Requirements for chemotaxis protein localization in Rhodobacter sphaeroides

Many proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium. Using genomic replacement of the wild-type chemotaxis genes in R. sphaeroides with their corresponding fluorescent protein fusions in conjunction with in frame deletions of other chemotaxis genes, we have investigated which proteins are required for the formation of the polar and cytoplasmic chemotaxis protein clusters. As in E. coli, the polarly targeted CheA and CheW homologues are required for the formation of the polar cluster. However, the formation of the cytoplasmic cluster requires the cytoplasmic chemoreceptors and CheW but not the CheAs. Interestingly, even when deletion of a component resulted in the chemotaxis proteins of one pathway becoming delocalized and diffuse in the cytoplasm, in no case were any chemotaxis proteins seen to localize to the other signalling cluster.     

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.     

Follicular dendritic cells produce IL-15 that enhances germinal center B cell proliferation in membrane-bound form

Factors that control the survival and proliferation of Ag-stimulated B cells within the germinal center (GC) are crucial for humoral immune responses with high affinity Abs against infectious agents. The follicular dendritic cell (FDC) is known as a key cellular component of the GC microenvironment for GC-B cell survival and proliferation. In this study, we report that IL-15 is produced by human FDC in vivo and by an FDC cell line, FDC/HK cells, in vitro. IL-15 is captured by IL-15Ralpha on the surface of FDC/HK cells. The surface IL-15 is functionally active and augments GC-B cell proliferation. Because GC-B cells have the signal-transducing components (IL-2/15Rbetagamma), but not a receptor for binding of soluble IL-15 (IL-15Ralpha), IL-15 signaling is possibly transduced by transpresentation from FDCs to GC-B cells via cell-cell contact. Together, these results suggest that IL-15 from FDC, in membrane-bound form, plays an important role in supporting GC-B cell proliferation, proposing a new target for immune modulation as well as treatment of B cell tumors of GC origin.     

Modulation of sexual signalling by immune challenged male mealworm beetles (Tenebrio molitor, L.): evidence for terminal investment and dishonesty

Organisms partition resources into life-history traits in order to maximise fitness over their expected lifespan. For the males of many species fitness is determined by qualitative and quantitative aspects of costly sexual signals: The notion that epigamic traits are costly forms the cornerstone of those theories that propose parasites drive sexual selection. Consequently studies examining this notion assume sexual signalling is honest (i.e. driven by cost) when they seek to identify correlations or causal links between male immune function and attractiveness. We demonstrate that immune challenged males of the mealworm beetle, Tenebrio molitor, increased their investment in epigamic pheromone signals: these males became significantly more attractive to females whilst increasing the activity of a key immune effector system. In other words males increase terminal reproductive effort (invest in attractiveness) in response to a survival threat (immune insult). Consequently the signal preferred by the female is dishonest when considering the male's condition.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Variable responses within epiphytic and benthic microalgal communities to nutrient enrichment

We evaluated how changes in nutrient supply altered the composition of epiphytic and benthic microalgal communities in a Thalassia testudinum (turtle grass) bed in Florida Bay. We established study plots at four sites in the bay and added nitrogen (N) and phosphorus (P) to the sediments in a factorial design. After 18, 24, and 30 months of fertilization we measured the pigment concentrations in the epiphytic and benthic microalgal assemblages using high performance liquid chromatography. Overall, the epiphytic assemblage was P-limited in the eastern portion of the bay, but each phototrophic group displayed unique spatial and temporal responses to N and P addition. Epiphytic chlorophyll a, an indicator of total microalgal load, and epiphytic fucoxanthin, an indicator of diatoms, increased in response to P addition at one eastern bay site, decreased at another eastern bay site, and were not affected by P or N addition at two western bay sites. Epiphytic zeaxanthin, an indicator of the cyanobacterial/coralline red algae complex, and epiphytic chlorophyll b, an indicator of green algae, generally increased in response to P addition at both eastern bay sites but did not respond to P or N addition in the western bay. Benthic chlorophyll a, chlorophyll b, fucoxanthin, and zeaxanthin showed complex responses to N and P addition in the eastern bay, suggesting that the benthic assemblage is limited by both N and P. Benthic assemblages in the western bay were variable over time and displayed few responses to N or P addition. The contrasting nutrient limitation patterns between the epiphytic and benthic communities in the eastern bay suggest that altering nutrient input to the bay, as might occur during Everglades restoration, can shift microalgal community structure, which may subsequently alter food web support for upper trophic levels.