Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence.

AUTOR(ES)

Krause, D C

RESUMO

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA-) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA+ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5' end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 Mr cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 Mr and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.

Documentos Relacionados

• HMW1 Is Required for Stability and Localization of HMW2 to the Attachment Organelle of Mycoplasma pneumoniae
• Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle.
• Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.
• Stability of Mycoplasma pneumoniae Cytadherence-Accessory Protein HMW1 Correlates with Its Association with the Triton Shell
• Nucleotide sequence analysis reveals novel features of the phase-variable cytadherence accessory protein HMW3 of Mycoplasma pneumoniae.