Transcriptional regulation of type 4 pilin genes and the site-specific recombinase gene, piv, in Moraxella lacunata and Moraxella bovis.

Moraxella lacunata and Moraxella bovis use type 4 pili to adhere to epithelial tissues of the cornea and conjunctiva. Primer extension analyses were used to map the transcriptional start sites for the genes encoding the major pilin subunits (tfpQ/I) and the DNA invertase (piv), which determines pilin type expression. tfpQ/I transcription starts at a sigma54-dependent promoter (tfpQ/Ip2) and, under certain growth conditions, this transcription is accompanied by weaker upstream transcription that starts at a potential sigma70-dependent promoter (tfpQ/Ip1). piv is expressed in both M. lacunata and M. bovis from a putative sigma70-dependent promoter (pivp) under all conditions assayed. Sigma54-dependent promoters require activators in order to initiate transcription; therefore, it is likely that tfpQ/Ip2 is also regulated by an activator in Moraxella. Primer extension assays with RNA isolated from Escherichia coli containing the subcloned pilin inversion region from M. lacunata showed that pivp is used for the expression of piv; however, tfpQ/Ip2 is not used for the transcription of tfpQ/I. Transcription from tfpQ/Ip2 was activated in E. coli when the sensor (PilS) and response regulator (PilR) proteins of type 4 pilin transcription in Pseudomonas aeruginosa were expressed from a plasmid. These results suggest that the expression of the type 4 pilin in M. lacunata and M. bovis is regulated not only by a site-specific DNA inversion system but also by a regulatory system which is functionally analogous to the PilS-PilR two-component system of P. aeruginosa.

Amino acid sequence homology between Piv, an essential protein in site-specific DNA inversion in Moraxella lacunata, and transposases of an unusual family of insertion elements.

Deletion analysis of the subcloned DNA inversion region of Moraxella lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tfpQ/tfpI pilin segment. The predicted amino acid sequence of Piv shows significant homology solely with the transposases/integrases of a family of insertion sequence elements, suggesting that Piv is a novel site-specific recombinase.

Bacteriophage Mu sites and functions involved in the inhibition of lambda::mini-Mu growth.

To better understand the nature of the mini-Mu-directed process which results in inhibition of lambda::mini-Mu growth we characterized spontaneous deletion mutants of the lambda::mini-Mu phage. On the basis of analysis of the deletion endpoints, mini-Mu replication functions, and integration and inhibition properties, the lambda::mini-Mu deletion mutants were divided into five classes which define the Mu sites and functions involved in lambda::mini-Mu growth inhibition. Class 1 mutants, which still exhibit lambda::mini-Mu growth inhibition, collectively delete all the Mu late functions encoded by the mini-Mu. Class 2 and 5 mutants, which show cis-dominant defects in inhibition and integration, delete the right and left mini-Mu attachment sites, respectively. Phages of Classes 3 and 4, which delete the Mu B or A and B genes, respectively, show recessive defects in growth inhibition. The properties of these mutants define the Mu replication functions, A and B, and the Mu attachment sites as essential for the inhibition of lambda::mini-Mu growth. The observation that the sites and functions essential for Mu replication also have requisite roles in the inhibition of lambda::mini-Mu growth suggests that inhibition results from mini-Mu-promoted replicative interference of lambda::mini-Mu development.

Spatial relationship of the Fis binding sites for Hin recombinational enhancer activity.

Site-specific recombination reactions involve the joining or rearrangement of discrete DNA segments in a highly precise manner. A site-specific DNA inversion regulates the expression of flagellin genes in Salmonella by switching the orientation of a promoter. Analysis of the reaction has shown that, in addition to DNA sequences at the two boundaries of the 1-kilobase invertible segment where strand exchange occurs, another cis acting sequence is required for efficient inversion. This 60-base-pair enhancer-like sequence can function at many different locations and in either orientation in a plasmid substrate. It includes two binding sites for a host protein called Factor II or Fis (refs 4 and 5). Here we have investigated the importance of the spatial relationship between the two Fis binding sites for enhancer activity and have found that the correct helical positioning of the binding sites on the DNA is critical. However, this result could not be accounted for by effects on Fis binding. We propose a model for enhancer function in which the enhancer region acts to align the recombination sites into a specific conformation required for productive synapsis.

Location, degree, and direction of DNA bending associated with the Hin recombinational enhancer sequence and Fis-enhancer complex.

The Fis protein of Escherichia coli and Salmonella typhimurium stimulates several site-specific DNA recombination reactions, as well as transcription of a number of genes. Fis binds to a 15-bp core recognition sequence and induces DNA bending. Mutations in Fis which alter its ability to bend DNA have been shown to reduce the stimulatory activity of Fis in both site-specific recombination and transcription systems. To examine the role of DNA bending in the activity of the Fis-recombinational enhancer complex in Hin-mediated site-specific DNA inversion, we have determined the locations, degrees, and directions of DNA bends associated with the recombinational enhancer and the Fis-enhancer complex. Circular-permutation assays demonstrated that a sequence-directed DNA bend is associated with the Fis binding sites in the proximal and distal domains of the recombinational enhancer. Binding of Fis to its core recognition sequence significantly increases the degree of DNA bending associated with the proximal and distal domains. The degree of DNA bending induced by Fis binding depended on the DNA sequences flanking the core Fis binding site, with angles ranging from 42 to 69 degrees. Phasing analyses indicate that both the sequence-directed and the Fis-induced DNA bends associated with the proximal and distal domains face the minor groove of the DNA helix at the center of the Fis binding site. The positions and directions of DNA bends associated with the Fis-recombinational complex support a direct role for Fis-induced DNA bending in assembly of the active invertasome.

DNA-binding properties of the Hin recombinase.

The recombinase of the Salmonella inversion system, Hin, mediates site-specific recombination between two 26 base pairs (bp) inverted repeat sequences (hixL and hixR) which flank a 993-bp DNA segment. We have investigated Hin recognition of, and association with, the hix recombination sites. Nuclease and chemical protection studies with linear and supercoiled DNA substrates demonstrate that Hin initially binds hixL and hixR independently of binding of the other protein components of the inversion system, Fis and HU. DNA-binding assays with mutant recombination sites and methylation interference experiments indicate that the critical bases for Hin recognition of its DNA-binding site are within an 8-bp sequence covering adjacent major and minor grooves of the DNA helix in each of the 12-bp half-sites of the hix recombination sites. The nature of the Hin-hix complexes in these binding studies and the results of gel filtration assays with purified Hin suggests that Hin binds the recombination sites as a dimer. The implications of the nature of the interactions of Hin with its recombination sites on the mechanism of the recombination reaction and on the novel features of DNA recognition by Hin are discussed.

Excision of IS492 requires flanking target sequences and results in circle formation in Pseudoalteromonas atlantica.

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

Development of a nurse-led chronic pain clinic in UK primary care.

We describe the development of an innovative, nurse-led chronic pain clinic in primary care. Benefit of the structured intervention was seen in terms of overall patient pain (as measured by the short form McGill-Melzack pain scale) with no overall impact on drug costs. A significant proportion (54%) of patients taking non-steroidal anti-inflammatory drugs were deemed to be at risk of gastropathy and in need of gastroprotection as defined by the clinic protocol. Areas worthy of further study are discussed.

Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata.

The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata.

Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed. Using an M. bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M. lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M. lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli. Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations. Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids. These deletions and insertions thus localize a probable invertase necessary for the inversion event. The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M. bovis and called ORF2 appeared to be a strong candidate for the invertase. This conclusion was confirmed when a plasmid containing the M. bovis ORF2 supplied, in trans, the inversion function missing from one of the M. lacunata phase-locked inversion mutants. We have named these putative invertase genes piv(ml) (pilin inversion of M. lacunata) and piv(mb) (pilin inversion of M. bovis). Despite previously noted sequence similarities between the M. bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases.

Fis binding to the recombinational enhancer of the Hin DNA inversion system.

The recombinational enhancer of the Hin inversion system in Salmonella stimulates recombination in vitro 150-fold in the presence of the Escherichia coli host factor Fis. To gain an understanding of the roles of the enhancer and Fis in stimulating the Hin-mediated inversion reaction, we have used nuclease and chemical protection/interference studies and gel retardation assays to examine the interactions between Fis and the recombinational enhancer. These studies combined with mutational analysis defined the enhancer sequences required for Fis binding and function. Fis binds with different affinities to two domains within the enhancer sequence. The binding of Fis at each domain is independent of the occupancy of the other domain and appears to be to opposite faces of the DNA helix. These results support a model for the role of the recombinational enhancer in Hin-mediated inversion in which the interaction between Hin bound at recombination sites and Fis bound to each domain of the recombinational enhancer results in a structure with the proper alignment and topology to promote DNA inversion.

Importance of minor-groove contacts for recognition of DNA by the binding domain of Hin recombinase.

Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.