Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD ؉ , a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.Evolution has produced a diverse array of metabolic pathways and regulatory mechanisms that reflect the adaptation of an immense variety of microorganisms to different environments and nutritional requirements. A prominent example is the metabolism of glucose, the primary and preferred fuel for eukaryotic microorganisms. Although glucose is metabolized by a highly conserved series of connected enzymatic reactions, the mechanisms that regulate its fate and the properties of the ATP-producing pathways have been subjected to selection pressure during evolution. Aerobic (respiration) and anaerobic (fermentation) pathways are used by microorganisms to obtain energy from glucose, in the form of ATP. These pathways allow organisms to produce ATP at different rates and with different efficiencies; respiration proceeds at a lower rate and with a high yield, whereas fermentation operates at higher rates but with lower yield. Selection pressure imposed by energy limitation and the high ATP yield of respiration has been implicated in facilitating the evolutionary transition from unicellular to undifferentiated multicellular organisms (1).Unicellular microorganisms, such as the yeast Saccharomyces cerevisiae, use both pathways depending on the metabolic state of the cell, whereas multicellular microorganisms, such as filamentous fungi, preferentially use respiration (2). Mucor racemosus, a dimorphic fungus that can grow either in a unicellular (yeast-like) or a multicellular (mycelial) form, also uses both; the unicellular form exploits fermentation, whereas the multicellular form is capable of respiration (3-5).S. cerevisiae preferentially ferments glucose, even in the presence of oxygen, producing ethanol and CO 2 by anaerobic metabolism. Only after exhaustion of the available glucose...

Oxygen is essential for the survival of obligatorily aerobic eukaryotic microorganisms, such as the multicellular fungus Trichoderma reesei. However, the molecular basis for the inability of such cells to survive for extended periods under anoxic conditions is not fully understood. Using cDNA microarray analysis, we show that changes in oxygen availability have a drastic effect on gene expression in T. reesei. The expression levels of 392 (19.6%) out of 2000 genes examined changed significantly in response to hypoxia, transient anoxia, and reoxygenation. In addition to modulating many genes with no previously assigned function, cells respond to hypoxia by readjusting the balance of expression between genes required for energy production and consumption, and altering the expression of genes involved in protective mechanisms and signaling pathways. Moreover, we show that transient anoxia strongly represses genes for enzymes that are critical for glycolysis, and are essential for energy production under anaerobic conditions. Our study thus reveals crucial differences between the facultative anaerobe Saccharomyces cerevisiae and T. reesei with regard to the oxygen-dependent transcriptional control of the glycolytic pathway, which can account for the differential survival of the two species in the absence of oxygen.

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.

No abstract