The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution.

Although mycoplasmas have small genomes, many of them, including the HIV-associated opportunist Mycoplasma penetrans, construct a polar attachment organelle (AO) that is used for both adherence to host cells and gliding motility. However, the irregular phylogenetic distribution of similar structures within the mycoplasmas, as well as compositional and ultrastructural differences among these AOs, suggests that AOs have arisen several times through convergent evolution. We investigated the ultrastructure and protein composition of the cytoskeleton-like material of the M. penetrans AO with several forms of microscopy and biochemical analysis, to determine whether the M. penetrans AO was constructed at the molecular level on principles similar to those of other mycoplasmas, such as Mycoplasma pneumoniae and Mycoplasma mobile We found that the M. penetrans AO interior was generally dissimilar from that of other mycoplasmas, in that it exhibited considerable heterogeneity in size and shape, suggesting a gel-like nature. In contrast, several of the 12 potential protein components identified by mass spectrometry of M. penetrans detergent-insoluble proteins shared certain distinctive biochemical characteristics with M. pneumoniae AO proteins, although not with M. mobile proteins. We conclude that convergence between M. penetrans and M. pneumoniae AOs extends to the molecular level, leading to the possibility that the less organized material in both M. pneumoniae and M. penetrans is the substance principally responsible for the organization and function of the AO.IMPORTANCEMycoplasma penetrans is a bacterium that infects HIV-positive patients and may contribute to the progression of AIDS. It attaches to host cells through a structure called an AO, but it is not clear how it builds this structure. Our research is significant not only because it identifies the novel protein components that make up the material within the AO that give it its structure but also because we find that the M. penetrans AO is organized unlike AOs from other mycoplasmas, suggesting that similar structures have evolved multiple times. From this work, we derive some basic principles by which mycoplasmas, and potentially all organisms, build structures at the subcellular level.

Mutations in central carbon metabolism genes suppress defects in nucleoid position and cell division of replication mutants in Escherichia coli.

A genetic link of the carbon metabolism and DNA replication was recently reported for the representative of Gram-negative bacteria, Escherichia coli. Our studies showed that the viability of thermosensitive replication mutants at high temperature can be improved or fully recovered by deleting certain genes of central carbon metabolism (CCM). In order to improve our understanding of this phenomenon, in this study we analyzed the length and nucleoid distribution of suppressed thermosensitive replication mutants. The dysfunctions in the replication machinery generally lead to formation of elongated cells (termed filaments) that originate from an inhibition of cell division dependent on replication-stress, and to abnormal distribution and compaction of nucleoids. The results reported here provide evidence that deletion of the pta and ackA CCM genes significantly reduces observed cell length in the replication mutants dnaA46, dnaB8, dnaE486, dnaG(ts) and dnaN159. A weaker effect was shown in the tktB dnaE486 double mutant. The CCM enzyme dysfunction restored also the nucleoid shape and position in double mutants. The specificity of these effects was confirmed by overexpression of fully functional genes coding for relevant CCM enzymes, which caused the reversion to the initial filamentous and nucleoid phenotypes. These results indicate that CCM mutations can rescue (or reduce) the cell division defects resulting from various replication mutations. We thus suggest that the replication-metabolism connection may serve as a general mechanism affecting DNA duplication at various levels to adjust this process and the cell division to the status of cell physiology.

Assessing the structure and function of single biomolecules with scanning transmission electron and atomic force microscopes.

The scanning transmission electron microscope (STEM) and the atomic force microscope (AFM) have provided a wealth of useful information on a wide variety of biological structures. These instruments have in common that they raster-scan a probe over a sample and are able to address single molecules. In the STEM the probe is a focused electron beam that is deflected by the scan-coils. Detectors collecting the scattered electrons provide quantitative information for each sub-nanometer sized sample volume irradiated. These electron scattering data can be reconstituted to images of single macromolecules or can be integrated to provide the mass of the macromolecules. Samples need to be dehydrated for such quantitative STEM imaging. In contrast, the AFM raster-scans a sharp tip over a sample surface submerged in a buffer solution to acquire information on the sample's surface topography at sub-nanometer resolution. Direct observation of function-related structural changes induced by variation of temperature, pH, ionic strength, and applied force provides insight into the structure-function relationship of macromolecules. Further, the AFM allows single molecules to be addressed and quantitatively unfolded using the tip as nano-tweezers. The performance of these two scanning probe approaches is illustrated by several examples including the chaperonin GroEL, bacterial surface layers, protein crystals, and bacterial appendices.