Genome Sequences of Mycoplasma alligatoris A21JP2^T and Mycoplasma crocodyli MP145^T

Abstract

Mycoplasma alligatoris and Mycoplasma crocodyli are closely related siblings, one being highly virulent and the other relatively attenuated. We compared their genomes to better understand the mechanisms and origins of M. alligatoris' remarkable virulence amid a clade of harmless or much less virulent species. Although its chromosome was refractory to closure, M. alligatoris differed most notably by its complement of sialidases and other genes of the N-acetylneuraminate scavenging and catabolism pathway.

TEXT

Unlike the mycoplasmas that cause usually subtle mycoplasmosis, Mycoplasma alligatoris causes acute lethal multisystemic inflammatory disease in susceptible hosts (1, 2). We compared its genome to that of its attenuated sibling species Mycoplasma crocodyli, which causes only mild pneumonia or polyarthritis (5, 6). The DNA sequences were obtained through traditional random shotgun sequencing conducted at the University of Florida and the J. Craig Venter Institute (JCVI), assembled at JCVI using Celera Assembler, and analyzed using JCVI's automated functional annotation service, which included Glimmer, BLAST-Extend-Repraze, HMM/TMHMM searches, and SignalP predictions. The manual annotation tool Manatee was used to curate the output.

The closed circular M. crocodyli MP145^T genome was 934,379 nucleotides (nt) in length, had a 26% G+C content, and contained 81% coding sequences. Of 763 predicted open reading frames (ORFs), about two-thirds had assigned functions, including 43 structural RNAs; 43 proteins involved in metabolism of amino acids, purines, pyrimidines, nucleosides, nucleotides, cofactors, prosthetic groups, or carriers; 16 proteins involved in fatty acid or phospholipid metabolism; 87 proteins involved in central intermediary or energy metabolism; 83 proteins involved in transport or binding; 238 proteins involved in nucleic acid metabolism, transcription or translation, or protein fate; 12 proteins involved in regulatory functions, including HrcA, ArsR, GntR, LacI, MarR, and RpiR family transcription regulators; 28 proteins involved in processes including cell division and adaptation to atypical conditions, including two glycosyltransferases possibly involved in biofilm synthesis; and 64 putative cell envelope lipoproteins. About one-third of the ORFs remained hypothetical. The proportions of the genome assigned to the various functional role categories were generally similar to those of related glucose-fermenting mollicutes in the JCVI's Comprehensive Microbial Resource, but M. crocodyli had no identified genes for adhesins, variable surface antigens, or mobile element functions such as the four IS1634/ISMhp1-type transposases present in M. alligatoris A21JP2^T. Candidate M. crocodyli virulence factors included hyaluronidase, glucuronidase, one α- and two β-hexosaminidases, a broad-specificity polysaccharide lyase (chondroitinase), and α-amylase. In contrast to M. alligatoris, no sialidase (neuraminidase) or other genes of the N-acetylneuraminate scavenging and catabolism pathway were evident.

The noncontiguous finished draft M. alligatoris A21JP2^T genome was 973,030 nt in length, with a 26% G+C content and 89% coding sequences; however, the 32 remaining gaps were recalcitrant to closure. Twenty-one of the contigs are small (1,685 to 15,302 bp). These and even some of the larger contigs are likely the result of transposon jumping and genome inversions that made the sequenced DNA preparations contain a number of minor populations that confounded the assembly. Its complement of glycosidases, including five hyaluronidases, glucuronidase, two sialidases, three β-galactosidases, β-hexosaminidase, α-amylase (glycogenase), and two glycosyltransferases, is unprecedented among mollicutes (3). MUMer alignments found only limited synteny between the two species. Site-specific gene knockout methods were ineffective in M. alligatoris, but a library of M. alligatoris knockout mutants was created by random transposon insertion using the mini-Tn4001tetM plasmid pTF20 (4). Unique stable knockouts were obtained for ∼1,100 random Tet^r clones mapped using direct genomic DNA sequencing via extension from primers complementary to the transposon. Isogenic knockouts of the extracellular hyaluronidase, sialidase, galactosidase, and hexosaminidase genes are available to test specific hypotheses regarding the roles of glycosidases in virulence of M. alligatoris and other mycoplasmas.

Nucleotide sequence accession numbers.

The master record for the M. alligatoris A21JP2^T whole-genome shotgun project is GenBank accession no. NZ_ADNC01000000; the version described in this paper is the first version, NZ_ADNC00000000.1. The closed circular M. crocodyli MP145^T genome is GenBank accession no. CP001991; the version described in this paper is the first version, CP001991.1.