groEL expression in gyrB mutants of Borrelia burgdorferi

Abstract

GroEL protein and groEL mRNA transcript were up-regulated in gyrB mutants of Borrelia burgdorferi, a causative agent of Lyme disease. Furthermore, the protein and transcript levels in gyrB mutants were greater than those in experimentally heat-shocked cultures of wild-type B. burgdorferi. Circular DNA in the gyrB mutants was more relaxed than in wild-type cells, although groEL is on the linear chromosome of B. burgdorferi. To our knowledge, this is the first evidence, albeit indirect, for the effect of DNA topology on gene expression from a linear DNA molecule in a bacterium.

FIG. 1.

Purification of GroEL from a gyrB mutant of B. burgdorferi. (A) The 68-kDa protein (identified as GroEL) was purified by column chromatography. Lane 1, cell lysate; lane 2, flowthrough from a heparin column; lane 3, pooled fractions after elution from a phenyl Superose column; lane 4, pooled fractions after elution from a Mono-Q column. Molecular masses are in kilodaltons. (B) N-terminal sequence of the 68-kDa protein and alignment with GroEL (BB0649; GenBank accession no. AE001166) (6).

FIG. 2.

Increased GroEL synthesis and groEL expression in a gyrB mutant of B. burgdorferi. Immunoblot (A) and Northern blot (B) analyses of wild-type B31 (wt), experimentally heat-shocked wild-type B31 (hs), and the gyrB mutant X32 were carried out.

FIG. 3.

Effect of a gyrB mutation on DNA topology. Plasmid pGOΔ15 was transiently introduced into wild-type B31 and the gyrB mutant X32. The topology of the plasmid was determined by one-dimensional 0.8% agarose gel electrophoresis in the absence (native) or presence of 15 μM chloroquine (CQ). The more negatively supercoiled molecules migrate faster through the gel; chloroquine intercalates into the plasmid DNA, introducing positive supercoiling that retards migration of negatively supercoiled DNA (sc) and expedites migration of relaxed DNA (r). The migration of the linearized (l) form of the plasmid is not affected in the mutant. Molecular size standards are in kilobase pairs.