Integration Host Factor Protein Research Articles

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA.

Timely initiation of chromosomal DNA replication in Escherichia coli is achieved by cell cycle-coordinated regulation of the replication origin, oriC, and the replication initiator, ATP-DnaA. Cellular levels of ATP-DnaA increase and peak at the time for initiation at oriC, after which hydrolysis of DnaA-bound ATP causes those to fall, yielding initiation-inactive ADP-DnaA. This hydrolysis is facilitated by the chromosomal locus datA located downstream of the tRNA-Gly (glyV-X-Y) operon, which possesses a cluster of DnaA-binding sequences and a single binding site (IBS) for the DNA bending protein IHF (integration host factor). While IHF binding activates the datA function and is regulated to occur specifically at post-initiation time, the underlying regulatory mechanisms remain obscure. Here, we demonstrate that datA-IHF binding at pre-initiation time is down-regulated depending on the read-through transcription of datA IBS initiated at the glyV-X-Y promoter. During the cell cycle, the level of read-through transcription, but not promoter activity, fluctuated in a manner inversely related to datA-IHF binding. Transcription from the glyV-X-Y promoter was predominantly interrupted at datA IBS by IHF binding. The terminator/attenuator sequence of the glyV-X-Y operon, as well as DnaA binding within datA overall, contributed to attenuation of transcription upstream of datA IBS, preserving the timely fluctuation of read-through transcription. These findings provide a mechanistic insight of tRNA transcription-dependent datA-IHF regulation, in which an unidentified factor is additionally required for the timely datA-IHF dissociation, and support the significance of datA for controlling the cell cycle progression as a connecting hub of tRNA production and replication initiation.

Quantitation of DNA Binding Affinity Using Tethered Particle Motion.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered particle motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to measure binding constants of proteins binding to DNA reliably, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This change in bead motion makes it possible to measure the DNA-binding properties of proteins. We use the bacterial protein integration host factor (IHF) and the archaeal histone HMfA as examples to show how specific binding to DNA can be measured. Moreover, we show how the end-to-end distance can provide structural insights into protein-DNA binding.

Z-Form Extracellular DNA in Pediatric CRS May Provide a Mechanism for Recalcitrance to Treatment.

We examined sinus mucosal samples recovered from pediatric chronic rhinosinusitis (CRS) patients for the presence of Z-form extracellular DNA (eDNA) due to its recently elucidated role in pathogenesis of disease. Further, we immunolabeled these specimens for the presence of both members of the bacterial DNA-binding DNABII protein family, integration host factor (IHF) and histone-like protein (HU), due to their known role in converting common B-DNA to the rare Z-form. Sinus mucosa samples recovered from 20 patients during functional endoscopic sinus surgery (FESS) were immunolabelled for B- and Z-DNA, as well as for both bacterial DNABII proteins. Nineteen of 20 samples (95%) included areas rich in eDNA, with the majority in the Z-form. Areas positive for B-DNA were restricted to the most distal regions of the mucosal specimen. Labeling for both DNABII proteins was observed on B- and Z-DNA, which aligned with the role of these proteins in the B-to-Z DNA conversion. Abundant Z-form eDNA in culture-positive pediatric CRS samples suggested that bacterial DNABII proteins were responsible for the conversion of eukaryotic B-DNA that had been released into the luminal space by PMNs during NETosis, to the Z-form. The presence of both DNABII proteins on B-DNA and Z-DNA supported the known role of these bacterial proteins in the B-to-Z DNA conversion. Given that Z-form DNA both stabilizes the bacterial biofilm and inactivates PMN NET-mediated killing of trapped bacteria, we hypothesize that this conversion may be contributing to the chronicity and recalcitrance of CRS to treatment. NA Laryngoscope, 134:1564-1571, 2024.

Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara.

Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara. These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara. Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin.

Nucleoid-associated proteins (NAPs) crucially contribute to organizing bacterial chromatin and regulating gene expression. Among the most highly expressed NAPs are the HU and integration host factor (IHF) proteins, whose functional homologues, HupB and mycobacterial integration host factor (mIHF), are found in mycobacteria. Despite their importance for the pathogenicity and/or survival of tubercle bacilli, the role of these proteins in mycobacterial chromosome organization remains unknown. Here, we used various approaches, including super-resolution microscopy, to perform a comprehensive analysis of the roles of HupB and mIHF in chromosome organization. We report that HupB is a structural agent that maintains chromosome integrity on a local scale, and that the lack of this protein alters chromosome morphology. In contrast, mIHF is a highly dynamic protein that binds DNA only transiently, exhibits susceptibility to the chromosomal DNA topology changes and whose depletion leads to the growth arrest of tubercle bacilli. Additionally, we have shown that depletion of Mycobacterium smegmatis integration host factor (msIHF) leads to chromosome shrinkage and replication inhibition.

The integration host factor regulates multiple virulence pathways in bacterial pathogen Dickeya zeae MS2.

Dickeya zeae is an aggressive bacterial phytopathogen that infects a wide range of host plants. It has been reported that integration host factor (IHF), a nucleoid‐associated protein consisting of IHFα and IHFβ subunits, regulates gene expression by influencing nucleoid structure and DNA bending. To define the role of IHF in the pathogenesis of D. zeae MS2, we deleted either and both of the IHF subunit encoding genes ihfA and ihfB, which significantly reduced the production of cell wall‐degrading enzymes (CWDEs), an unknown novel phytotoxin and the virulence factor‐modulating (VFM) quorum‐sensing (QS) signal, cell motility, biofilm formation, and thereafter the infection ability towards both potato slices and banana seedlings. To characterize the regulatory pathways of IHF protein associated with virulence, IHF binding sites (consensus sequence 5′‐WATCAANNNNTTR‐3′) were predicted and 272 binding sites were found throughout the genome. The expression of 110 tested genes was affected by IHF. Electrophoretic mobility shift assay (EMSA) showed direct interaction of IhfA protein with the promoters of vfmE, speA, pipR, fis, slyA, prtD, hrpL, hecB, hcp, indA, hdaA, flhD, pilT, gcpJ, arcA, arcB, and lysR. This study clarified the contribution of IHF in the pathogenic process of D. zeae by controlling the production of VFM and putrescine QS signals, phytotoxin, and indigoidine, the luxR‐solo system, Fis, SlyA, and FlhD transcriptional regulators, and secretion systems from type I to type VI. Characterization of the regulatory networks of IHF in D. zeae provides a target for prevention and control of plant soft rot disease.

Negative feedback forDARS2–Fis complex by ATP–DnaA supports the cell cycle-coordinated regulation for chromosome replication

In Escherichia coli, the replication initiator DnaA oscillates between an ATP- and an ADP-bound state in a cell cycle-dependent manner, supporting regulation for chromosome replication. ATP–DnaA cooperatively assembles on the replication origin using clusters of low-affinity DnaA-binding sites. After initiation, DnaA-bound ATP is hydrolyzed, producing initiation-inactive ADP–DnaA. For the next round of initiation, ADP–DnaA binds to the chromosomal locus DARS2, which promotes the release of ADP, yielding the apo-DnaA to regain the initiation activity through ATP binding. This DnaA reactivation by DARS2 depends on site-specific binding of IHF (integration host factor) and Fis proteins and IHF binding to DARS2 occurs specifically during pre-initiation. Here, we reveal that Fis binds to an essential region in DARS2 specifically during pre-initiation. Further analyses demonstrate that ATP–DnaA, but not ADP–DnaA, oligomerizes on a cluster of low-affinity DnaA-binding sites overlapping the Fis-binding region, which competitively inhibits Fis binding and hence the DARS2 activity. DiaA (DnaA initiator-associating protein) stimulating ATP–DnaA assembly enhances the dissociation of Fis. These observations lead to a negative feedback model where the activity of DARS2 is repressed around the time of initiation by the elevated ATP–DnaA level and is stimulated following initiation when the ATP–DnaA level is reduced.

Whole-Genome Analysis Reveals That the Nucleoid Protein IHF Predominantly Binds to the Replication Origin oriC Specifically at the Time of Initiation.

The structure and function of bacterial chromosomes are dynamically regulated by a wide variety of nucleoid-associated proteins (NAPs) and DNA superstructures, such as DNA supercoiling. In Escherichia coli, integration host factor (IHF), a NAP, binds to specific transcription promoters and regulatory DNA elements of DNA replication such as the replication origin oriC: binding to these elements depends on the cell cycle but underlying mechanisms are unknown. In this study, we combined GeF-seq (genome footprinting with high-throughput sequencing) with synchronization of the E. coli cell cycle to determine the genome-wide, cell cycle-dependent binding of IHF with base-pair resolution. The GeF-seq results in this study were qualified enough to analyze genomic IHF binding sites (e.g., oriC and the transcriptional promoters of ilvG and osmY) except some of the known sites. Unexpectedly, we found that before replication initiation, oriC was a predominant site for stable IHF binding, whereas all other loci exhibited reduced IHF binding. To reveal the specific mechanism of stable oriC–IHF binding, we inserted a truncated oriC sequence in the terC (replication terminus) locus of the genome. Before replication initiation, stable IHF binding was detected even at this additional oriC site, dependent on the specific DnaA-binding sequence DnaA box R1 within the site. DnaA oligomers formed on oriC might protect the oriC–IHF complex from IHF dissociation. After replication initiation, IHF rapidly dissociated from oriC, and IHF binding to other sites was sustained or stimulated. In addition, we identified a novel locus associated with cell cycle-dependent IHF binding. These findings provide mechanistic insight into IHF binding and dissociation in the genome.

Antibodies against the DNABII protein integration host factor (IHF) inhibit sinus implant biofilms.

Chronic rhinosinusitis is a common, costly condition often treated with endoscopic sinus surgery and intraoperative placement of intranasal sinus implant materials. Whereas these materials aid in postoperative healing, they also support bacterial biofilm formation and thus contribute to negative outcomes. This study examined pretreatment of sinus implant materials with antibody against an essential bacterial biofilm structural component, the DNABII family of DNA-binding proteins, as a strategy to prevent biofilm formation. Sinus implant materials were equilibrated in immunoglobulin G (IgG)-enriched antiserum against the DNABII protein integration host factor (IHF), individually or in combination with amoxicillin-clavulanate prior to inoculation with nontypeable Haemophilus influenzae (NTHI), a predominant pathogen of chronic rhinosinusitis. After 16 hours, the bacterial burden was quantitated and compared to pretreatment with saline, IgG-enriched naive serum, or amoxicillin-clavulanate alone. NTHI readily formed biofilms on all three materials in vitro. However, pretreatment of each material with IgG-enriched anti-IHF resulted in a significant decrease in bacterial burden compared to controls (P ≤ 0.05). Moreover, a significant and synergistic outcome was achieved with a cocktail of anti-IHF plus amoxicillin-clavulanate (P ≤ 0.05) with complete inhibition of NTHI biofilm formation on all three materials. Biofilm formation was well supported in vitro on three sinus implant materials that vary in composition and resorption characteristics; however, pretreatment of each with DNABII protein targeted antibodies in combination with a previously ineffective antibiotic was highly effective to prevent the formation NTHI biofilms. These data demonstrate the potential for clinical utility of pretreatment of sinus implant and additional surgical materials with anti-DNABII antibodies. NA Laryngoscope, 130:1364-1371, 2020.

Characterization of balofloxacin-stressed proteomics and identification of balofloxacin-binding proteins pre-peptidase and integration host factor in Edwardsiella tarda

The overuse of antibiotics to control bacterial pathogens leads to the generation of their antibiotic-resistant strains including Edwardsiella tarda. Understanding of mechanisms of the antibiotic resistance is crucial to develop novel methods to manage the infection. Here, two-dimensional electrophoresis-based proteomics was used to characterize balofloxacin-responsive proteins. The altered proteome consisted of 19 proteins with differential abundance, where six metabolic pathways were enriched. The metabolic modulation activated the central carbon metabolism with elevation of NADH, PMF, and ATP. Among the 19 proteins, ETAE_1987 (pre-peptidase) and ETAE_2174 (integration host factor beta subunit) were bound with balofloxacin directly. This was further confirmed by the binding of balofloxacin with recombinant ETAE_1987 and ETAE_2174 using Oxford cup method. Compared with bovine serum albumin, a known balofloxacin-binding protein, ETAE_1987 and ETAE_2174 increased the binding capability by 3.3- and 22-fold, respectively. The combination was validated by microscale thermophoresis. These data characterize the balofloxacin-stressed proteome as a result of the increased central carbon metabolism and energy metabolism and determine ETAE_1987 and ETAE_2174 as balofloxacin-binding proteins. These findings have significant implications in understanding bacterial antibiotic-resistant and drug action mechanisms based on balofloxacin-binding proteins. SignificanceAntibiotic-resistant Edwardsiella tarda strains are frequently isolated and cause a great loss in aquaculture since these bacterial strains are insensitivity to antibiotics. The present study showed that the increased central carbon metabolism forms a characteristic feature of the balofloxacin-stressed proteomics. Furthermore, two proteins, ETAE_1987 (pre-peptidase) and ETAE_2174, of the balofloxacin-stressed proteome were identified as balofloxacin-binding proteins. The binding capability is 0.39 ± 0.017 and 2.67 ± 0.066 ng/μg proteins for ETAE_1987 and ETAE_2174, respectively. These results reveal the elevated central carbon metabolism as a key feature of the balofloxacin-stressed proteomics and pre-peptidase and integration host factor as balofloxacin-binding proteins in E. tarda. These findings are useful in the understanding of bacterial balofloxacin-stressed mechanisms and providing new targets for controlling antibiotic-resistant bacteria.

Characterizing Watson-Crick versus Hoogsteen Base Pairing in a DNA-Protein Complex Using Nuclear Magnetic Resonance and Site-Specifically 13C- and 15N-Labeled DNA.

A( syn)-T and G( syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF ), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interproton nuclear Overhauser effects indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (∼2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.

To Kink or Not to Kink: Sequence-Dependent DNA Flexibility Unveiled in Complex with DNA-Bending Protein IHF

Site-specific recognition by DNA-binding proteins often involves severe DNA deformations including sharp kinks. Yet, it has been unclear how rigid or flexible these protein-induced kinks are. Here we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H’) into a U-turn by sharply kinking DNA at two sites by ∼90°. We utilized fluorescence lifetime-based FRET spectroscopy and revealed a surprisingly dynamic IHF-H’ specific complex: while 78% of the IHF-H’ population exhibited FRET efficiency consistent with the crystal structure, 22% exhibited FRET efficiency indicative of partially bent DNA, with one side or the other unkinked. This conformational flexibility was modulated by sequence variations in the cognate site. Notably, IHF bound to another cognate site, H1, that differs from H’ primarily by the lack of an A-tract on one side, exhibited a significantly higher population (68%) in the partially bent conformations. These differences in their dynamics reflect the different roles of these complexes in their natural context, in the phage lambda “intasome”. The population distribution between bent/unbent conformations could be further tuned by introducing mismatches in the DNA at one of the kink sites, to enhance DNA “kinkability” at that site. We propose to take advantage of this FRET assay to further investigate sequence-dependent flexibility at the kink sites by performing SELEX studies for tight-binders to IHF, selected from a pool of semi-randomized sequences, with segments that encompass the kink sites chosen to be randomized for selection of the most “kinkable” (by IHF) DNA sequences. These SELEX studies are expected to go beyond similar studies on nucleosomes and reveal sequence patterns that significantly enhance DNA flexibility.

Reconstitution of CRISPR adaptation in vitro and its detection by PCR.

CRISPR adaptation is the initial step in CRISPR-Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1-Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Structural and biochemical studies from reconstituted complexes have provided mechanistic insight into how CRISPR adaptation occurs; however, these studies have been limited to a narrow subset of CRISPR-Cas types and may not be representative of the other types. Here we describe methods for the purification of the type I-F CRISPR adaptation complex (Cas1:Cas2-3) from Pectobacterium atrosepticum, purification of the DNA architectural protein integration host factor (IHF), and a sensitive PCR-based in vitro integration assay. This assay could easily be used to investigate mechanisms of CRISPR adaptation in other CRISPR-Cas systems, including the roles of accessory proteins.

RpoN and the Nps and Npa two-component regulatory system control pilE transcription in commensal Neisseria.

Over 20 genes are involved in the biogenesis and function of the Neisseria Type IV pilus (Tfp). In the pathogenic species, RpoD and the integration host factor (IHF) protein regulate expression of pilE, encoding the Tfp structural subunit. We previously reported that in commensal species, pilE transcription is regulated by RpoN, IHF, and activator Npa. Npa has many hallmarks of response regulators in two‐component regulatory systems, leading us to search for its response regulator partner. We report that Npa partners with sensor kinase Nps to control pilE transcription. Among the genes involved in Tfp biogenesis and function, only pilE is controlled by RpoN and Npa/Nps. We summarize our findings in a model, and discuss the implications of the differential regulation of pilE the context of Neisseria Tfp biogenesis.

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered Particle Motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM.

Physical and Functional Characterization of a Viral Genome Maturation Complex

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Structural and evolutionary analyses reveal determinants of DNA binding specificities of nucleoid-associated proteins HU and IHF

Nucleoid-associated proteins (NAPs) are chromosome-organizing factors, which affect the transcriptional landscape of a bacterial cell. HU is an NAP, which binds to DNA with a broad specificity while homologous IHF (Integration Host Factor), binds DNA with moderately higher specificity. Specificity and differential binding affinity of HU/IHF proteins towards their target binding sites play a crucial role in their regulatory dynamics. Decades of biochemical and genomic studies have been carried out for HU and IHF like proteins. Yet, questions related to their DNA binding specificity, and differential ability to bend DNA thus affecting the binding site length remained unanswered. In addition, the problem has not been investigated from an evolutionary perspective. Our phylogenetic analysis revealed three major clades belonging to HU, IHFα and IHFβ like proteins with reference to E. coli. We carried out a comparative analysis of three-dimensional structures of HU/IHF proteins to gain insight into the structural basis of clade division. The present study revealed three major features which contribute to differential DNA binding specificity of HU/IHF proteins, (I) conformational restriction of DNA binding residues due to salt-bridge formation, (II) the enrichment of alanine in the DNA binding site increasing conformational space of flexible side chains in its vicinity and (III) nature of DNA binding residue (Arg to Lys bias in different clades) which interacts differentially to DNA bases. We observed an extended electropositive surface at the DNA draping site for IHF clade proteins compared to HU, which stabilizes the DNA bend. Differences in the dimer stabilization strategies between HU and IHF were also observed. Our analysis reveals a comprehensive evolutionary picture, which rationalizes the origin of multi-specificity of HU/IHF proteins using sequence and structure-based determinants, which could also be applied to understand differences in binding specificities of other nucleic acid binding proteins.

CRISPR Immunological Memory Requires a Host Factor for Specificity

Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci invivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E.coli integration host factor (IHF) protein is required for spacer acquisition invivo and for integration into linear DNA invitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.

Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzT Promoter.

PatzT is an internal promoter of the atzRSTUVW operon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzT is σ(N) dependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzT promoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzT promoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σ(N) and PatzT that lead to open complex formation. Here, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σ(N)-dependent promoter PatzT Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model.

Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.