Stability of Mycoplasma pneumoniae cytadherence-accessory protein HMW1 correlates with its association with the triton shell.

Mycoplasma pneumoniae adsorbs to host respiratory epithelium primarily by its attachment organelle, the proper function of which depends upon mycoplasma adhesin and cytoskeletal proteins. Among the latter are the cytadherence-associated proteins HMW1 and HMW2, whose specific roles in this process are unknown. In the M. pneumoniae cytadherence mutant I-2, loss of HMW2 results in accelerated turnover of HMW1 and other cytadherence-accessory proteins, probably by proteolysis. However, both the mechanism of degradation and the means by which these proteins are rendered susceptible to it are not understood. In this study, we addressed whether HMW1 degradation is a function of its presence among specific subcellular fractions and established that HMW1 is a peripheral membrane protein that is antibody accessible on the outer surfaces of both wild-type and mutant I-2 M. pneumoniae but to a considerably lesser extent in the mutant. Quantitation of HMW1 in Triton X-100-fractionated extracts from cells pulse-labeled with [(35)S]methionine indicated that HMW1 is synthesized in a Triton X-100-soluble form that exists in equilibrium with an insoluble (cytoskeletal) form. Pulse-chase analysis demonstrated that over time, HMW1 becomes stabilized in the cytoskeletal fraction and associated with the cell surface in wild-type M. pneumoniae. The less efficient transition to the cytoskeleton and mycoplasma cell surface in mutant I-2 leads to accelerated degradation of HMW1. These data suggest a role for HMW2 in promoting export of HMW1 to the cell surface, where it is stable and fully functional.

Transcriptional analysis of the hmw gene cluster of Mycoplasma pneumoniae.

Mycoplasma pneumoniae adherence to host cells is a multifactorial process that requires the cytadhesin P1 and additional accessory proteins. The hmw gene cluster consists of the genes p30, hmw3, and hmw1, the products of which are known to be essential for cytadherence, the rpsD gene, and six open reading frames of unknown function. Putative transcriptional terminators flank this locus, raising the possibility that these genes are expressed as a single transcriptional unit. However, S1 nuclease protection and primer extension experiments identified probable transcriptional start sites upstream of the p32, p21, p50, and rpsD genes. Each was preceded at the appropriate spacing by the -10-like sequence TTAAAATT, but the -35 regions were not conserved. Analysis of the M. pneumoniae genome sequence indicated that this promoter-like sequence is found upstream of only a limited number of open reading frames, including the genes for P65 and P200, which are structurally related to HMW1 and HMW3. Promoter deletion studies demonstrated that the promoter-like region upstream of p21 was necessary for the expression of p30 and an hmw3-cat fusion in M. pneumoniae, while deletion of the promoter-like region upstream of p32 had no apparent effect. Analysis by reverse transcription-PCR confirmed transcriptional linkage of all the open reading frames in the hmw gene cluster. Taken together, these findings suggest that the genes of this locus constitute an operon expressed from overlapping transcripts.

Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally.

The genomic sequence of Mycoplasma pneumoniae establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication. However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species. This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia. Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation. However, the organization, regulation, and assembly of the attachment organelle in M. pneumoniae are poorly understood, and no counterparts have been identified among the walled bacteria. M. pneumoniae possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle. The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle. Disruptions in hmw2 result in the loss of HMW1 and HMW3. However, the hmw1 and hmw3 genes were transcribed and translated at wild-type levels in hmw2 mutants. HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period. Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover. Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW- mutants.

Water Relation Alterations Observed during Hypersensitive Reaction Induced by Bacteria.

Upon exposure to pathogenic bacteria, resistant and nonhost plants undergo a hypersensitive reaction (HR) that is expressed as rapid plant cell death. If sufficient concentrations of these bacteria are inoculated to such plant tissue, then that portion of the tissue rapidly collapses and becomes necrotic. As the tissue collapses the water relations of inoculated tissues become markedly disturbed. We measured a decline in the relative water content (RWC) in the leaf-like cotyledons of cotton (Gossypium hirsutum cv Immune 216) within the first 4 h (cut at 1 h) after inoculation with Pseudomonas syringae pv tabaci. However, the decrease in RWC was not caused by a decrease in initial fresh weight but by increased water uptake during incubation in water. By 8 h after inoculation, cotyledons still on the plant had lost turgidity, and their area decreased. K+ efflux was also observed concurrently with the decrease in RWC, providing a reason for the loss of turgidity in the tissue. These observations suggest that cells lose turgor and change shape from cylinders with large intercellular spaces to those of a more tabular shape. During this change cell walls come closer together, providing an avenue for increased water uptake through capillary action. The stomatal diffusive resistance of intact cotyledons increased; hence, water loss through stomata is not the cause of the observed wilting and RWC decline. An increase in K+ per dry weight suggests that phloem loading or movement may also be impaired during bacterially induced HR.

Use of Dimethyl Sulfoxide to Detect Hydroxyl Radical during Bacteria-Induced Hypersensitive Reaction.

Excess active oxygen is generated during the hypersensitive reaction (HR), an incompatible reaction of plants to bacterial pathogens. During HR, lipid peroxidation correlates chronologically with production of the oxygen species, superoxide (O(2) (.-)). However, O(2) (.-) may not be the active oxygen species that initiates lipid peroxidation. Evidence from other systems suggest that O(2) (.-) is converted to the hydroxyl radical (HO(.)) before lipid peroxidation is initiated. Until recently, HO(.) could not be detected directly in vivo. This study utilizes a newly reported method to directly detect and quantify the formation of HO(.)in vivo. Dimethyl sulfoxide (DMSO), used as a molecular probe, is oxidized by HO(.), forming the stable compound methanesulfinic acid. The methanesulfinic acid can be easily extracted from plant tissues and measured with a colorimetric assay. This study demonstrates significant increases in HO(.) concentration after simultaneous infiltration of cucumber (Cucumis sativa L.) plants with paraquat and DMSO. The concentration of HO(.) did not increase significantly when cucumber plants were infiltrated simultaneously with the HR-inducing bacteria, Pseudomonas syringae pv. pisi, and with DMSO. Lipid peroxidation, however, could be measured at times when HO(.) was not detectable. It appears that HO(.) is not generated during bacteria-induced HR; therefore, HO(.) is not responsible for the initiation of lipid peroxidation.