The observation that a cis-acting DNA sequence can be separated from its site of action by long distances has been made in many different biological systems, but is not yet understood at the molecular level (for reviews, see Dynan and Tjian 1985;Ptashne 1986). The Hin sitespecific recombination system found in Salmonella typhimurium provides a simple model for understanding this phenomenon. The Hin protein mediates a site-specific DNA inversion that is promoted by the presence of a DNA sequence, a recombinational enhancer, which acts in an orientation-and distance-independent manner (Johnson and Simon 1985). Elucidation of the mechanism of action of the recombinational enhancer may help our understanding of the function of the large variety of enhancer sequences found in other systems.Inversion of the 996-bp DNA segment by Hin alters the expression of flagellin type in Salmonella (Zieg et al. 1977). The recombination reaction requires three DNA sequences: two recombination sites, hixL and hixR, which mark the boundary of the inversion sequence , and the recombinational enhancer (Johnson et al. 1986). Each recombination site is composed of two half-sites with approximate dyad symmetry. The hix sites share a common sequence which must be in inverted configuration for recombination to 3Current address:

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.

Deletion analysis of the subcloned DNA inversion region of MoraxeUla lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tlpQIip'I 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.Moraxella lacunata and Moraxella bovis are human and bovine ocular pathogens, respectively (4,17,27). Both of these gram-negative pathogens produce type 4 (MePhe) pili, which are important virulence factors mediating adherence of the bacilli to corneal tissue (7,22,23,29). M. bovis and M. lacunata utilize site-specific DNA inversion of a chromosomal segment to alter the expression of their type 4 pilin genes (22, 28). M. bovis alternately expresses Q and I pilin, and M. lacunata apparently exhibits an on/off pilin phase variation (23, 30). The 2.1-kb invertible DNA segment in M. bovis contains the carboxyl-terminal portion of the tfpQ and tfpI pilin genes and an additional open reading frame, ORF1 or tfpB ( Fig. 1) (21).The promoter and constant region of the pilin genes is located upstream of the invertible segment. The DNA inversion region of M. lacunata is very similar in sequence and organization to that of M. bovis, with the notable exceptions of a 19-bp duplication within the coding sequence for the carboxylterminal region of the tfpI pilin gene, resulting in no active pilin expression in one orientation of the invertible segment, and a 2-bp deletion in tfpB which shortens the ORF by one-third (28).A subcloned 6-kb fragment containing the inversion region of M. lacunata in a pBluescript vector (pMxLl; Fig. 1 (22), has the inversion region from M. lacunata (same region encoded on pMxLl), but piv has been inactivated by insertion of a segment from the fl interposon encoding streptomycin/spectinomycin resistance. The deletion of tfpB was accomplished by restriction of pMxL5 with AfllI and SnaBI (New England Biolabs) (Fig. 1) and ligation of the annealed oligonucleotides 5'-TTAAGATCGATGACGTCAGATCTG AGCTCGATACTCGAG-3' and 5'-CTCGAGTATCGAGCT CAGATCT'GACGTCATCGATC-3' into the Aflll and SnaBI sites, using T4 DNA ligase (Boehringer) to create pAG701. Inversion assays were then performed by transforming E. coli DH5a containing pMxL5 or pAG701 with a compatible plasmid, pACYCpiv-1 (provided by Meredith Hackel and Carl Marrs), which is pACYC184 containing the M. bovis piv gene under control of its own promoter. The transformants and DH5a strains containing pMxL5 or pAG701 were grown in culture overnight selecting for chloramphenicol (pACYC piv-1) and/or ampicillin (pMxL5/pAG701). Plasmid DNA was then isolated and digested with KpnI, which cuts once asymmetrically within the invertible segment and once in the pBluescript vector DNA (Fig. 1); electrophoresis of restriction fragments was done as described by Marrs et al. (22). Digestion of pMxL5 with KpnI in the absence of Piv results in fragments of 9.4 and 0.5 k...

MoraxeUal lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I p_n ( Both similarities and differences in the genetic organization of type 4 pilin genes occur in different bacterial species. One similarity is that P. aeruginosa, N. gonorrhoeae, and M. bovis pilin genes all appear to use rpoN (glnF, ntrA)-dependent promoters (18,19). RpoN has been shown to be the alternative sigma factor, cr4, required for transcriptional activation of some genes (reviewed in reference 22). Differences exist in the copy numbers of pilin genes among type 4 pili. P. aeruginosa strains only have a single copy of the pilin gene in each genome (29,35 (25,32). The switch in expression between Q and I pilin is due to an inversion of a 2.1-kilobase (kb) region of DNA (13,24).Among the best-characterized families of bacterial DNA inversion systems are the Hin system of Salmonella typhimurium, Gin and Cin of bacteriophages Mu and P1, and Pin of Escherichia coli (reviewed in reference 14); another is the newly described Min of plasmid pl5B (33

No abstract

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.

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 IS492insertion 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 intoEscherichia 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 inE. 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.

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 54 -dependent promoter (tfpQ/Ip 2 ) and, under certain growth conditions, this transcription is accompanied by weaker upstream transcription that starts at a potential 70 -dependent promoter (tfpQ/Ip 1 ). piv is expressed in both M. lacunata and M. bovis from a putative 70 -dependent promoter (pivp) under all conditions assayed. 54 -dependent promoters require activators in order to initiate transcription; therefore, it is likely that tfpQ/Ip 2 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/Ip 2 is not used for the transcription of tfpQ/I. Transcription from tfpQ/Ip 2 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.Moraxella lacunata and Moraxella bovis are gram-negative human and bovine pathogens, respectively, that primarily infect the conjunctiva and cornea (3,15,29,32). Attachment to the epithelial tissues of these areas is mediated by type 4 pili (28). Other bacteria that express type 4 pili include Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, Aeromonas hydrophila, and Dichelobacter nodosus (10,13,23,25,37). In bacteria expressing type 4 pili, the major pilin subunit is expressed as a pre-pilin protein with an unusually short 6-amino-acid leader sequence followed by a predominantly hydrophobic domain (35). The hydrophobic domain and the leader sequence are required to deliver the protein to an ATP-dependent, Sec-independent secretory pathway (34). During the process of secretion, a pre-pilin peptidase cleaves the leader sequence and methylates the first amino acid of the mature protein, creating N-methylphenylalanine (36). Thus, type 4 pili have also been classified as MePhe pili.M. bovis exhibits antigenic and phase variations of its type 4 pili, alternately expressing Q-or I-type pili and switching between P ϩ (piliated) and P Ϫ (nonpiliated) at a frequency as high as 10 Ϫ4 per cell per generation (21). M. bovis cells expressing Q-type pili are more efficient at establishing infection than those expressing I-type pili (27, 29), and nonpiliated M. bovis cells cannot establish infection (15). Therefore, pili are important virulence factor...