A family of secreted pathogenesis-related proteins in Candida albicans.

Analysing culture supernatants of yeast and hyphal cells of Candida albicans, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome. Due to sequence homology, three additional, yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, together with RBE1 and RBT4 were assigned to a novel family of CaPRY proteins. In a Δrbe1/Δrbt4 deletion strain, genome-wide transcriptional analysis revealed differential transcription of only a limited set of genes implicated in virulence and oxidative stress response. Single deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in a Δrbe1/Δrbt4 double deletion strain, where virulence was strongly affected. Remarkably, transcription of RBT4 and RBE1 was each upregulated in blastospores of Δrbe1 or hyphae of Δrbt4 deletion strains respectively, indicating functional complementation thereby compensating a potential virulence defect in the single deletion strains. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leucocytes. Therefore, the crucial contribution of both C. albicans pathogenesis-related proteins to virulence might be vested in protection against phagocyte attack.

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains.

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography-mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector.

Global transcription and metabolic flux analysis of Escherichia coli in glucose-limited fed-batch cultivations.

A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic "work mode." sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.