Phylogenetic Analysis of Mycoplasma Strain ISM1499 and Its Assignment to the Acholeplasma oculi Strain Cluster

Abstract

A mycoplasma strain designated ISM1499 was used to develop a mycoplasma genetic system (G. G. Mahairas and F. C. Minion, J. Bacteriol. 171:1775-1780,1989; G. G. Mahairas, C. Jian, and F. C. Minion, Gene 93:61-65, 1990), but phenotypic inconsistencies led to the conclusion that this organism had been classified incorrectly as a member of the species Mycoplasma pulmonis. Studies were initiated to determine the proper taxonomic position of ISM1499, and on the basis of the results of our genetic analysis, this strain was assigned to the AchoZephsma oculi strain cluster. The base composition of strain ISM1499 was identical to the base composition of A. oculi 19L, but not to the base composition of AchoZephsma hidhwii PG8 (28.3 and 30.7 mol% G+C, respectively). The taxonomic position of ISM1499 was examined by performing a parsimony analysis with 16s ribosomal DNA sequence data, and the results were compared with previous phylogenetic reconstructions. Our results indicated that ISM1499 is more closely related phylogenetically to A. oculi 19L than to A. hidlawii PG8 and JAl. Heterogeneity in the 16s ribosomal DNA sequences of A. oculi 19L and ISM1499 and in the 16s ribosomal DNA sequences of A. hidhwii PG8 and JAl may indicate that unusual dissimilarities occur in the 16s ribosomal DNA sequences of members of the genus AchoZephsma.Mycoplasma strain ISM1499 was originally isolated from a Mycoplasma pulmonis culture as a high-frequency transforming isolate. This organism was used to study mycoplasma transformation and to establish a mycoplasmal cloning system (9-12). On the basis of the results of these studies and Western blot (immunoblot) analyses performed with antisera raised in rabbits against a M. pulmonis stock strain by Minion and coworkers, strain ISM1499 was assumed to be a M. pulmonis strain, possibly a membrane or restriction mutant with a mutation which conveyed upon it the high-frequency transforming phenotype.Dybvig and coworkers first noticed differences between ISM1499 and other M. pulmonis strains when they examined recA homology and phage typing data (4, 5). A serological examination by J. G. Tully (Mycoplasmology Section, Laboratory of Medical Microbiology, National Institute of Allergy and Infectious Diseases confirmed that ISM1499 is an Acholeplasma strain rather than an M. pulmonis strain; this resulted in author's corrections being placed in both the Journal of Bacteriology and Plasmid (11, 12). The serological typing results indicated that ISM1499 is antigenically related to Acholeplasma oculi and Acholeplasma laidlawii; the strongest reactivity was obtained with an anti-A. oculi 19L antiserum.The class Mollicutes comprises a unique group of eubacteria that lack cell walls (17). In a newly proposed classification scheme Tully et al. (24) divided these organisms into eight genera on the basis of the phylogenetic data of Weisburg et al. (26) and various other characteristics, including a sterol growth requirement (the genera Mycoplasma, Ureaplasma, Entomoplasma, Spiroplasma, and Anaeroplasma), a ...

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

“…Furthermore, to select the supposedly rare events of integration, we are currently trying to counter-select the replication of the oriC plasmid, as proposed for other bacteria (36). Although a case of homologous recombination with M. pulmonis using suicide vectors has been reported (28), further studies indicated that these results were obtained with a strain that did not belong to the species M. pulmonis but belonged to the other mollicute species Acholeplasma oculi (1,9,28). Our results confirmed that homologous recombination for M. pulmonis using suicide vectors is difficult, if not impossible, to detect.…”

Phylogeny-Based Rapid Identification of Mycoplasmas and Ureaplasmas from Urethritis Patients

“…Until now, the phylogenetic analysis of mycoplasmas and ureaplasmas has been based on approximately 1,000 bp of 16S rRNA gene (2,3,21). The phylogenetic analysis based on these sequences enables the prototype strains to be classified into species; however, this size, approximately 1,000 bp of a nucleotide sequence, is too long for the phylogeny-based rapid identification of PCR products to be applied with clinical materials.…”

“…However, using several species-specific PCRs to determine all human mycoplasmas and ureaplasmas associated with urethritis would be a complicated undertaking. To date, amplification of the genome by PCR, followed by determination of the nucleotide sequences and phylogenetic analysis has become a generalized technique for both the classification and identification of etiological agents (2,3,12,21,28). When the nucleotide sequences of 16S rRNA genes were compared by van Kuppeveld et al (33), the 5Ј flanking region of the variable V3 region and 3Ј flanking region of the variable V6 region were well conserved among the 15 prototype strains of mycoplasmas and ureaplasmas.…”

The Genome Sequence ofMycoplasma hyopneumoniaeStrain 232, the Agent of Swine Mycoplasmosis

“…Strain 232 of M. hyopneumoniae was isolated from a pig infected with strain 11 (4,30) and has been commonly used to study virulence and vaccination regimens in the United States (49,54,55). Chromosomal DNA was isolated by the method of Artiushin et al (1), and two libraries were constructed. Chromosomal DNA was incompletely digested with Tsp509I and size fractionated on sucrose gradients, and 2-to 4-kb fragments were ligated into EcoRI-digested, dephosphorylated pZERO vector (Invitrogen) with selection for kanamycin resistance (library 1).…”