Development of shuttle vectors for spirochetes.

Constructions of Escherichia coli-spirochete shuttle vectors are based on naturally occurring plasmids, broad host range plasmids or bacteriophages. This review primarily focuses on genetic tools for Treponema denticola which is associated with periodontal diseases. The T. pallidum FlaA protein, E. coli beta-galactosidase, and the green fluorescent protein were successfully expressed in T. denticola from a shuttle vector system.

The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.

Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.

A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus Ixodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with MluI were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only fragments greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgdorferi sensu stricto isolates were recognized by a band at 135 kbp, each of the 12 Borrelia garinii isolates by two bands (220 and 80 kbp), and each of the 20 Borrelia afzelii isolates by three bands (460, 320, and 90 kbp). Whereas differences in the PFGE patterns among B. burgdorferi sensu stricto isolates and B. garinii isolates were noted, B. afzelii isolates were all similar. Identification of isolates by PFGE correlates with their belonging to a given species within B. burgdorferi sensu lato.

Scattering of the rRNA genes on the physical map of the circular chromosome of Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.

Linear chromosome of Borrelia burgdorferi.

The DNA organization of several European and American isolates of Borrelia burgdorferi, the aetiological agent of Lyme disease, was analysed in pulse-field agarose gel electrophoresis. The results of in situ cell lysis in agarose plugs demonstrated a unique arrangement for the DNA of this spirochete. The chromosome of Borrelia behaved as a eukaryotic linear chromosome with a size of around 1,000 kb. The genome also comprised several circular and linear plasmids which varied in size from 15 to 60 kb.

Circular dichroism investigation of Escherichia coli adenylate kinase.

We examined by circular dichroism (CD) spectroscopy in far- and near-ultraviolet three different molecular forms of Escherichia coli adenylate kinase: the wild type protein, the enzyme carboxymethylated at a single cysteine residue (Cys-77), and the thermosensitive adenylate kinase. The thermosensitive enzyme differs from the wild type protein in that a serine is substituted for a proline residue at position 87 (Gilles, A.-M., Saint Girons, I., Monnot, M., Fermandjian, S., Michelson, S., and Bârzu, O. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 5798-5802). We also examined the CD spectra of isolated peptides resulting from chemical cleavage of adenylate kinase at Cys-77 (C1, residues 1-76; C2, residues 77-214). The secondary structure composition of wild type bacterial adenylate kinase (50% alpha-helix and 15% beta-sheet) was close to that derived from x-ray analysis of pig muscle enzyme (Schulz, G.E., Elzinga, M., Marx, F., and Schirmer, R. H. (1974) Nature 250, 120-123). Carboxymethylation of wild type protein did not greatly affect the CD spectrum. The secondary structure of the thermosensitive adenylate kinase was observed to be significantly different from that of the wild type enzyme (reduction in alpha-helix content to 39%). Changes in ellipticities at 222 nm as a function of temperature indicated that the melting temperature for thermosensitive adenylate kinase was 38 degrees C and that for the wild type enzyme was 54 degrees C. Isolated C1 and C2 peptides had a large proportion of unordered structures. When mixed, C1 and C2 fragments reassociated into structures resembling native, uncleaved adenylate kinase. The recovery of ordered structures, indicated by CD spectroscopy, paralleled the recovery of catalytic activity.