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Freeze-fracture electron microscopy was used to investigate the membrane architectures of high-passage Borrelia burgdorferi B31 and low- and high-passage isolates of B. burgdorferi N40. In all three organisms, fractures occurred almost exclusively through the outer membrane (OM), and the large majority of intramembranous particles were distributed randomly throughout the concave OM leaflet. The density of intramembranous particles in the concave OM leaflet of the high-passage N40 isolate was significantly greater than that in the corresponding leaflet of the low-passage N40 isolate. Also noted in the OMs of all three organisms were unusual structures, designated linear bodies, which typically were more or less perpendicular to the axis of the bacterium. A comparison of freeze-fractured B. burgdorferi and Treponema pallidum, the syphilis spirochete, revealed that the OM architectures of these two pathogens differed markedly. All large membrane blebs appeared to be bounded by a membrane identical to the OM of B. burgdorferi whole cells; in some blebs, the fracture plane also revealed a second bilayer closely resembling the B. burgdorferi cytoplasmic membrane. Aggregation of the lipoprotein immunogens outer surface protein A (OspA) and OspB on the bacterial surface by incubation of B. burgdorferi B31 with specific polyclonal antisera did not affect the distribution of OM particles, supporting the contention that lipoproteins do not form particles in freeze-fractured OMs. The expression of poorly immunogenic, surface-exposed proteins as virulence determinants may be part of the parasitic strategy used by B. burgdorferi to establish and maintain chronic infection in Lyme disease.

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The major potato tuber protein, patatin, is a family of 40kd glycoproteins that constitutes forty per cent of the soluble protein in tubers but is generally undetectable in other tissues. Fused rocket immunoelectro-phoresis was used to detect in roots patatin that is immunologically different from tuber patatin. Western blots of SDS-polyacrylamide gels show root patatin to have a different molecular weight distribution than tuber patatin isoforms, but immunoprecipitation of in vitro translation products shows the patatin precursors to be of similar molecular weight in both tissues. This suggests that post-translational processing may differ in tubers and roots. Northern blots show that tuber and root patatin mRNAs are of similar size, but tuber transcripts are about 100-fold more abundant. 5' S1 nuclease and primer extension mapping suggests the class of patatin transcripts expressed in roots (class II transcripts) to be a subset of patatin transcripts expressed in tubers (classes I and II). Class II patatin mRNAs differ from class I transcripts by the presence of a 22 nucleotide insertion just upstream of the initiation codon. These data demonstrate that expression of the patatin multigene family is differentially regulated in tubers and roots.

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.

Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracelhularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins.Lyme disease and syphilis are infectious disorders caused by the spirochetal pathogens Borrelia burgdorferi and Treponema pallidum, respectively. These diseases share many clinical and microbiological features, including the abilities of their respective pathogens to persist for prolonged periods in individuals with high titers of specific antibodies (19,31). In the case of syphilis, efforts to elucidate this phenomenon revealed that the outer membrane of T. pallidum is a fragile phospholipid bilayer with a paucity of integral membrane proteins (23,25,35). The major membrane immunogens of T. pallidum, molecules formerly thought to be surface exposed (1,17,21), are now believed to be lipoproteins anchored by fatty acids to the periplasmic leaflet of the cytoplasmic membrane (8,22,23,28,33). The recognition that the outer membrane of B. burgdorferi is similarly labile (2) and therefore easily disrupted during experimental manipulation led us to consider the possibility that the major lipoprotein immunogens of B. burgdorferi, outer surface proteins (Osps) A and B (7), also have been incorrectly characterized as exclusively surface-exposed molecules (3,4,18). Interestingly, a reevaluation of published data provides some support for this contention. Periplasmic constitutents, namely, endoflagella (15) washing to remove this material could disrupt or even remove the outer membranes. We therefore focused our analyses on intact cells and protoplasmic cylinders, reasoning that obvious reductions of putative outer membrane proteins should be detectable in the cylinder fractions following selective removal of the outer membranes.Three preparations of B. burgdorferi B31 (high passage) were investigated. One consisted of viable organisms pelleted directly from BSK II medium at 13,700 x g for 10 min. In the other two, organisms were incubated on ice for 30 min with or without 0.1% Triton X-114; this incubation was followed by three successive centrifugations (each at 13,700 x g for 10 min at 4°C) and resuspensions (washes) in phosphate-buffered saline (PBS), pH 7.4. Portions of each preparation were removed for SDS-polyacrylamide gel electrophoresis (PAGE) or prepared for whole-mount and ultrathin-section electron microscopy.Nearly all of the organisms taken directly from BSK II medium were intact ( Fig. la and b). Repeated centrifugation and resuspension, manipulations comparable to those used in surface localization studies (3,4,18), disrupted many of the organisms (Fig. lc and ld); scattered membrane vesicles also were seen (data not shown). Outer membranes were uniformly absent from the detergent-incubated organisms, and membrane vesicles were not seen despite extensive ...

The 3' region of the Anabaena variabilis nifD gene contains an li-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional ni/D gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the ni/K-proximal end of the ni/D element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xis4 gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.

A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.