Molecular toolbox for genetic manipulation of the stalked budding bacterium Hyphomonas neptunium.

The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species.

Function and localization dynamics of bifunctional penicillin-binding proteins in Caulobacter crescentus.

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the noncanonical amino acid d-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.

The effect of six years of water storage on resin composite bonding to human dentin.

The aim of this in vitro study was to evaluate dentin bond strength and marginal adaptation of direct resin composite fillings after different storage times. Three hundred sixty cavities were prepared in discs of freshly extracted human third molars and filled with resin composites. Multistep self-etching adhesives (Syntac Classic, A.R.T. Bond, both with and without total etching), three-step etch-and-rinse adhesives (Scotchbond Multi-Purpose Plus, EBS), and two-step etch-and-rinse adhesives (Prime and Bond 2.0, Syntac Single-Component) were used for bonding. After 1, 90, and 2190 days of water storage and 24 h thermocycling (1150 cycles), push-out testing was performed. From the 6-year group, replicas were made after 1 day, 90 days, and 1, 2, 3, 4, 5, and 6 years, and examined regarding marginal adaptation under an SEM (x 200 magnification). In all groups under investigation, push-out bond strengths remained stable after 90 days; however, the strengths significantly decreased after 6 years of water storage. The two-step systems exhibited lower bond strengths than three-step systems after 6 years. Marginal analysis revealed a significant loss regarding the percentage of perfect margins having been stable after 2 years for the three-step etch-and-rinse systems. Overall, the older three-step systems proved to be more effective than the simplified adhesives Syntac Single-Component and Prime and Bond 2.0 with regard to bond strength and marginal adaptation.

Evaluation of the fatigue behavior of the resin-dentin bond with the use of different methods.

The clinical performance of directly bonded resin composites is fundamentally dependent on durable adhesion to prevent gap formation over time. The goal of this investigation was to evaluate the effectiveness of various dentin adhesives by means of quasistatic and dynamic dentin bond strengths, and also to determine marginal and internal gap formation after loading in an artificial oral environment. Three hundred thirty human third molars were used within four weeks of extraction. Adhesives used were A.R.T. Bond, OptiBond FL, Scotchbond Multi-Purpose Plus, Single Bond, Prime & Bond NT, and One Up Bond F for bonding of one resin composite (Z 250). Buccal and lingual aspects of 90 teeth were ground flat to expose dentin, then resin composite cylinders were bonded. Initial bond strengths (n = 10) and adhesive fatigue limits (n = 20) were determined with the use of a shear test apparatus. One hundred eighty conical cavities were prepared into dentin discs and filled with the same materials. After 21 days of storage, initial push-out bond strengths (n = 10) and adhesive fatigue limits (n = 20) were measured. Sixty molars with MO cavities (n = 10) with margins below the cement-enamel junction were filled. Before and after thermomechanical loading (100000 x 50 N and 2500 x thermocycling between + 5 and + 55 degrees C), marginal gap formation and internal adaptation (only after loading) were analyzed under a SEM (x 200). The one-bottle systems showed higher shear bond strengths when evaluated statically and dynamically. However, cyclic fatigue push-out bond strengths resulted in higher values for older multistep systems. Marginal and internal gap analysis confirmed the results, in favor of older adhesive systems (p <.05; Mann-Whitney U test).