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.Members of the genus Mycoplasma are distinguished by their lack of a cell wall or cell wall precursors, the incorporation of cholesterol in their membranes, the use of UGA as a codon for tryptophan rather than termination, and their extremely small genomes (Ն580 kbp). In addition, several mycoplasma species possess a complex terminal structure that directs the colonization of vertebrate host cells and is thought to function in cell division and gliding motility. The presence of this complex organelle stands in marked contrast to the perception of mycoplasmas as otherwise simple wall-less prokaryotes (1). In Mycoplasma pneumoniae, a major cause of primary atypical community-acquired pneumonia, the terminal (attachment) organelle is seen by transmission electron microscopy of thin sections as a membrane-bound extension of the cell body, with an electron-dense core associated with a cytoskeleton-like network of mycoplasma proteins (2). The organization, regulation, and assembly of the terminal organelle of M. pneumoniae is poorly understood, largely due to the stringent nutritional requirements and poor growth yields of mycoplasmas, as well as a general lack of genetic tools for use in these prokaryotes. Finally, no counterparts to the terminal organelle have been identified among the walled bacteria.Adherence of M. pneumoniae to host respiratory epithelium (cytadheren...
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.Adherence of the human pathogen Mycoplasma pneumoniae to host respiratory epithelial cells constitutes a critical step in the pathway leading to tracheobronchitis and atypical (walking) pneumonia. A polar extension of the mycoplasma cell membrane, the terminal organelle, is the major site of attachment and contains proteins responsible both directly and indirectly for attachment. Although the M. pneumoniae adhesin P1 mediates receptor binding (1, 5, 13), Triton X-100 (TX)-insoluble (hereafter referred to as triton shell or cytoskeletal) cytadherence-accessory proteins are required for both the proper formation of the attachment organelle and the localization of P1 to the attachment organelle (reviewed in reference 17). Loss of some of these proteins results in the failure to accumulate P1 at the attachment organelle and confers irregular cell shapes (9,21,34,35), though the means by which this is manifested is unknown. Therefore, characterization of the biochemical properties and the order of assembly of the cytadherence-accessory proteins is necessary for a fuller understanding of both the regulation of adherence and the structure and formation of the attachment organelle.The cytadherence-accessory protein HMW1 is a 112-kDa phosphoprotein (3, 4) that is concentrated along mycoplasma cell filaments, including the attachment organelle, as revealed by immunoelectron microscopic analyses (34). HMW1 has a modular structure (Fig. 1), including a l...
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, therpsD 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 thep32, p21, p50, and rpsDgenes. 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 ofp21 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 thehmw gene cluster. Taken together, these findings suggest that the genes of this locus constitute an operon expressed from overlapping transcripts.
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 (02-). However MO) seedlings were grown in a growth chamber at 23 to 270C with a photoperiod of 14 h/l0 h light/ dark. The seedlings were grown in vermiculite in plastic trays with cheesecloth covering aeration holes in the bottom. The plants were watered daily from below with 1 mM CaSO4. On days 3 and 5, the CaSO4 was replaced with a nutrient solution consisting of the following: 4 mm CaCl2 2H20, 0.45 mM K2HPO4.3H20, 0.5 mM MgSO4.7H20, 1.25 mM K2SO4, 10 mM NH4NO3, 26 AM Fe citrate, 2.3 AM H3BO3, 0.9 ,AM MnSO4. H20, 0.6 ,uM ZnSO4.7H20, 0.15 ,M CuSO4.5H20, 0.1 M Na2MoO4. 2H20, 0.01 ,uM CoCl2 * 6H20, and 0.1 1 AM NiCl2 * 6H20 (D.G. Blevins, Department of Agronomy, University of Missouri, personal communication). Experiments were performed on 9-to 1 -d-old plants. BacteriaPseudomonas syringae pv. pisi, a bacterium that induces HR on cucumber, was used for all experiments. The bacteria were stored at -70TC in 12% glycerol. When needed, bacteria were removed from the freezer and streaked onto a nutrient agar plate. The bacteria from the plate were transferred to a nutrient agar slant 48 h before inoculation and then were transferred from the slant to nutrient broth 12 to 18 h before inoculation. Bacteria were incubated at 25°C on a reciprocal shaker to obtain a high concentration of cells in the log phase of growth. Preceding inoculation, bacteria were collected by centrifugation at l0,OOOg for 5 min. The bacteria were resuspended in distilled water and collected by centrifugation at l0,OOOg for 5 min. The bacteria were again resuspended in distilled water, and the suspension was adjusted spectrophotometrically to 108 colony forming units mL-'. Plants were inoculated by piercing the abaxial epidermis of the cotyledons 1157 www.plantphysiol.org on April 3, 2019 -Published by Downloaded from
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