucidates the genomic characteristics of PD-L1-induced resistance to EGFR-TKIs. For patients with concurrent mutations in EGFR and PD-L1 expression, a first-line treatment strategy combining EGFR-TKIs with chemotherapy may offer a more effective alternative.

Lineage- and stage-specific activity of antigen receptor gene enhancers during lymphocyte development

Lymphocyte development culminates with generation of mature B and T cells that express unique antigen receptors on the cell surface. Genes that encode the two chains of B or T cell receptors are generated via DNA recombination and expressed sequentially during development, guided by locus activating enhancer sequences. In this review we summarize our understanding of molecular mechanisms that activate these enhancers in a lineage and developmental stage-specific manner. We draw attention to 1) the distinction between chromatin accessibility and transcriptional activation of these loci, 2) incomplete understanding of mechanisms that regulate B versus T cell-specific enhancer activity and 3) transcription factors that contribute to stage-specific enhancer activation within each lineage.

Appelmans Protocol for in vitro Klebsiella pneumoniae phage host range expansion leads to induction of the novel temperate linear plasmid prophage vB_KpnS-KpLi5.

Adjuvant therapy with bacteriophage (phage) cocktails in combination with antibiotics is a therapeutic approach currently considered for treatment of infections with encapsulated, biofilm forming, and multidrug-resistant Klebsiella pneumoniae (Kp). Klebsiella phage are highly selective in targeting a bacterial capsule type. Considering the numerous Kp capsule types and other host restriction factors, phage treatment could be facilitated when generating phages with a broad host range. A modified 'Appelmans protocol' was used to create phages with an extended host range via invitro forced DNA recombination. Three T7-like Kp phages with highly colinear genomes were subjected to successive propagation on their susceptible host strains representing the capsule types K64, K27, and K23, and five Kp isolates of the same capsule types initially unsusceptible for phage lysis. After 30 propagation cycles, five phages were isolated via plaque assay. Four output phages represented the original input phages, while the fifth lysed a previously non-permissible Kp isolate, which was not lysed by any of the input phages. Surprisingly, sequence analysis revealed a novel N15/phiKO2-like phage genome (vB_KpnS_KpLi5) lacking substantial homologies to any of the used T7-like phages. This phage is not a chimeric recombinant of the applied T7-like phages, but represents a temperate phage that was induced from Kp due to the application of the input phages phages (cocktail), but not by any of them individually. Adapted phages with chimeric genomes and extended host range derived from input phages were not observed. Induction of temperate phages may be a stress response caused by using multiple phages simultaneously (i.e., by destabilization of the cell wall due to an unspecific binding of the phages). Successive use of different phages for therapeutic purposes may be preferable over simultaneous application in cocktail formulations to avoid undesired induction of temperate phages.

DNA duplication-mediated activation of a two-component regulatory system serves as a bet-hedging strategy for Burkholderia thailandensis.

Burkholderia thailandensis strain E264 (BtE264) and close relatives stochastically duplicate a 208.6 kb region of chromosome I via RecA-dependent recombination between two nearly identical insertion sequence elements. Because homologous recombination occurs at a constant, low level, populations of BtE264 are always heterogeneous, but cells containing two or more copies of the region (Dup+) have an advantage, and hence predominate, during biofilm growth, while those with a single copy (Dup-) are favored during planktonic growth. Moreover, only Dup+ bacteria form 'efficient ' biofilms within 24 hours in liquid medium. We determined that duplicate copies of a subregion containing genes encoding an archaic chaperone-usher pilus (aplFABCDE) and a two-component regulatory system (bubSR) are necessary and sufficient for generating efficient biofilms and for conferring a selective advantage during biofilm growth. BubSR functionality is required, as deletion of either bubS or bubR, or a mutation predicted to abrogate phosphorylation of BubR, abrogates biofilm formation. However, duplicate copies of the aplFABCDE genes are not required. Instead, we found that BubSR controls expression of aplFABCDE and bubSR by activating a promoter upstream of aplF during biofilm growth or when the 208.6 kb region, or just bubSR, are duplicated. Single cell analyses showed that duplication of the 208.6 kb region is sufficient to activate BubSR in 75% of bacteria during planktonic (BubSR 'OFF') growth conditions. Together, our data indicate that the combination of deterministic two-component signal transduction and stochastic, duplication-mediated activation of that TCS form a bet-hedging strategy that allows BtE264 to survive when conditions shift rapidly from those favoring planktonic growth to those requiring biofilm formation, such as may be encountered in the soils of Southeast Asia and Northern Australia. Our data highlight the positive impact that transposable elements can have on the evolution of bacterial populations.