Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

Abstract

The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf-X-spiralin gene-pfl-pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.Spiroplasmas are mollicutes (mycoplasmas) (class Mollicutes) with motility and helical morphology (33). Analysis of the Spiroplasma genome by classical genetic techniques has been difficult. The dependence of these organisms on complex growth media and their poorly defined metabolic pathways explain the paucity of auxotrophic mutants and hence of genetic markers in these procaryotes. In the absence of such markers, characterization of cloned DNA fragments provides the means for analyzing the structural organization of Spiroplasma genes.The gene for spiralin seemed to be of particular interest. Spiralin (35) is the major membrane protein of Spiroplasma citri, an important plant pathogen (28). It is apparently a transmembrane amphiphilic protein (34), and it is acylated (37). Acylation seems to be a characteristic of several membrane proteins of mollicutes. The role of spiralin is not known. It must, however, be a key protein of the spiroplasmal membrane, since spiralinlike proteins occur in Spiroplasma species other than S. citri (37) and probably in all spiroplasmas. Elucidation of the amino acid sequence of spiralin as deduced from the nucleotide sequence of its gene would undoubtly contribute to a better understanding of its structure and function, as well as the organization of its gene. Sequence determination of the spiralin gene was made possible by the fact that we have previously cloned the gene.

Nucleotide sequence of the Spiroplasma citri fibril protein gene

“…A preferential use of A-and T-rich codons was observed, especially when A or T occurred at the 3' end of codons specifying the same amino acid. Such a situation has already been described for other spiroplasma proteins (5,22). The amino acid sequence of the fibril protein contains 515 amino acids, including four cysteine residues and seven tryptophan residues.…”

“…We used information available on spiroplasma codon usage (preference of codons with A or T in the first or third position) (5,22) to construct the oligonucleotides shown in Fig. 3.…”

Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli

“…Chevalier et al [39] cloned the gene for spiralin, a major membrane protein of SpiropZusmu citri, and found the phosphofructokinase gene downstream of the spiralin gene. They also found part of an open reading frame downstream of the phosphofructokinase gene.…”

Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus

“…Figure 2 shows the alignment of the amino acid sequences of the five bacterial ATP-dependent PFKs known at present (4,7,8,37). The obvious resemblance between these sequences makes it likely that their tertiary structures are very similar and that they derive from a common ancestor.…”