The wall-less prokaryote Mycoplasma pneumoniae, a common cause of chronic respiratory tract infections in humans, is considered to be among the smallest and simplest known cells capable of self-replication, yet it has a complex architecture with a novel cytoskeleton and a differentiated terminal organelle that function in adherence, cell division, and gliding motility. Recent findings have begun to elucidate the hierarchy of protein interactions required for terminal organelle assembly, but the engineering of its gliding machinery is largely unknown. In the current study, we assessed gliding in cytadherence mutants lacking terminal organelle proteins B, C, P1, and HMW1. Furthermore, we screened over 3,500 M. pneumoniae transposon mutants individually to identify genes associated with gliding but dispensable for cytadherence. Forty-seven transformants having motility defects were characterized further, with transposon insertions mapping to 32 different open reading frames widely distributed throughout the M. pneumoniae genome; 30 of these were dispensable for cytadherence. We confirmed the clonality of selected transformants by Southern blot hybridization and PCR analysis and characterized satellite growth and gliding by microcinematography. For some mutants, satellite growth was absent or developed more slowly than that of the wild type. Others produced lawn-like growth largely devoid of typical microcolonies, while still others had a dull, asymmetrical leading edge or a filamentous appearance of colony spreading. All mutants exhibited substantially reduced gliding velocities and/or frequencies. These findings significantly expand our understanding of the complexity of M. pneumoniae gliding and the identity of possible elements of the gliding machinery, providing a foundation for a detailed analysis of the engineering and regulation of motility in this unusual prokaryote.The cell wall-less prokaryote Mycoplasma pneumoniae establishes chronic infections of the human respiratory tract that result in bronchitis and atypical or "walking" pneumonia and account for up to 30% of all cases of community-acquired pneumonia (49). With a minimal genome lacking genes for typical transcriptional regulators, two-component systems, and pathways for de novo synthesis of most macromolecular building blocks, including nucleotides and amino acids (8, 21), M. pneumoniae is considered to be among the simplest organisms capable of self-replication; yet this unusual species exhibits remarkable architectural complexity, with a dynamic cytoskeleton and a specialized, membrane-bound polar cell extension or terminal organelle that has an elaborate macromolecular core (2,12,20,33). The terminal organelle functions in diverse cellular processes that include adherence to host epithelium (cytadherence) and cell division (7,17,40) and engages as the leading end as M. pneumoniae cells glide over solid surfaces (3). Recent studies have begun to establish the assembly sequence in terminal organelle development and the hierarchy of protein interact...

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plantspecific accessory components such as the H3K27me3 reader LIKE HETEROCHROMA-TION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF

Mycoplasma pneumoniae exhibits a novel form of gliding motility that is mediated by the terminal organelle, a differentiated polar structure. Given that genes known to be involved in gliding in other organisms are absent in M. pneumoniae, random transposon mutagenesis was employed to generate mutants with gliding-deficient phenotypes. Transposon insertions in the only annotated Ser/Thr protein kinase gene (prkC; MPN248) and its cognate phosphatase gene (prpC; MPN247) in M. pneumoniae resulted in significant and contrasting effects on gliding frequencies. prkC mutant cells glided at approximately half the frequency of wild-type cells, while prpC mutant cells glided more than twice as frequently as wild-type cells. Phosphoprotein staining confirmed the association between phosphorylation of the cytoskeletal proteins HMW1 and HMW2 and membrane protein P1 and the gliding phenotype. When the prpC mutant was complemented by transposon delivery of a wild-type copy of the prpC allele, gliding frequencies and phosphorylation levels returned to the wild-type standard. Surprisingly, delivery of the recombinant wild-type prkC allele dramatically increased gliding frequency to a level approximately 3-fold greater than that of wild-type in the prkC mutant. Collectively, these data suggest that PrkC and PrpC work in opposition in M. pneumoniae to influence gliding frequency.M ycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract causing primary atypical pneumonia and tracheobronchitis (1). Mycoplasmas lack major biosynthetic pathways, classical transcriptional regulators, chemotactic and other two-component systems, and the prototypical prokaryotic cell division apparatus (2, 3). The limited biosynthetic capabilities of mycoplasmas are generally explained by their evolution as obligate parasites of diverse eukaryotic hosts (4). Colonization of the host respiratory epithelium by M. pneumoniae requires gliding motility (5), which might facilitate access to receptors on the host cell surface and subsequent lateral spread.The gliding apparatus of M. pneumoniae is a polar terminal structure (6) that also functions in cell division (7) and adhesion to host receptors (2, 5). While the cytoskeletal protein HMW1 (MPN447) and membrane proteins P1, B/C, and P30 (MPN141, MPN142, and MPN453, respectively) localize to the terminal organelle and are required for gliding (5,(8)(9)(10), these proteins are also essential for cytadherence and attachment to surfaces. Accordingly, their distinct functions in gliding motility are difficult to define by mutagenesis alone. Given that the M. pneumoniae genome exhibits no homology to elements of defined gliding mechanisms (2, 3), including those of other gliding mycoplasmas (11-14), it was necessary to perform saturating transposon mutagenesis in order to identify the components specific to gliding (15). Transposon insertions in the genes encoding the cytoskeletal proteins P41 and P65 (MPN311 and MPN309, respectively) (16), which are known to localize to the termina...

We report the whole-genome sequences and annotations of 42 Lactobacillales strains isolated from commercial cucumber fermentations performed in North Carolina ( n = 34) and Minnesota ( n = 9), USA. The isolates include representatives from 12 acid-producing species.

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