The type IVa pilus machinery is pre-installed during cell division

ABSTRACTType IV pili (T4aP) are ubiquitous microbial appendages used for adherence, twitching motility, DNA uptake, and electron transfer. Many of these functions depend on dynamic assembly and disassembly of the pilus by a megadalton-sized, cell envelope-spanning protein complex located at the poles of rod-shaped bacteria. How the T4aP assembly complex becomes integrated into the cell envelope in the absence of dedicated peptidoglycan (PG) hydrolases is unknown. After ruling out potential involvement of housekeeping PG hydrolases in installation of the T4aP machinery in P. aeruginosa, we discovered that key components of inner (PilMNOP) and outer (PilQ) membrane subcomplexes are recruited to future sites of cell division. Mid-cell recruitment of a fluorescently tagged alignment subcomplex component, mCherry-PilO, depended on PilQ secretin monomers – specifically, their N-terminal PG-binding AMIN domains. PilP, which connects PilO to PilQ, was required for recruitment, while PilM, which is structurally similar to divisome component FtsA, was not. Recruitment preceded secretin oligomerization in the outer membrane, as loss of the PilQ pilotin, PilF, had no effect on localization. These results were confirmed in cells chemically blocked for cell division prior to outer membrane invagination. The hub protein FimV and a component of the Polar Organelle Coordinator complex – PocA – were independently required for mid-cell recruitment of PilO and PilQ. Together, these data reveal an integrated, energy-efficient strategy for the targeting and pre-installation – rather than retrofit – of the T4aP system into nascent poles, without the need for dedicated PG-remodelling enzymes.

PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens

Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole.

Novel Cellular Organization in a Gliding Mycoplasma, Mycoplasma insons

ABSTRACT Mycoplasmas that are known to exhibit gliding motility possess a differentiated tip structure. This polar organelle mediates cytadherence and gliding motor activity and contains a cytoskeleton-like component that provides structural support. Here, we describe gliding motility and a unique cytoskeleton in Mycoplasma insons, which lacks any obviously differentiated tip structure.

Deefgea rivuli gen. nov., sp. nov., a member of the class Betaproteobacteria

Two strains, designated WB 3.4-79T and WB 3.3-25, were isolated from a hard-water sample collected from the Westerhöfer Bach, Lower Saxony, Germany. The strains shared 100 % DNA–DNA relatedness, indicating membership of the same genospecies. This close relationship was supported by identical 16S rRNA gene sequences and high similarities in fatty acid composition and biochemical characteristics. The G+C content of the genomic DNA of strain WB 3.4-79T was 48.5 mol% and the predominant ubiquinone was Q-8. Major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Major fatty acids (>10 %) were C16 : 0 and C16 : 1 ω7c. Polyhydroxybutyrate and polyphosphate granules as well as unidentified enterosomes and a polar organelle are visible by electron microscopy. Comparative 16S rRNA gene sequence analysis indicated that the isolates were placed within the class Betaproteobacteria, remotely related to Chitinibacter tainanensis DSM 15459T, Silvimonas terrae KCTC 12358T, Formivibrio citricus DSM 6150T and Iodobacter fluviatilis DSM 3764T. On the basis of phylogenetic and phenotypic distinctness, we propose a novel genus, Deefgea gen. nov., with Deefgea rivuli sp. nov. as the type species. The type strain of Deefgea rivuli is strain WB 3.4-79T (=DSM 18356T=CIP 109326T).

Identification of Genes Required for Synthesis of the Adhesive Holdfast in Caulobacter crescentus

ABSTRACT Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the “holdfast,” which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export.

Regulation of podJ Expression during theCaulobacter crescentus Cell Cycle

ABSTRACT The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of theCaulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJresults in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors.

Unorthodox male meiosis in Trichosia pubescens (Sciaridae). Chromosome elimination involves polar organelle degeneration and monocentric spindles in first and second division

Male meiosis in Trichosia pubescens (Sciaridae) was investigated by means of serial section electron microscopy and immunofluorescence light microscopy. From earlier studies of another sciarid fly, Sciara coprophila (Phillips (1967) J. Cell. Biol. 33, 73–92), it is known that the spindle poles in sciarid spermatogonia are characterized by pairs of ‘giant centrioles’, ring-shaped organelles composed of large numbers of singlet microtubules. In the present study spermatocytes in early prophase of Trichosia were found to possess single giant centrioles at opposite sides of the nucleus. The obvious reduction in centriole number from the spermatogonial to the spermatocyte stage is suggested to be the result of a suppression of daughter centriole formation. In late prophase, a large aster is developed around the centriole at one pole. At the opposite pole no comparable aster is formed. Instead, a number of irregular centriolar components appear in this region, a process that is understood to be a degeneration of the polar organelle. The components of the degenerate pole migrate into a cytoplasmic protrusion (‘bud’), which later is also utilized for the elimination of paternal chromosomes. The existence of only one functional polar centre is the reason for the formation of a monopolar monocentric spindle in first meiotic division, which in turn is one of the prerequisites for the elimination of paternal chromosomes. While the set of maternal and L chromosomes orientates and probably moves towards the pole, paternal chromosomes seem to be unable to contact the pole, possibly due to an inactivation of their kinetochores. Retrograde (‘away from the pole’) chromosome motion not involving kinetochores is assumed. Eventually, paternal chromosomes move into the pole-distal bud and are eliminated by casting off, together with the components of the degenerate polar organelle. Chromosome elimination can be delayed until the second meiotic division. The spindle of the second meiotic division is bipolar and monocentric. One spindle pole is marked by the polar centre of first division. The opposite spindle apex is devoid of a polar centre. It is assumed that spindle bipolarity in the second division is induced by the amphi-orientated chromosomes themselves. The maternal and L chromosome set (except the non-disjunctional X chromosome, which is found near the polar centre) congress in a metaphase plate, divide and segregate. Of the two daughter nuclei resulting from the second meiotic division, the one containing the X chromatids is retained as the nucleus of the future spermatozoon. The other nucleus becomes again eliminated within a second cytoplasmic bud.