Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.     

Experimental murine hypersensitivity pneumonitis

To establish a model of experimental hypersensitivity pneumonitis (EHP) in mice and to examine the influence of genetic background on the pulmonary inflammatory response to Micropolyspora faeni, we determined the responses of C57BL/6, SJL/J, and C3H/HeJ mice to intratracheal (i.t.) injections of M. faeni. Recipient animals received lymph node cells (LNC), peritoneal exudate cells (PEC), and spleen cells (SC) from sensitized mice cultured in vitro with M. faeni. Controls included serum containing anti-M. faeni antibody; uncultured SC from M. faeni-sensitized donors, and M. faeni-cultured SC from ovalbumin (OA)-sensitized donors. Recipients were challenged i.t. with M. faeni or normal saline 48 hr after the cell or serum transfer. We developed a model of EHP in mice. Increasing amounts of i.t. M. faeni were associated with increasing extent of pulmonary inflammation with no difference between the mouse strains. There was substantial increase of the extent of pulmonary abnormalities in the animals receiving cultured SC. The number of transferred cells and the M. faeni concentration correlated with the extent of pulmonary histologic abnormalities. Cultured PEC and LNC could transfer EHP in C3H/HeJ mice only. Serum containing anti-M. faeni antibody, cultured SC from OA-sensitized donors, and noncultured SC from sensitized donors could not transfer EHP. We conclude that it is possible to adoptively transfer EHP.     

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.     

Multilevel selection 3: modeling the effects of interacting individuals as a function of group size

Bijma et al. (2007a,b) presented a quantitative genetic theory of multilevel selection and showed how to estimate the relevant parameters using standard restricted maximum-likelihood (REML) methodology. Extending their results we develop a wider class of models that provide a more realistic framework for capturing the effects of interacting individuals. These models also make use of standard REML techniques and include the original model as a special case.     

Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ΔAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ΔAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.     

Design and analysis of nanoscale bioassemblies

Bionanotechnology is an emerging field in nanotechnology. In general, it uses concepts from chemistry, biochemistry, and molecular biology to identify components and processes for the construction of self-assembling materials and devices. Distant goals of the science of bionanotechnology range from developing programmable nanoscale devices that can sample or alter their environments to developing assemblies capable of Darwinian evolution. At the heart of these approaches is the concept of the production of supramolecular assemblies (SMAs; also known as supramolecular aggregates) by programmed self-assembly in an aqueous medium. Ordered arrays, planar and closed-shell tilings, dynamic machines, and switches have been designed and constructed by using DNA-DNA, protein-protein, and protein-nucleic acid biospecificities. We review the designs and the analytical techniques that have been employed in the production of SMAs that do not occur in nature.     

Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78

White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.