Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions

Abstract

The first and, to date, only extrachromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Since the LE1 prophage replicates autonomously as a circular replicon in L. biflexa, we were able to engineer an L. biflexa-Escherichia coli shuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol. 182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Theta-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of a rep encoding a protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both parA and parB are present. These results enable the construction of new shuttle vectors for studying the genetics of Leptospira spp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.

FIG. 1.

Schematic representation of the LE1 genome. A. Genome sequence of leptophage LE1. The position and direction of transcription of the predicted ORFs are indicated by arrows. The filled circle is a potential rho-independent terminator. B. AT, GC, and cumulative skew diagrams of the LE1 genome. The analysis began at the designated nucleotide sequence position 1, corresponding to the LE1 replication region.

FIG. 2.

Restriction map of the LE1 virion DNA for the enzymes EagI, BglII, and BssHII (from the outer to the inner arc). The positions of ends of linear DNA molecules are indicated.

FIG. 3.

Identification of replication and partition functions of bacteriophage LE1. A. The ability of each plasmid construct to yield L. biflexa transformants and the stability of the plasmid without selective pressure are indicated on the right. ≪-≫ indicates that the plasmid was not replicative in L. biflexa. Plasmid stability in L. biflexa was evaluated as described in Material and Methods. The primers used to generate each DNA fragment are indicated. Boxes indicate site-directed mutagenesis of direct repeats (DR) and inverted repeats (IR), and nucleotide substitutions are written in uppercase. ND, not determined. B. Sequence alignment of Burkholderia cepacia phage Bcep22 protein (Bcep2), Lactococcus lactis phage phi31.1 (phi31), Lactococcus phage BK5-T (BK5), Salmonella enterica serovar Typhimurium phage ST64B (ST64B), Shigella flexneri phage V (phage V), and L. biflexa phage LE1 Rep (LE1) proteins. Residues conserved in at least four homologous proteins are shaded.

FIG. 4.

Schematic of the L. biflexa-E. coli shuttle vectors pGKBLe24, pGSBLe24, and pGKBLe94. KmR, kanamycin resistance cassette; SpcR, spectinomycin-resistance cassette. Unique restriction sites are indicated.

FIG. 5.

LE1 transcription analysis by RT-PCR. The numbers above each lane represent a selection of potential ORFs (see Table 1) used for RT-PCR. The three different stages of the LE1 life cycle (lysogen, early, and late steps of the lytic stage) for which mRNA was extracted are indicated above the panels.