The structure of the oligomerization domain of Lsr2 from Mycobacterium tuberculosis reveals a mechanism for chromosome organization and protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 ? resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ?-?-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.     

Lsr2 of Mycobacterium represents a novel class of H-NS-like proteins

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Our previous in vitro biochemical studies showed that Lsr2 is a DNA-bridging protein, a property shared by H-NS-like proteins in gram-negative bacteria. Here we present in vivo evidence based on genetic complementation experiments that Lsr2 is a functional analog of H-NS, the first such protein identified in gram-positive bacteria. We show that lsr2 can complement the phenotypes related to hns mutations in Escherichia coli, including β-glucoside utilization, mucoidy, motility, and hemolytic activity. We also show that Lsr2 binds specifically to H-NS-regulated genes and the repression of hlyE by Lsr2 can be partially eliminated by overexpression of slyA, suggesting that the molecular mechanisms of Lsr2 repression and depression are similar to those of H-NS. The functional equivalence of these two proteins is further supported by the ability of hns to complement the lsr2 phenotype in Mycobacterium smegmatis. Taken together, our results demonstrate unequivocally that Lsr2 is an H-NS-like protein.     

Mechanics of DNA bridging by bacterial condensin MukBEF in vitro and in singulo

Structural maintenance of chromosome (SMC) proteins comprise the core of several specialized complexes that stabilize the global architecture of the chromosomes by dynamically linking distant DNA fragments. This reaction however remains poorly understood giving rise to numerous proposed mechanisms of the proteins. Using two novel assays, we investigated real‐time formation of DNA bridges by bacterial condensin MukBEF. We report that MukBEF can efficiently bridge two DNAs and that this reaction involves multiple steps. The reaction begins with the formation of a stable MukB–DNA complex, which can further capture another protein‐free DNA fragment. The initial tether is unstable but is quickly strengthened by additional MukBs. DNA bridging is modulated but is not strictly dependent on ATP and MukEF. The reaction revealed high preference for right‐handed DNA crossings indicating that bridging involves physical association of MukB with both DNAs. Our data establish a comprehensive view of DNA bridging by MukBEF, which could explain how SMCs establish both intra‐ and interchromosomal links inside the cell and indicate that DNA binding and bridging could be separately regulated.     

Phosphoenolpyruvate phosphotransferase system regulates detection and processing of the quorum sensing signal autoinducer-2

Autoinducer‐2 (AI‐2) a signal produced by a range of phylogenetically distant microorganisms, enables inter‐species cell–cell communication and regulates many bacterial phenotypes. Certain bacteria can interfere with AI‐2‐regulated behaviours of neighbouring species by internalizing AI‐2 using the Lsr transport system (encoded by the lsr operon). AI‐2 imported by the Lsr is phosphorylated by the LsrK kinase and AI‐2‐phosphate is the inducer of the lsr operon. Here we show that in Escherichia coli the phosphoenolpyruvate phosphotransferase system (PTS) is required for Lsr activation and is essential for AI‐2 internalization. Although the phosphorylation state of Enzyme I of PTS is important for this regulation, LsrK is necessary for the phosphorylation of AI‐2, indicating that AI‐2 is not phosphorylated by PTS. Our results suggest that AI‐2 internalization is initiated by a PTS‐dependent mechanism, which provides sufficient intracellular AI‐2 to relieve repression of the lsr operon and, thus induce depletion of AI‐2 from the extracellular environment. The fact that AI‐2 internalization is not only controlled by the community‐dependent accumulation of AI‐2, but also depends on the phosphorylation state of PTS suggests that E. coli can integrate information on the availability of substrates with external communal information to control quorum sensing and its interference.     

Proteins as supramolecular building blocks: Nterm‐Lsr2 as a new protein tecton

Proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics, and pharmaceuticals. The potential to produce nanomaterials from proteins is well supported by the numerous examples of self‐assembling proteins found in nature. We have explored self‐assembling proteins for use as supramolecular building blocks, or tectons, specifically the N‐terminal domain of Lsr2, Nterm‐Lsr2. A key feature of this protein is that it undergoes self‐assembly via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. Herein, we report the effects of pH and protein concentration on the oligomerization of Nterm‐Lsr2. Furthermore, via protein engineering, we have introduced a new trigger for oligomerization via enteropeptidase cleavage. The new construct of Nterm‐Lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 260–270, 2015.     

Impact of Xenogeneic Silencing on Phage–Host Interactions

Phages, viruses that prey on bacteria, are the most abundant and diverse inhabitants of the Earth. Temperate bacteriophages can integrate into the host genome and, as so-called prophages, maintain a long-term association with their host. The close relationship between host and virus has significantly shaped microbial evolution and phage elements may benefit their host by providing new functions. Nevertheless, the strong activity of phage promoters and potentially toxic gene products may impose a severe fitness burden and must be tightly controlled. In this context, xenogeneic silencing (XS) proteins, which can recognize foreign DNA elements, play an important role in the acquisition of novel genetic information and facilitate the evolution of regulatory networks. Currently known XS proteins fall into four classes (H-NS, MvaT, Rok and Lsr2) and have been shown to follow a similar mode of action by binding to AT-rich DNA and forming an oligomeric nucleoprotein complex that silences gene expression. In this review, we focus on the role of XS proteins in phage–host interactions by highlighting the important function of XS proteins in maintaining the lysogenic state and by providing examples of how phages fight back by encoding inhibitory proteins that disrupt XS functions in the host. Sequence analysis of available phage genomes revealed the presence of genes encoding Lsr2-type proteins in the genomes of phages infecting Actinobacteria. These data provide an interesting perspective for future studies to elucidate the impact of phage-encoded XS homologs on the phage life cycle and phage–host interactions.     

Defining a temporal order of genetic requirements for development of mycobacterial biofilms

Most mycobacterial species spontaneously form biofilms, inducing unique growth physiologies and reducing drug sensitivity. Biofilm growth progresses through three genetically programmed stages: substratum attachment, intercellular aggregation and architecture maturation. Growth of Mycobacterium smegmatis biofilms requires multiple factors including a chaperonin (GroEL1) and a nucleoid‐associated protein (Lsr2), although how their activities are linked remains unclear. Here it is shown that Lsr2 participates in intercellular aggregation, but substratum attachment of Lsr2 mutants is unaffected, thereby genetically distinguishing these developmental stages. Further, a suppressor mutation in a glycopeptidolipid synthesis gene (mps) that results in hyperaggregation of cells and fully restores the form and functions of Δlsr2 mutant biofilms was identified. Suppression by the mps mutation is specific to Δlsr2; it does not rescue the maturation‐deficient biofilms of a ΔgroEL1 mutant, thereby differentiating the process of aggregation from maturation. Gene expression analysis supports a stepwise process of maturation, highlighted by temporally separated, transient inductions of iron and nitrogen import genes. Furthermore, GroEL1 activity is required for induction of nitrogen, but not iron, import genes. Together, the findings begin to define molecular checkpoints during development of mycobacterial biofilms.     

The LuxS-dependent autoinducer AI-2 controls the expression of an ABC transporter that functions in AI-2 uptake in Salmonella typhimurium

In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi, is made by many species of Gram-negative and Gram-positive bacteria. In every case, production of AI-2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known. Here, we describe the identification and characterization of AI-2-regulated genes in Salmonella typhimurium. We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium. A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells. It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.     

Structure,dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD⁺ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.     

Patterning protein complexes on DNA nanostructures using a GFP nanobody

DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies.     

Cancer-related diseases of the eye: the role of calcium and calcium-binding proteins

The eye provides unique opportunities to study complex biochemical pathways and to describe how components of these pathways contribute to the molecular basis of disease. In this article, the role of calcium-binding proteins in cancer-related diseases of the eye is reviewed. First, paraneoplastic syndromes, or so-called remote effects of cancer, arise from damage to tissues distant from any tumor or its metastases. Many of these syndromes are believed to be immune-mediated. Cancer-associated retinopathy (CAR), a blinding disease due to the degeneration of retinal photoreceptor cells, is one of the best characterized of the paraneoplastic syndromes. The CAR autoantigen has been identified as recoverin, a calcium-binding protein of the EF-hand superfamily. Its features as a calcium-binding protein, along with its function in photoreceptor cells and its role as the CAR autoantigen, are discussed. Next, unlike visual symptoms instigated by a distant tumor, ocular melanoma is the primary malignancy originating in the eye. ALG-2 encodes a pro-apoptotic calcium-binding protein that is down-regulated in ocular melanoma, thus providing these tumor cells with a selective advantage. In addition to background discussion of ALG-2, data describing the expression, cellular localization, and dimerization characteristics of ALG-2 in melanoma cells are presented. Biochemical studies of ALG-2 and its interactions with its target Alix/AIP1 also are presented. Finally, the function of ALG-2 in calcium-induced cell death is discussed. Additional calcium-binding proteins in retina and in ocular tumors are described in relation to different disease entities. Such proteins and their expression in the eye provide valuable examples bridging studies of protein chemistry, cellular function, and human disease.     

H-NS promotes looped domain formation in the bacterial chromosome

The bacterial chromosome is organized into loops, which constitute topologically isolated domains. It is unclear which proteins are responsible for the formation of the topological barriers between domains. The abundant DNA-binding histone-like nucleoid structuring protein (H-NS) is a key player in the organization and compaction of bacterial chromosomes [1,2]. The protein acts by bridging DNA duplexes [3], thus allowing for the formation of DNA loops. Here, genome-wide studies of H-NS binding suggest that this protein is directly involved in the formation or maintenance of topological domain barriers.