In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, >256 g/ml) and 1 was weakly resistant (MIC, 8 g/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.Mycoplasma pneumoniae is a pathogen causing human respiratory infections such as atypical pneumonia, mainly in children and younger adults. In the chemotherapy of M. pneumoniae infection in children, erythromycin (ERY) and clarithromycin (CLR) among 14-membered macrolides and the 15-membered macrolide azithromycin (AZM) are usually considered the first-choice agents in Japan. Although there was no report on the isolation of ERY-resistant M. pneumoniae before 2000 in Japan, we found that ca. 20% of M. pneumoniae strains isolated from patients from 2000 to 2003 were ERY resistant. These results are consistent with pediatricians' impression that antibiotics such as ERY, CLR, and clindamycin (CLI) are not effective for some patients with M. pneumoniae infection.It is well known that the macrolide-lincosamide-streptogramin B (MLS) antibiotics inhibit protein synthesis by binding to domain II and/or domain V of 23S rRNA (3, 26). Lucier et al. (10) and Okazaki et al. (17) found that an A-to-G transition or A-to-C transversion at position 2063 (corresponding to 2058 in Escherichia coli numbering) or 2064 of the 23S rRNA gene resulted in high resistance to macrolide antibiotics. No point mutation was found in domain II of 23S rRNA of the ERYresistant M. pneumoniae strains used in the present study.We report here the prevalence of macrolide-resistant M. pneumoniae infection in Japan. By using 13 ERY-resistant M. pneumoniae strains, we investigated the mechanisms of resistance to MLS antibiotics. Furthermore, we established restriction fragment length polymorphism (RFLP) techniques to detect point mutations in domain V of 23S rRNA...

Jasmonates mediate various physiological events in plant cells such as defense responses, flowering, and senescence through intracellular and intercellular signaling pathways, and the expression of a large number of genes appears to be regulated by jasmonates. In order to obtain information on the regulatory network of jasmonate-responsive genes (JRGs) in Arabidopsis thaliana (Arabidopsis), we screened 2880 cDNA clones for jasmonate responsiveness by a cDNA macroarray procedure. Since many of the JRGs reported so far have been identified in leaf tissues, the cDNA clones used were chosen from a non-redundant EST library that was prepared from above-ground organs. Hybridization to the filters was achieved using α-33 P-labeled single-strand DNAs synthesized from mRNAs obtained from methyl jasmonate (MeJA)-treated and untreated Arabidopsis seedlings. Data analysis identified 41 JRGs whose mRNA levels were changed by more than three fold in response to MeJA. This was confirmed by Northern blot analysis by using eight representatives. Among the 41 JRGs identified, 5 genes were JA biosynthesis genes and 3 genes were involved in other signaling pathways (ethylene, auxin, and salicylic acid). These results suggest the existence of a positive feedback regulatory system for JA biosynthesis and the possibility of crosstalk between JA signaling and other signaling pathways.

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.

Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, c1arithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 238 rRNA gene. Furthermore, 141 EM-sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 fLglml of erythromycin (EM). Among 11 EM-resistant strains that grew in the medium, point mutations in the 238 rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM-resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.

Mycoplasma pneumoniae lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins-P65, HMW2, P41, and P24-that are encoded in the crl operon (for "cytadherence regulatory locus"), which is known to be essential for the adherence of M. pneumoniae to host cells. When the P65 and HMW2 proteins were fused to Mycoplasma pneumoniae, one of the smallest self-replicating bacteria known, is a causative agent of bronchitis and primary atypical pneumonia in humans (43,44). M. pneumoniae lacks a cell wall and hence has a pleomorphic cell shape. However, a majority of M. pneumoniae cells in cultures are filamentous and have a differentiated terminal structure at one pole. This terminal structure, the attachment organelle, is a tapered membrane protrusion responsible for the adherence of M. pneumoniae to host respiratory epithelium (cytadherence) (23, 24). The attachment organelle renders M. pneumoniae cells asymmetric and functions as a leading end for gliding motility. This organelle also may have a role in initiating cell division in M. pneumoniae, because the bifurcation of the attachment organelle seems to occur prior to the binary fission of M. pneumoniae (2,6,7,25,26,34,36,48).The attachment organelle and polar filamentous cell shape of M. pneumoniae are thought to be stabilized by intracellular cytoskeleton-like structures, which have been observed in electron micrographs of M. pneumoniae (5,25,33). The most remarkable architectural feature of the cytoskeleton-like structures is the electron-dense core, a rod-like structure that exists longitudinally at the center of the attachment organelle (33). This rod-like structure, measuring about 300 nm long and 80 nm thick, has a knob at the distal end (terminal button) (33,45). A network of fibrous structures is also observed in the cytoplasm of M. pneumoniae (33). These cytoskeleton-like structures are major components of the Triton X-100-insoluble fraction of M. pneumoniae cells (Triton shell) and are thought to have a scaffold-like function upon which other cell components construct M. pneumoniae cells (45,51).A recent report indicated that the Triton X-100-insoluble fraction contains about 100 proteins, including most of the known proteins required for cytadherence (P1, B, C, HMW1, HMW2, and HMW3) (45). These cytadherence-related proteins are believed to be the main components of the attachment organelle and are encoded in three operons, designated p1, hmw, and crl, in the genome (24,25). Protein P1 (encoded in the p1 operon) is a major adhesin molecule responsible for cytadherence and is densely clustered at the surface of the attachment organelle (9,18,26,48). Proteins B, C, HMW1, HMW2, and HMW3, called cytadherence accessory proteins,...