NUCLEOTIDE-SEQUENCE OF THE SPIROPLASMA-CITRI FIBRIL PROTEIN GENE

Electron microscopic observation of spiroplasmas lysed by detergent (sodium deoxycholate) revealed the release of bundles of fibrils from the cells. Individual fibrils are 4 nm in diameter and possess a 9-nm periodicity along their length. These fibrils are thought to function as cytoskeletal structures involved in the shape and motility of spiroplasmas. Polyacrylamide gel electrophoresis of density gradient-purified fibrils showed a protein of approximately 55 kDa. Oligonucleotide probes were constructed from the N-terminal amino acid sequence of two peptides obtained after V8 protease hydrolysis of the fibril protein. The probes were used to identify the clones in a genomic DNA library of Spiroplasma citri that contained inserts carrying the probe sequence. Sequencing of a 3.3-kbp fragment yielded the full open reading frame of the fibril protein gene and the start of a second open reading frame of an unknown protein. The fibril protein is composed of 515 amino acids, which have a computed molecular mass of 59 kDa. Northern (RNA) blot hybridization and primer extension experiments showed that transcription of the fibril protein gene starts from a promoter located 100 nucleotides upstream of the initiation codon and stops at a rho-independent type terminator, leading to a 1.7-kbp transcript. Southern blot hybridization of genomic DNA using the fibril protein gene as the probe showed that a single copy of the gene is present in the chromosomes of both S. citri and Spiroplasma melliferum. The genotypic symbol fib is proposed for the spiroplasma fibril protein gene.