Our current understanding of Antarctic soils is derived from direct culture on selective media, biodiversity studies based on clone library construction and analysis, quantitative PCR amplification of specific gene sequences and the application of generic microarrays for microbial community analysis. Here, we investigated the biodiversity and functional potential of a soil community at Mars Oasis on Alexander Island in the southern Maritime Antarctic, by applying 454 pyrosequencing technology to a metagenomic library constructed from soil genomic DNA. The results suggest that the commonly cited range of phylotypes used in clone library construction and analysis of 78–730 OTUs (de-replicated to 30–140) provides low coverage of the major groups present (∼5%). The vast majority of functional genes (>77%) were for structure, carbohydrate metabolism, and DNA/RNA processing and modification. This study suggests that prokaryotic diversity in Antarctic terrestrial environments appears to be limited at the generic level, with Proteobacteria, Actinobacteria being common. Cyanobacteria were surprisingly under-represented at 3.4% of sequences, although ∼1% of the genes identified were involved in CO2 fixation. At the sequence level there appeared to be much greater heterogeneity, and this might be due to high divergence within the relatively restricted lineages which have successfully colonized Antarctic terrestrial environments.

The plant pathogen Streptomyces scabies 87-22 has a functional pyochelin biosynthetic pathway that is regulated by TetR-and AfsR-family proteins Siderophores are high-affinity iron-chelating compounds produced by bacteria for iron uptake that can act as important virulence determinants for both plant and animal pathogens. Genome sequencing of the plant pathogen Streptomyces scabies 87-22 revealed the presence of a putative pyochelin biosynthetic gene cluster (PBGC). Liquid chromatography (LC)-MS analyses of culture supernatants of S. scabies mutants, in which expression of the cluster is upregulated and which lack a key biosynthetic gene from the cluster, indicated that pyochelin is a product of the PBGC. LC-MS comparisons with authentic standards on a homochiral stationary phase confirmed that pyochelin and not enantio-pyochelin (ent-pyochelin) is produced by S. scabies. Transcription of the S. scabies PBGC occurs via~19 kb and~3 kb operons and transcription of the~19 kb operon is regulated by TetR-and AfsR-family proteins encoded by the cluster. This is the first report, to our knowledge, of pyochelin production by a Gram-positive bacterium; interestingly regulation of pyochelin production is distinct from characterized PBGCs in Gram-negative bacteria. Though pyochelin-mediated iron acquisition by Pseudomonas aeruginosa is important for virulence, in planta bioassays failed to demonstrate that pyochelin production by S. scabies is required for development of disease symptoms on excised potato tuber tissue or radish seedlings. INTRODUCTIONThe genus Streptomyces is comprised of hundreds of species, most of which are soil-dwelling and saprophytic. The ability of these filamentous actinobacteria to produce biologically active secondary metabolites is well known; streptomycetes produce nearly two-thirds of the world's naturally occurring antibiotics (Bentley et al., 2002). Since soil is poor in nutrients but rich in microbial competitors, most secondary metabolites are thought to serve as antimicrobial agents. However, secondary metabolites have other roles, one of which is iron acquisition. Iron is relatively unavailable to soil bacteria due to the low solubility of the Fe 3+ ion under aerobic conditions at neutral pH. The most common way that bacteria cope with low bioavailability of iron is the production of ironchelating compounds called siderophores (Guerinot, 1994). Production of siderophores by saprophytic Streptomyces species has been known since the 1960s; desferrioxamine siderophores are produced by multiple Streptomyces species ( Barona-Gó mez et al., 2004Bickel et al., 1960;Imbert et al., 1995;Schupp et al., 1988). Recently, streptomycetes have been found to produce siderophores other than desferrioxamines. Streptomyces sp. Tü 6125 produces enterobactin, a catecholate-type siderophore typically produced by members of the family Enterobacteriaceae (Fiedler et al., 2001). Streptomyces sp. ATCC 700974 and Streptomyces griseus produce griseobactin, a siderophore containing catechol, threonine and arginine t...

f Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due to the lack of reliable treatment options. This pathogen requires uptake of zinc from host tissues in order to successfully grow and cause virulence. Reducing the availability of that micronutrient could help treat A. fumigatus infections. In this study, we examined the in vitro effects of seven chelators using a bioluminescent strain of A. fumigatus. 1,10-Phenanthroline and N,N,N=,N=-tetrakis(2-pyridylmethyl) ethane-1,2-diamine (TPEN) proved to be the chelators most effective at inhibiting fungal growth. Intraperitoneal administration of either phenanthroline or TPEN resulted in a significant improvement in survival and decrease of weight loss and fungal burden for immunosuppressed mice intranasally infected with A. fumigatus. In vitro both chelators had an indifferent effect when employed in combination with caspofungin. The use of TPEN in combination with caspofungin also significantly increased survival compared to that when using these drugs individually. Our results suggest that zinc chelation may be a valid strategy for dealing with A. fumigatus infections and that both phenanthroline and TPEN could potentially be used either independently or in combination with caspofungin, indicating that their use in combination with other antifungal treatments might also be applicable.A spergillus fumigatus is a widespread soil-dwelling fungal saprotroph (1). It is one of the most ubiquitous fungal species with airborne conidia, and it is estimated that all humans inhale several hundred conidia each day (2). These are completely innocuous to immunocompetent hosts. However, the conidia are able to develop and cause invasive pulmonary aspergillosis (IPA) in immunocompromised individuals (3). This disease is difficult to treat, with a mortality rate of 45.6% (4). Commonly used drug treatment options include triazoles such as voriconazole, which inhibit ergosterol synthesis, amphotericin B, which binds to ergosterol and thus results in increased permeability of the cell membrane, and echinocandins such as caspofungin, which inhibit glucan synthesis (5).Both fungal and bacterial pathogens require cations to grow within their hosts and utilize specialized mechanisms in order to obtain them (6). Zinc is considered essential for all organisms, including pathogens (7). The average concentration of free Zn 2ϩ in human serum is 0.08 M, which is about 150 times lower than the minimal concentration required for A. fumigatus to grow optimally in a defined liquid medium (8). A. fumigatus possesses three genes, zrfA, zrfB, and zrfC, encoding plasma membrane zinc transporters (8). The zrfA and zrfB genes are required for zinc uptake in acidic Zn-limited environments (7), while zrfC is required for zinc uptake in alkaline environments (9) The zrfC gene is primarily responsible for zinc acquisition within a host's lungs and is required for virulence; zrfA and zrfB contribute to fungal pathogenesis to a lesser extent than zrfC and are not r...

Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and causes invasive pulmonary aspergillosis. The concentration of free zinc in living tissues is much lower than that required for optimal fungal growth in vitro because most of it is tightly bound to proteins. To obtain efficiently zinc from a living host A. fumigatus uses the zinc transporters ZrfA, ZrfB, and ZrfC. The ZafA transcriptional regulator induces the expression of all these transporters and is essential for virulence. Thus, ZafA could be targeted therapeutically to inhibit fungal growth. The ZrfC transporter plays the major role in zinc acquisition from the host whereas ZrfA and ZrfB rather have a supplementary role to that of ZrfC. In addition, only ZrfC enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mn-chelating protein synthesized and released by neutrophils within the fungal abscesses of immunosuppressed non-leucopenic animals. Hence, fungal survival in these animals would be undermined upon blocking therapeutically the function of ZrfC. Therefore, both ZafA and ZrfC have emerged as promising targets for the discovery of new antifungals to treat Aspergillus infections.

Cyclosporin A (CsA) is widely used as an immunosuppressive agent for organ transplant recipients. CsA inhibits calcineurin, which is highly conserved in mammals and fungi, and thus affects both types of organism. In mammals, the immunosuppressive effect of CsA is via hampering T cell activation. In fungi, the growth inhibitory effect of CsA is via interference with hyphal growth. The aim of this study was to determine whether CsA renders mice susceptible to invasive pulmonary aspergillosis (IPA) and whether it can protect immunosuppressed mice from infection. We therefore examined both the antifungal and the immunosuppressive activity of CsA in immunosuppressed and in immunocompetent mice infected with Aspergillus fumigatus to model IPA. We found that daily injections of CsA could not produce an antifungal effect sufficient to rescue immunosuppressed mice from lethal IPA. However, a 100% survival rate was obtained in non-immunosuppressed mice receiving daily CsA, indicating that CsA did not render the mice vulnerable to IPA. The lymphocyte subset was significantly suppressed by CsA, while the myeloid subset was not. Therefore, we speculate that CsA does not impair the host defense against IPA since the myeloid cells are preserved.