Nucleotide sequence of the Spiroplasma citri fibril protein gene

Abstract

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.