Replacement of the bacteriophage Mu strong gyrase site and effect on Mu DNA replication

Abstract

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.

FIG. 1

Lysis and replication without a central gyrase site (■), with the Mu SGS in the positive (○) or negative (●) orientation, or with the D108 gyrase site (□). Cultures of lysogens were grown in L broth at 30°C to a density of about 10⁸ cells/ml, diluted threefold in L broth, and then induced by shifting to 42°C. Growth was monitored by Klett readings, and samples were taken for quantitative PCR analysis to measure DNA replication. Replication is expressed as the ratio of the amount of an amplified Mu DNA fragment to the amount of a chromosomal malF fragment. (Top panel) Growth and lysis of the cultures. (Bottom panel) Mu DNA replication.

FIG. 2

Sequence of a region of the D108 genome containing the central strong gyrase site. Mismatches with the Mu sequence are noted, and the locations of the gyrase site consensus sequence (denoted as SGS) and the restriction sites for MluI and ScaI are underlined. (The GenBank accession no. is Bank It 253889 AF 128885.)

FIG. 3

Gyrase cleavage of Mu and D108 DNA. pBR322 Δ(EcoRI-BamHI) plasmids with cloned 1.4-kb central fragments from Mu (lanes 2 and 3) and D108 (lanes 4 and 5) were linearized by PvuII restriction digestion and then incubated with DNA gyrase in the presence of enoxacin. Following treatment with SDS and proteinase K, the DNA fragments were separated on a 1% agarose gel and stained with ethidium bromide. The expected fragment sizes for cleavage at the Mu gyrase site are ∼2.9 and 2.6 kb. Lane 1, molecular size markers. −, no gyrase; +, with gyrase.

FIG. 4

Lysis and replication without a central gyrase site (■), with the Mu SGS in the positive orientation (○), with the pSC101 gyrase site (●), or with the pBR322 gyrase site (□). Growth and replication were monitored as in Fig. 1. (Top panel) Growth and lysis of the cultures. (Bottom panel) Mu DNA replication.

FIG. 5

In vitro gyrase cleavage. pBR322 Δ(EcoRI-BamHI) plasmids without (lanes 2 and 3) or with cloned fragments carrying the Mu SGS (lanes 4 and 5), the pSC101 gyrase site (lanes 6 and 7), or the nrdAB BIME (lanes 8 and 9) were linearized with PvuII restriction digestion and then incubated with DNA gyrase in the presence of enoxacin. Following treatment with SDS and proteinase K, the DNA fragments were separated on a 1% agarose gel and stained with ethidium bromide. The expected fragment sizes for cleavage at the gyrase site in question were as follows: pBR322, 2.8 + 1.2 kb; Mu, 2.9 + 2.6 kb; pSC101, 2.5 + 1.9 kb; nrd BIME, 2.4 + 1.8 kb. Lanes 1 and 10, molecular size markers. −, no gyrase; +, with gyrase.

FIG. 6

In vivo gyrase cleavage. Lysogens with prophages carrying either the Mu SGS or the pSC101 gyrase site were grown in L broth, enoxacin was added to 300 μg/ml for 5 min, and the cells were rapidly lysed in hot SDS. DNA was isolated, treated with proteinase K, and digested with the appropriate restriction enzyme. One-directional PCR with primers near the gyrase sites yielded shorter fragments from templates that were cleaved by gyrase and longer fragments from templates not cleaved by gyrase. A sequencing ladder provided size markers (base pairs).