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
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with đź’™ for researchers
Part of the Research Solutions Family.