The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3 end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRSbinding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.Spiroplasmas are wall-free bacteria belonging to the class Mollicutes, a group of microorganisms phylogenetically related to Gram-positive bacteria with low guanine ϩ cytosine contents (1). Sequence analysis (70) of a 6.8-kbp 1 DNA fragment (GenBank TM accession number AF012877) of the phytopathogenic mollicute Spiroplasma citri (2) made it possible to identify eight putative ORFs that encode ribosomal protein S2, elongation factor Ts, spiralin, 6-phosphofructokinase, pyruvate kinase, and three unidentified proteins (A, B, and X) (Fig. 1). Ribosomal protein S2 and the translational elongation factor Ts (Ef-Ts), respectively, encoded by rpsB and tsf genes, are both components of the translational apparatus in prokaryotes. These genes are adjacent in Escherichia coli (3, 4) and Bacillus subtilis (5), whereas in the genome of the two mollicutes Mycoplasma genitalium (6) and Mycoplasma pneumoniae (7) they reside at different locations, and thus each of them may constitute monocistronic transcriptional units or be part of two different polycistronic operons.The organization and relative orientation of rpsB and tsf of S. citri ( Fig. 1) are analogous to those reported for E. coli (3, 4) and B. subtilis (5). In E. coli, rpsB and tsf form a single transcriptional unit, and an attenuation mechanism was proposed to explain the 1 to 3 ratio of Ef-Ts to S2 (3). In B. subtilis a potential terminator is found between rpsB and tsf. In S. citri, the absence of a rho-independent termination signal in the spacer region between rpsB and tsf, and between tsf and x, indicates that rpsB, ...