"I'm attached, and I'm a good guy/gal!": how character attachment influences pro- and anti-social motivations to play massively multiplayer online role-playing games.

One's feelings of intimacy and connectedness with distal, fictional media characters are referred to as parasocial interactions. Video games have challenged this concept, as the distance between game players and characters is greatly reduced, if not completely removed, in virtual environments. Games encourage the internalization and psychological merging of a player's and a character's mind, a multidimensional concept known as character attachment (CA). Data from our study suggest that dimensions of CA are useful in understanding both pro- and anti-social gaming motivations. Pro-social gamers feel a greater sense of control over their characters, while anti-social gamers are more likely to suspend their disbelief of the game environment and not take responsibility for their virtual actions. Pro-social gaming was more prevalent in older gamers, and younger male game characters were motivated by anti-social reasons.

The presumptive magnetosome protein Mms16 is a poly(3-hydroxybutyrate) granule-bound protein (phasin) in Magnetospirillum gryphiswaldense.

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested. Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in Rhodospirillum rubrum sharing 55% identity to Mms16. Fusions of Mms16 and ApdA to enhanced green fluorescent protein (EGFP) or EYFP were colocalized in vivo with the PHB granules but not with the magnetosome particles after conjugative transfer to M. gryphiswaldense. Although the Mms16-EGFP fusion protein became detectable by Western analysis in all cell fractions upon cell disruption, it was predominantly associated with isolated PHB granules. Contrary to previous suggestions, our results argue against an essential role of Mms16 in magnetosome formation, and the previously observed magnetosome localization is likely an artifact due to unspecific adsorption during preparation. Instead, we conclude that Mms16 in vivo is a PHB granule-bound protein (phasin) and acts in vitro as an activator of PHB hydrolysis by R. rubrum PHB depolymerase PhaZ1. Accordingly, we suggest renaming the Mms16 protein of Magnetospirillum species to ApdA, as in R. rubrum.

Inactivation of the flagellin gene flaA in Magnetospirillum gryphiswaldense results in nonmagnetotactic mutants lacking flagellar filaments.

Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines. The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations. Here we report a method for knockout mutagenesis by homologous recombination in Magnetospirillum gryphiswaldense. Multiple flagellin genes, which are unlinked in the genome, were identified in M. gryphiswaldense. The targeted disruption of the flagellin gene flaA was shown to eliminate flagella formation, motility, and magnetotaxis. The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of M. gryphiswaldense and other magnetotactic bacteria.

Unraveling the function of the Rhodospirillum rubrum activator of polyhydroxybutyrate (PHB) degradation: the activator is a PHB-granule-bound protein (phasin).

Efficient hydrolysis of native poly(3-hydroxybutyrate) (nPHB) granules in vitro by soluble PHB depolymerase of Rhodospirillum rubrum requires pretreatment of nPHB with an activator compound present in R. rubrum cells (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). Edman sequencing of the purified activator (17.4 kDa; matrix-assisted laser desorption ionization-time of flight mass spectrometry) revealed identity to a hypothetical protein deduced from a partially sequenced R. rubrum genome. The complete activator gene, apdA (activator of polymer degradation), was cloned from genomic DNA, expressed as a six-His-tagged protein in recombinant Escherichia coli (M(r), 18.3 x 10(3)), and purified. The effect of ApdA on PHB metabolism was studied in vitro and in vivo. In vitro, the activity of the activator could be replaced by trypsin, but recombinant ApdA itself had no protease activity. Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein patterns of trypsin- and ApdA-treated nPHB granules isolated from different PHB-accumulating bacteria showed that trypsin activated nPHB by removing proteins of the surface layer of nPHB regardless of the origin of nPHB, but ApdA bound to and interacted with the surface layer of nPHB in a nonproteolytic manner, thereby transforming nPHB into an activated form that was accessible to the depolymerase. In vivo, expression of ApdA in E. coli harboring the PHB biosynthetic genes, phaCBA, resulted in significant increases in the number and surface/volume ratio of accumulated PHB granules, which was comparable to the effect of phasin proteins, such as PhaP in Ralstonia eutropha. The amino acid sequence of ApdA was 55% identical to the amino acid sequence of Mms16, a magnetosome-associated protein in magnetotactic Magnetospirillum species. Mms16 was previously reported to be a GTPase with an essential function in magnetosome formation (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276:48183-48188, 2001). However, no GTPase activity of ApdA could be demonstrated. We obtained evidence that Mms16 of Magnetospirillum gryphiswaldense can functionally replace ApdA in R. rubrum. Fusions of apdA and mms16 to gfp or yfp were functionally expressed, and both fusions colocalized with PHB granules after conjugative transfer to R. rubrum. In conclusion, ApdA in vivo is a PHB-bound, phasin-like protein in R. rubrum. The function of Mms16 in magnetotactic bacteria requires further clarification.

Development of a genetic system for Magnetospirillum gryphiswaldense.

Genetic analysis of bacterial magnetosome biomineralization has been hindered by the lack of an appropriate methodology for cultivation and genetic manipulation of most magnetotactic bacteria. In this report, a genetic system for Magnetospirillum gryphiswaldense is described. The system includes a plating technique that allows the screening of magnetic vs non-magnetic colonies, and a protocol for the transfer of foreign DNA by electroporation and high-frequency conjugation. Various broad-host-range vectors of the IncQ, IncP, and pBBR1 groups were found to be capable of replication in M. gryphiswaldense. Several antibiotic resistance markers that can be expressed in M. gryphiswaldense were identified. Tn 5 transposons delivered on a suicide plasmid showed transpositional insertion into random chromosomal sites.