Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

Abstract

Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium, are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn4001 was not localized in or near the hmw1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

“…Regardless of this, the intermediate cytadherence displayed by our mg317 2 mutants is in accordance with the phenotype described for the M. pneumoniae hmw3 mutant (Willby & Krause, 2002). Thus, the partial cytadherence activity displayed by mg317 2 and mg218 2 mutants provides an explanation for the previous failure to isolate M. genitalium haemadsorptionnegative mutants showing transposon insertions in these two cytadherence-related genes (Reddy et al, 1996). Interestingly, a similar situation could be hampering the isolation of Mycoplasma gallisepticum haemadsorption-negative mutants bearing transposon insertion in genes different from gapA and crmA (Mudahi-Orenstein et al, 2003).…”

“…Two independent factors have probably hindered the study of TO formation and the whole cytadherence scenario in the smallest mycoplasma. First, screening for haemadsorption-negative colonies has not allowed the isolation of transposon-generated mutants involving any of the cytadherence-accessory genes from M. genitalium (Reddy et al, 1996). Second, the high rates of spontaneous occurrence of class I and class II haemadsorption-negative mutants (Mernaugh et al, 1993), which probably reflect a subjacent cytadherence phase variation mechanism (Burgos et al, 2006), hampers the screening process and jeopardizes the characterization of the mutants isolated.…”

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

“…The cytadherence regulatory locus (crl) was identified by transposon mutagenesis and maps about 160 kbp from the hmw operon, yet insertions therein result in loss of HMW1-HMW3 (16). Recently, Reddy et al (35) confirmed our finding that transposon insertion in this locus results in loss of cytadherence.…”

Functional Analysis of the Mycoplasma genitalium MG312 Protein Reveals a Specific Requirement of the MG312 N-Terminal Domain for Gliding Motility

“…This fact suggests differences regarding the role of MG312/HMW1 in the cytadherence properties of M. genitalium and M. pneumoniae or the presence of additional mutations in the M. pneumoniae M6 strain (10). On the other hand, the cytadherence activity remaining in the ⌬MG_312 mutants justifies the absence of M. genitalium MG312 mutants after screening for nonadherent mutants (24,28).…”