Phage Attachment Site Research Articles

Fluoroquinolone resistance does not facilitate phage Φ13 integration or excision in Staphylococcus aureus.

Prophages of the ΦSa3int family are commonly found in human-associated strains of Staphylococcus aureus where they encode factors for evading the human innate immune system. In contrast, they are usually absent in livestock-associated methicillin-resistant S. aureus (LA-MRSA) strains where the phage attachment site is mutated compared to the human strains. However, ΦSa3int phages have been found in a subset of LA-MRSA strains belonging to clonal complex 398 (CC398), including a lineage that is widespread in pig farms in Northern Jutland, Denmark. This lineage contains amino acid changes in the DNA topoisomerase IV and the DNA gyrase encoded by grlA and gyrA, respectively, which have been associated with fluoroquinolone (FQ) resistance. As both of these enzymes are involved in DNA supercoiling, we speculated that the mutations might impact recombination between the ΦSa3int phage and the bacterial chromosome. To examine this, we introduced the FQ resistance mutations into S. aureus 8325-4attBLA that carry the mutated CC398-like bacterial attachment site for ΦSa3int phages. When monitoring phage integration and release of Φ13, a well-described representative of the ΦSa3int phage family, we did not observe any significant differences between the FQ-resistant mutant and the wild-type strain. Thus our results suggest that mutations in grlA and gyrA do not contribute to the presence of the ΦSa3int phages in LA-MRSA CC398.

Integration of Multiple Phage Attachment Sites System to Create the Chromosomal T7 System for Protein Production in Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic used to treat gastrointestinal diseases. The probiotic and endotoxin-free characteristics of EcN support its potential to be developed into a microbial expression system. With this aim, in this study, the powerful T7 expression system was constructed in the cryptic plasmid-free EcN (EcNP) to generate the T7 expression host ENL6P. The concept of multiple copies of gene expression cassettes regulated by the chromosomal T7 promoter was promoted due to plasmid instability issues with protein production in ENL6P. The integration of multiple phage attachment sites (IMPACT) system, which combined Cre-lox72, CRIM, and lambda red recombinase systems, was designed to simplify the manipulation and achieve the multiple φ80 bacterial attachment sites (attB) in ENL6P to generate the new strain ENL6PP4 with four φ80 attB sites. The strain can simultaneously integrate four copies of gene expression cassettes in the chromosome to produce recombinant proteins. The IMPACT systems incorporated several tools in gene editing to rapidly achieve more robust and stable microbial strains for research and various industrial applications.

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli.

We describe a new set of tools for inserting DNA into the bacterial chromosome. The system uses site-specific recombination reactions carried out by bacteriophage integrases to integrate plasmids at up to eight phage attachment sites in E. coli MG1655. The introduction of mutant loxP sites in the integrating plasmids allows repeated removal of antibiotic resistance genes and other plasmid sequences without danger of inducing chromosomal rearrangements. The protocol for Cre-mediated antibiotic resistance gene removal is greatly simplified by introducing the Cre plasmid by phage infection. Finally, we have also developed a set of four independently inducible expression modules with tight control and high dynamic range which can be inserted at specific chromosomal locations.

The Site-Specific Recombination System of the Escherichia coli Bacteriophage Φ24B.

Stx bacteriophages are members of the lambdoid group of phages and are responsible for Shiga toxin (Stx) production and the dissemination of Shiga toxin genes (stx) across shigatoxigenic Escherichia coli (STEC). These toxigenic bacteriophage hosts can cause life-threatening illnesses, and Stx is the virulence determinant responsible for the severe nature of infection with enterohemorrhagic E. coli, a subset of pathogenic STEC. Stx phages are temperate, and in the present study, the identification of what is actually required for Stx phage Φ24B and bacterial DNA recombination was tested using both in vitro and in situ recombination assays. It is well established that phage λ, which underpins most of what we understand about lambdoid phage biology, requires its own encoded phage attachment site (attP) of 250 bp, a host-encoded attachment site (attB) of 21 bp, and a host-encoded DNA binding protein known as integration host factor (IHF). The assays applied in this study enabled the manipulation of the phage attachment site (attP) and the bacterial attachment site (attB) sequences and the inclusion or exclusion of a host-encoded accessory element known as integration host factor. We were able to demonstrate that the minimal attP sequence required by Φ24B phage is between 350 and 427 bp. Unlike phage λ, the minimal necessary flanking sequences for the attB site do not appear to be equal in size, with a total length between 62 and 93 bp. Furthermore, we identified that the Φ24B integrase does not require IHF to drive the integration and the recombination process. Understanding how this unusual Stx phage integrase works may enable exploitation of its promiscuous nature in the context of genetic engineering.

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase.

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186.

The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

A novel approach for T7 bacteriophage genome integration of exogenous DNA

BackgroundThe comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ΦC31 integrase is one of the most robust site-specific recombination systems. ΦC31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ΦC31 integrase.MethodsPlasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7∆G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7∆G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7∆G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths.ResultsT7∆G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ΦC31 integrase (attB) was confirmed to be present in the T7∆G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7∆G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7∆G10G11-attB was explored, and it’s found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably.ConclusionWe advanced and established a novel approach for gene knock-in into the T7 genome using ΦC31 integrase.

CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

BackgroundSynthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites.ResultsBy combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated.ConclusionsThe CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis

BackgroundSite-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901–1 integrating system.ResultsThe constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901–1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.ConclusionThe engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters.

The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.

The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif.

To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase.

Commercial Biocides Induce Transfer of Prophage Φ13 from Human Strains of Staphylococcus aureus to Livestock CC398.

Human strains of Staphylococcus aureus commonly carry the bacteriophage ΦSa3 that encodes immune evasion factors. Recently, this prophage has been found in livestock-associated, methicillin resistant S. aureus (MRSA) CC398 strains where it may promote human colonization. Here, we have addressed if exposure to biocidal products induces phage transfer, and find that during co-culture, Φ13 from strain 8325, belonging to ΦSa3 group, is induced and transferred from a human strain to LA-MRSA CC398 when exposed to sub-lethal concentrations of commercial biocides containing hydrogen peroxide. Integration of ΦSa3 in LA-MRSA CC398 occurs at multiple positions and the integration site influences the stability of the prophage. We did not observe integration in hlb encoding β-hemolysin that contains the preferred ΦSa3 attachment site in human strains, and we demonstrate that this is due to allelic variation in CC398 strains that disrupts the phage attachment site, but not the expression of β-hemolysin. Our results show that hydrogen peroxide present in biocidal products stimulate transfer of ΦSa3 from human to LA-MRSA CC398 strains and that in these strains prophage stability depends on the integration site. Knowledge of ΦSa3 transfer and stability between human and livestock strains may lead to new intervention measures directed at reducing human infection by LA-MRSA strains.

Coiled-coil interactions mediate serine integrase directionality.

Serine integrases are bacteriophage enzymes that carry out site-specific integration and excision of their viral genomes. The integration reaction is highly directional; recombination between the phage attachment site attP and the host attachment site attB to form the hybrid sites attL and attR is essentially irreversible. In a recent model, extended coiled-coil (CC) domains in the integrase subunits are proposed to interact in a way that favors the attPxattB reaction but inhibits the attLxattR reaction. Here, we show for the Listeria innocua integrase (LI Int) system that the CC domain promotes self-interaction in isolated Int and when Int is bound to attachment sites. Three independent crystal structures of the CC domain reveal the molecular nature of the CC dimer interface. Alanine substitutions of key residues in the interface support the functional significance of the structural model and indicate that the same interaction is responsible for promoting integration and for inhibiting excision. An updated model of a LI Int•attL complex that incorporates the high resolution CC dimer structure provides insights that help to explain the unusual CC dimer structure and potential sources of stability in Int•attL and Int•attR complexes. Together, the data provide a molecular basis for understanding serine integrase directionality.

A novel genetic tool for metabolic optimization of Corynebacterium glutamicum: efficient and repetitive chromosomal integration of synthetic promoter-driven expression libraries.

Fine-tuning the expression level of multiple genes is usually pivotal for metabolic optimization. We have developed a tool for this purpose for the important industrial workhorse Corynebacterium glutamicum that allows for the introduction of synthetic promoter-driven expression libraries of arbitrary genes. We first devised a method for introducing genetic elements into the chromosome repeatedly, relying on site-specific recombinases and the vector pJS31 serving as the carrier. The pJS31 vector contains a synthetic cassette including a phage attachment site attP for integration, a bacterial attachment site attB for subsequent integration, a multiple cloning site, and two modified loxP sites to facilitate easy removal of undesirable vector elements. Meanwhile, we constructed a derivative of the wild-type strain ATCC 13032 carrying an attB site in its chromosome (JS34) and demonstrated that pJS31 readily could integrate into the attB site in this strain providing expression of the corresponding integrase. Subsequent expression of the Cre recombinase promoted recombination between the modified loxP sites, resulting in a strain only retaining the target insertions and an attB site. To simplify the procedure, non-replicating circular expression units for the phage integrase and the Cre recombinase were used. As a showcase, we used the tool to construct a battery of strains simultaneously expressing the two reporter genes, lacZ (encoding β-galactosidase) and gusA (encoding β-glucuronidase), to arbitrary levels. In principle, an unlimited number of genes, whether native, heterologous, or synthetic, can be introduced using the developed approach, and this should greatly facilitate metabolic optimization of this important platform organism.

Molecular characterization of the activity and requirements of a novel and promiscuous bacteriophage integrase

Stx bacteriophages are responsible for the dissemination and production of Shiga toxin genes (stx) across the Shigatoxigenic E. coli (STEC). These toxigenic bacteriophage hosts can cause severe, life-threatening illness, and Shiga toxin (Stx) is responsible for the severe nature of EHEC infection, a subset of pathogenic STEC. At the point of Stx phage infection, the injected phage DNA can direct its integration into the bacterial chromosome becoming a prophage; the host cell is then known as a lysogen. Unusually, our model Stx phage, Φ24B, can integrate into at least four distinct sites within the E. coli genome that shared no easily identifiable recognition sequence pattern. The identification of what are actually required for phage and bacterial DNAs recombination has been tested using both in vitro and in situ recombination assays. These assays enabled the simple manipulation of bacterial attachment site (attB) and phage attachment site (attP) sequences. The aim of the study is to fully characterize the requirements of this promiscuous integrase, carried by the Stx phage Φ24B (IntΦ24B), to drive integration. These assays enabled us to identify the minimal necessary flanking sequences for attB site identified (21 bp and 49 bp from the right and left the cross over region, respectively) and the attP site (200 bp each side). Furthermore, we identified that the Φ24B integrase does not need Integration Host Factor (IHF) to drive integration. Fi

Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli.

Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli. We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis.

Brujita Integrase: A Simple, Arm-Less, Directionless, and Promiscuous Tyrosine Integrase System

Mycobacteriophage Brujita is an unusual temperate phage in which establishment of superinfection immunity is dependent on chromosomal integration. Integration is mediated by a non-canonical tyrosine integrase (Int) lacking an N-terminal domain typically associated with binding to arm-type sites within the phage attachment site (attP). This raises the question as to how these Ints bind their DNA substrates, if they form higher-order protein DNA complexes, and how site selection and recombinational directionality are determined. Here we show that Brujita Int is a simple recombinase, whose properties more closely resemble those of FLP and Cre than it does the canonical phage Ints. Brujita Int uses relatively small DNA substrates, fails to discriminate between attP and attB, cleaves attachment site DNA to form a 6-base overlap region, and lacks directional control. Brujita Int also has an unusual pattern of binding to its DNA substrates. It binds to two half sites (B and B′) at attB, although binding to the B half site is strongly dependent on occupancy of B′. In contrast, binding to the P half site is not observed, even when Int is bound at P′. However, an additional Int binding site (P1) is displaced to the left of the crossover site at attP, is required for recombination and is predicted to facilitate binding of Int to the P half site during synapsis. These simple phage Int systems may reflect ancestral states of phage evolution with the complexities of higher-order complex formation and directional control representing subsequent adaptations.

Analysis of the First Temperate Broad Host Range Brucellaphage (BiPBO1) Isolated from B. inopinata.

Brucella species are important human and animal pathogens. Though, only little is known about mobile genetic elements of these highly pathogenic bacteria. To date, neither plasmids nor temperate phages have been described in brucellae. We analyzed genomic sequences of various reference and type strains and identified a number of putative prophages residing within the Brucella chromosomes. By induction, phage BiPBO1 was isolated from Brucella inopinata. BiPBO1 is a siphovirus that infects several Brucella species including Brucella abortus and Brucella melitensis. Integration of the phage genome occurs adjacent to a tRNA gene in chromosome 1 (chr 1). The bacterial (attB) and phage (attP) attachment sites comprise an identical sequence of 46 bp. This sequence exists in many Brucella and Ochrobactrum species. The BiPBO1 genome is composed of a 46,877 bp double-stranded DNA. Eighty-seven putative gene products were determined, of which 32 could be functionally assigned. Strongest similarities were found to a temperate phage residing in the chromosome of Ochrobactrum anthropi ATCC 49188 and to prophages identified in several families belonging to the order rhizobiales. The data suggest that horizontal gene transfer may occur between Brucella and Ochrobactrum and underpin the close relationship of these environmental and pathogenic bacteria.

An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection—lysogenic or lytic growth—as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33–34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.