Bacteriophage Mu Research Articles

Bacteriophage Mu C protein is a new member of unusual leucine zipper-HTH class of proteins

Transcription activator protein C of bacteriophage Mu activates transcription of the late genes, including mom, during the lytic cycle of the phage. C binding to its site leads to the alteration in DNA topology of the promoter elements resulting in RNA polymerase (RNAP) recruitment. At the next step, the transactivator enhances promoter clearance of RNAP from P(mom). The C protein binds DNA with a very high affinity using a carboxyl-terminal helix turn helix (HTH) motif which has similarity with the HTH from paired domain of Drosophila prd protein. Previous studies established that the protein is dimeric in free and DNA bound forms. We describe now the unique dimerization interface of the protein. Two heptad repeats of hydrophobic amino acids found in the protein were considered to be the candidates for dimerization region. Site-directed mutational analysis revealed that the amino-terminal coiled coil region is not the dimerization determinant. In contrast, similar mutagenesis studies indicated a role for the leucine zipper motif, located in the middle region of the protein, in dimerization. Mixed oligomerization assays confirmed the importance of leucine zipper in C dimer formation establishing the presence of an uncommon zipper-HTH domain in the transactivator.

Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-beta lactamase/beta-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

Characteristics of MuA transposase-catalyzed processing of model transposon end DNA hairpin substrates

Bacteriophage Mu uses non-replicative transposition for integration into the host's chromosome and replicative transposition for phage propagation. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that are known to employ a hairpin intermediate during the catalytic steps of transposition. Model transposon end DNA hairpin substrates were used in a minimal-component in vitro system to study their proficiency to promote Mu transpososome assembly and subsequent MuA-catalyzed chemical reactions leading to the strand transfer product. MuA indeed was able to assemble hairpin substrates into a catalytically competent transpososome, open the hairpin ends and accurately join the opened ends to the target DNA. The hairpin opening and transposon end cleavage reactions had identical metal ion preferences, indicating similar conformations within the catalytic center for these reactions. Hairpin length influenced transpososome assembly as well as catalysis: longer loops were more efficient in these respects. In general, MuA's proficiency to utilize different types of hairpin substrates indicates a certain degree of flexibility within the transposition machinery core. Overall, the results suggest that non-replicative and replicative transposition systems may structurally and evolutionarily be more closely linked than anticipated previously.

ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

Dual Role for Transactivator Protein C in Activation of mom Promoter of Bacteriophage Mu

Transactivator C protein of bacteriophage Mu activates the mom gene of the phage by an unusual mechanism. DNA binding by C to its site results in unwinding of the neighboring sequences, realigning the out-of-phase promoter elements to facilitate RNA polymerase (RNAP) binding. High level stimulation of a C-independent constitutive promoter mutant (where RNAP is already bound) by the transactivator suggested an additional mechanism of transcription activation at a step after RNAP recruitment. In this study, we have investigated the various steps of promoter-polymerase interactions during transcription initiation by using both the promoter mutant and a positive control (pc) mutant of C protein. The transactivator does not influence formation of the open complex or its stability after facilitating the RNAP binding. However, at a subsequent step, the protein exerts an important role, enhancing the promoter clearance by increasing the productive RNAP.promoter complex. The pc mutant of the transactivator C is compromised at this step, supporting the additional downstream role for C in mom transcription activation. We suggest that this unusual multistep activation of Pmom has evolved to ensure irreversibility of the switch during the late lytic cycle of the phage.

Chromosomal Integration Mechanism of Infecting Mu Virion DNA

DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.

A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts.

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac+ by prophage precise excision with a relatively high frequency (about 1 x 10(-6)). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced beta-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.

Dissection of the Bacteriophage Mu Strong Gyrase Site (SGS): Significance of the SGS Right Arm in Mu Biology and DNA Gyrase Mechanism

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.

Structural and Genetic Analyses Reveal a Key Role in Prophage Excision for the TorI Response Regulator Inhibitor

TorI (Tor inhibition protein) has been identified in Escherichia coli as a protein inhibitor acting through protein-protein interaction with the TorR response regulator. This interaction, which does not interfere with TorR DNA binding activity, probably prevents the recruitment of RNA polymerase to the torC promoter. In this study we have solved the solution structure of TorI, which adopts a prokaryotic winged-helix arrangement. Despite no primary sequence similarity, the three-dimensional structure of TorI is highly homologous to the (lambda)Xis, Mu bacteriophage repressor (MuR-DBD), and transposase (MuA-DBD) structures. We propose that the TorI protein is the structural missing link between the (lambda)Xis and MuR proteins. Moreover, in vivo assays demonstrated that TorI plays an essential role in prophage excision. Heteronuclear NMR experiments and site-directed mutagenesis studies have pinpointed out key residues involved in the DNA binding activity of TorI. Our findings suggest that TorI-related proteins identified in various pathogenic bacterial genomes define a new family of atypical excisionases.

Unwinding of the Nascent Lagging Strand by Rep and PriA Enables the Direct Restart of Stalled Replication Forks

During origin-independent replisome assembly, the replication restart protein PriC prefers to load the replication fork helicase, DnaB, to stalled replication forks where there is a gap in the nascent leading strand. However, this activity can be obstructed if the 5'-end of the nascent lagging strand is near the template branch point. Here we provide biochemical evidence that the helicase activities of Rep and PriA function to unwind the nascent lagging strand DNA at such stalled replication forks. PriC then loads the replicative helicase, DnaB, onto the newly generated, single-stranded template for the purposes of replisome assembly and duplex unwinding ahead of the replication fork. Direct rescue of replication forks by the Rep-PriC and PriA-PriC pathways in this manner may contribute to genomic stability by avoiding the potential dangers of fork breakage inherent to recombination-dependent restart pathways.

Control of Bacteriophage Mu Lysogenic Repression

The transposable and temperate phage Mu infects Escherichia coli where it can enter the lytic life-cycle or reside as a repressed and integrated prophage. The repressor protein Rep is the key element in the lysis-lysogeny decision. We have analyzed the fate of Rep in different mutants by Western blotting under two conditions that can induce a lysogen: high temperature and stationary phase. We show that, unexpectedly, Rep accumulates under all conditions where the prophage is completely derepressed, and that this accumulation is ClpX-dependent. An analysis of the degradation kinetics shows that Rep is a target of two protease systems: inactivation of either the clpP or lon gene results in a stabilization of Rep. Such a reaction scheme explains the counterintuitive observation that derepression is correlated with high repressor concentration. We conclude that under all conditions of phage induction the repressor is sequestered in a non-active form. A quantitative simulation accounts for our experimental data. It provides a model that captures the essential features of Mu induction and explains some of the mechanisms by which the physiological signals affecting the lysis-lysogeny decision converge onto Rep.

Structure of the Central Hub of Bacteriophage Mu Baseplate Determined by X-ray Crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.

Remodeling protein complexes: Insights from the AAA+ unfoldase ClpX and Mu transposase

Multiprotein complexes in the cell are dynamic entities that are constantly undergoing changes in subunit composition and conformation to carry out their functions. The protein-DNA complex that promotes recombination of the bacteriophage Mu is a prime example of a complex that must undergo specific changes to carry out its function. The Clp/Hsp100 family of AAA+ ATPases plays a critical role in mediating such changes. The Clp/Hsp100 unfolding enzymes have been extensively studied for the roles they play in protein degradation. However, degradation is not the only fate for proteins that come in contact with the ATP-dependent unfolding enzymes. The Clp/Hsp100 enzymes induce structural changes in their substrates. These structural changes, which we refer to as "remodeling", ultimately change the biological activity of the substrate. These biological changes include activation, inactivation (not associated with degradation), and relocation within the cell. Analysis of the interaction between Escherichia coli ClpX unfoldase and the Mu recombination complex, has provided molecular insight into the mechanisms of protein remodeling. We discuss the key mechanistic features of the remodeling reactions promoted by ClpX and possible implications of these findings for other biological reactions.

A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector.

Host factors that promote transpososome disassembly and the PriA‐PriC pathway for restart primosome assembly

Initiation of bacteriophage Mu DNA replication by transposition requires the disassembly of the transpososome that catalyses strand exchange and the assembly of a replisome promoted by PriA, PriB, PriC and DnaT proteins, which function in the host to restart stalled replication forks. Once the molecular chaperone ClpX weakens the very tight binding of the transpososome to the Mu ends, host disassembly factors (MRFalpha-DF) promote the dissociation of the transpososome from the DNA template and the assembly of a new nucleoprotein complex. Prereplisome factors (MRFalpha-PR) further alter the complex, allowing PriA binding and loading of major replicative helicase DnaB onto the template promoted by the restart proteins. MRFalpha-PR is essential for DnaB loading by restart proteins even on the deproteinized Mu fork whereas MRFalpha-DF is not required on the deproteinized template. When the transition from transpososome to replisome was reconstituted using MRFalpha-DF and MRFalpha-PR, initiation of Mu DNA replication was strictly dependent upon added PriC and PriA helicase. In contrast, initiation on the deproteinized template was predominantly dependent upon PriB and did not require PriA's helicase activity. The results indicate that transition mechanisms beginning with the transpososome disassembly can determine the pathway of replisome assembly by restart proteins.

Bacteriophage Mu Targets the Trinucleotide Sequence CGG

Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.

Expression and Characterization of a Baseplate Protein for Bacteriophage Mu, gp44

The gene product of gene 44 of Mu phage (gp44) is an essential protein for baseplate assembly and has been designated as gpP, a traditional genetic assignment. The function of gp44 during the assembly or infection process is not known. In the present study, we purified the recombinant gp44 and characterized it by analytical ultracentrifugation and differential scanning microcalorimetry. The results indicate that gp44 forms a trimer comprising a complex consisting of the 42 kDa and 40 kDa subunits that had been cleaved in the C-terminal region. Thermodynamic analysis also suggested that the C-terminal region forms a flexible domain.

Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes

Transposon mutagenesis is a powerful technique for generating collections of insertion mutants for genetic studies. This paper describes how phage Mu DNA transposition complexes, transpososomes, can be exploited for gene delivery to efficiently introduce selectable markers to genomes of Gram-positive bacteria. Mu transpososomes were assembled in vitro with custom-designed mini-Mu transposons, concentrated, and electroporated into cells of three Gram-positive bacterial species: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus suis. Within cells, the complexes reproduced an authentic DNA transposition reaction and integrated the delivered transposons into the bacterial genomes, yielding single-copy insertions. The integration efficiency among different species and strains of Gram-positive bacteria ranged from 1x10(1) to 2x10(4) c.f.u. (mug introduced transposon DNA)(-1). The strategy should be applicable to a variety of other Gram-positive species after initial optimization of certain key factors affecting transposon delivery, such as the preparation method of competent cells and physical parameters of electroporation. This study extends the scope of the Mu transpososome delivery-based genomic DNA integration strategy to Gram-positive bacteria. Thus, a straightforward generation of sizeable mutant banks is feasible for these bacteria, potentiating several types of genomic-level approaches for studies of a variety of important bacterial processes, such as pathogenicity.

Meox1Cre: A mouse line expressing Cre recombinase in somitic mesoderm

We developed a novel strategy based on in vitro DNA transposition of phage Mu to construct vectors for "knock-in" of the gene encoding Cre recombinase into endogenous loci in embryonic stem cells. This strategy was used to introduce Cre into the mouse Meox1 locus, which was expected to drive Cre expression in the presomitic and somitic mesoderm. In embryos heterozygous for both Meox1(Cre) and R26R or Z/AP reporter alleles, specific and efficient recombination of the reporter alleles was detected in the maturing somites and their derivatives, including developing vertebrae, skeletal muscle, back dermis, as well as endothelium of the blood vessels invading the spinal cord and developing limbs. In contrast to the somitic mesoderm, Cre activity was not observed in the cranial paraxial mesoderm. Thus, the Meox1(Cre) allele allows detailed fate-mapping of Meox1-expressing tissues, including derivatives of the somitic mesoderm. We used it to demonstrate dynamic changes in the composition of the mesenchyme surrounding the developing inner ear. Meox1(Cre) may also be used for tissue-specific mutagenesis in the somitic mesoderm and its derivatives.

Molecular evolution of Vibrio pathogenicity island-2 (VPI-2): mosaic structure among Vibrio cholerae and Vibrio mimicus natural isolates

Vibrio cholerae is a Gram-negative rod that inhabits the aquatic environment and is the aetiological agent of cholera, a disease that is endemic in much of Southern Asia. The 57.3 kb Vibrio pathogenicity island-2 (VPI-2) is confined predominantly to toxigenic V. cholerae O1 and O139 serogroup isolates and encodes 52 ORFs (VC1758 to VC1809), which include homologues of an integrase (VC1758), a restriction modification system, a sialic acid metabolism gene cluster (VC1773-VC1783), a neuraminidase (VC1784) and a gene cluster that shows homology to Mu phage. In this study, a 14.1 kb region of VPI-2 comprising ORFs VC1773 to VC1787 was identified by PCR and Southern blot analyses in all 17 Vibrio mimicus isolates examined. The VPI-2 region in V. mimicus was inserted adjacent to a serine tRNA similar to VPI-2 in V. cholerae. In 11 of the 17 V. mimicus isolates examined, an additional 5.3 kb region encoding VC1758 and VC1804 to VC1809 was present adjacent to VC1787. The evolutionary history of VPI-2 was reconstructed by comparative analysis of the nanH (VC1784) gene tree with the species gene tree, deduced from the housekeeping gene malate dehydrogenase (mdh), among V. cholerae and V. mimicus isolates. Both gene trees showed an overall congruence; on both gene trees V. cholerae O1 and O139 serogroup isolates clustered together, whereas non-O1/non-O139 serogroup isolates formed separate divergent branches with similar clustering of strains within the branches. One exception was noted: on the mdh gene tree, V. mimicus sequences formed a distinct divergent lineage from V. cholerae sequences; however, on the nanH gene tree, V. mimicus clustered with V. cholerae non-O1/non-O139 isolates, suggesting horizontal transfer of this region between these species.