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Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

ABSTRACT: DNA sequencing and Southern blot analyses of a Borrelia burgdorferi DNA fragment encoding a signal sequence led to the discovery of a genetic locus, designated 2.9, which appears to be present in at least seven copies in virulent B. burgdorferi 297. DNA sequence analysis of these regions revealed that each 2.9 locus contained an operon of four genes (ABCD) and open reading frames designated rep+ (positive strand) and rep- (negative strand) which encoded multiple repeat motifs. Downstream of the rep+ gene(s) in six of the completely cloned and sequenced 2.9 loci also were lipoprotein (LP) genes possessing highly similar signal sequences but encoding variable mature polypeptides. The lipoproteins could he separated into two classes on the basis of hydrophilicity profiles, sequence similarities, and reactivity with specific antibodies. The 2.9 loci were localized to two (20- and 30-kb) supercoiled plasmids in B. burgdorferi 297. Northern (RNA) blot analysis established that the 2.9 ABCD operon was only minimally expressed, whereas the rep- gene(s) and at least three of the seven LP genes were expressed by B. burgdorferi in vitro. A single putative promoter element was identified by RNA primer extension analysis upstream of the ABCD operon, whereas a number of potential promoter regions existed upstream of the LP genes. The combined data indicate that the ABCD operon, rep+ and rep- genes, and LP genes are separately transcribed during in vitro growth. The 2.9 loci possess a repetitiveness, diversity, and complexity not previously described for B. burgdorferi; differential expression of these genes may facilitate the spirochete's ability to survive in diverse host environments.

SUBMITTER: Porcella SF

PROVIDER: S-EPMC178083 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

Porcella S F SF Popova T G TG Akins D R DR Li M M Radolf J D JD Norgard M V MV

Journal of bacteriology 19960601 11

DNA sequencing and Southern blot analyses of a Borrelia burgdorferi DNA fragment encoding a signal sequence led to the discovery of a genetic locus, designated 2.9, which appears to be present in at least seven copies in virulent B. burgdorferi 297. DNA sequence analysis of these regions revealed that each 2.9 locus contained an operon of four genes (ABCD) and open reading frames designated rep+ (positive strand) and rep- (negative strand) which encoded multiple repeat motifs. Downstream of the ...[more]

PMID: 8655511

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Project description:Plasmid cp8.3 of Borrelia afzelii IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the Borrelia burgdorferi sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some B. afzelii isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field-pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of B. afzelii IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all B. burgdorferi sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the B. burgdorferi sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the B. burgdorferi sensu lato genome.

Project description:In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526-1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.

Project description:BackgroundUpstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood.MethodsWe examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs.ResultsWe provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses.ConclusionOur data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides.

Dataset Information

Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

Publications

Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

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