Search

Burkholderia pseudomallei, the causative agent of the severe disease melioidosis, can form persister cells, which can survive under high-dose antibiotic challenge complicating the treatment of the disease. In this review, we discuss our current knowledge on the formation of persister cells in B. pseudomallei. Recent studies suggest that growth-limiting conditions such as nutrient and oxygen limitation, as well as antibiotic challenge, can lead to formation of persister cells. Among the diverse set of genes that can contribute to persister cell formation, toxin-antitoxin pairs are the most studied and have been shown to contribute to persister cell formation in B. pseudomallei. B. pseudomallei infections result in high relapse rate after recovery from melioidosis. Such relapses may be facilitated by reactivation of the B. pseudomallei persister cells. Therefore, understanding the environmental and genetic factors that contribute to persistence is critical to develop effective treatment strategies for melioidosis.

The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser (PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95–99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis–suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.

We utilized RNAseq analysis of the Aspergillus fumigatus response to early hypoxic condition exposure. The results show that more than 89% of the A. fumigatus genome is expressed under normoxic and hypoxic conditions. Replicate samples were highly reproducible; however, comparisons between normoxia and hypoxia revealed that greater than 23% and 35% of genes were differentially expressed after 30 and 120 minutes of hypoxia exposure, respectively. Consistent with our previous report detailing transcriptomic and proteomic responses at later time points, the results here show major repression of ribosomal function and induction of ergosterol biosynthesis, as well as activation of alternate respiratory mechanisms at the later time point. RNAseq data was used to define thirty-two hypoxia-specific genes, which are not expressed under normoxic conditions. Transcripts of a C6 transcription factor and a histidine kinase response regulator were found only in hypoxia. In addition, several genes involved in the phosphoenyl pyruvate (PEP) and D-glyceraldehyde-3-phosphate (G3P) metabolism were only expressed in hypoxia. Interestingly, a 216 bp ncRNA Afu-182 in the 3’ region of insA (AFUB_064770), was significantly repressed under hypoxia with a 40-fold reduction in expression. A detailed analysis of Afu-182 showed similarity with several genes in the genome, many of which were also repressed in hypoxia. The results from this study show that hypoxia induces very early and widely drastic genome-wide responses in A. fumigatus that include expression of protein-coding and ncRNA genes. The role of these ncRNA genes in regulating the fungal hypoxia response is an exciting future research direction.

Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 β-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.

Genes encoding for glycosyl hydrolases (GH) in multiple families were recovered from an expression sequence tag library of Coptotermes formosanus, a xylophagous lower termite species. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, which may serve as strategic targets for designing molecular-based bio-pesticides, but also enrich discoveries of new cellulolytic enzymes for conversion of biomass into biofuel. Our study demonstrated that cellulose could be converted to glucose by two recombinant endogenous glycosyl hydrolases (endo-β-1,4 glucanase in GH9 and β-glucosidase in GH1). While the former cleaved cellulose to cellobiose and cellotriose, the resulting simple cellodextrins were digested to glucose. Both of the Escherichia coli-expressed recombinant proteins showed properties that could be incorporated in a glucose-based ethanol production program.

Aspergillus fumigatus, the most common airborne fungal pathogen of humans, employs two high-affinity iron uptake systems: iron uptake mediated by the extracellular siderophore triacetylfusarinine C and reductive iron assimilation. Furthermore, A. fumigatus utilizes two intracellular siderophores, ferricrocin and hydroxyferricrocin, to store iron. Siderophore biosynthesis, which is essential for virulence, is repressed by iron. Here we show that this control is mediated by the GATA factor SreA. During iron-replete conditions, SreA deficiency partially derepressed synthesis of triacetylfusarinine C and uptake of iron resulting in increased cellular accumulation of both iron and ferricrocin. Genome-wide DNA microarray analysis identified 49 genes that are repressed by iron in an SreA-dependent manner. This gene set, termed SreA regulon, includes all known genes involved in iron acquisition, putative novel siderophore biosynthetic genes, and also genes not directly linked to iron metabolism. SreA deficiency also caused upregulation of iron-dependent and antioxidative pathways, probably due to the increased iron content and iron-mediated oxidative stress. Consistently, the sreA disruption mutant displayed increased sensitivity to iron, menadion and phleomycin but retained wild-type virulence in a mouse model. As all detrimental effects of sreA disruption are restricted to iron-replete conditions these data underscore that A. fumigatus faces iron-depleted conditions during infection.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B(1) and B(2) biosynthesis, while A. parasiticus cultures had significantly increased B(1) and G(1) biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.

For a comprehensive evaluation of genes that have their expression modulated during exposure of the mycelia to voriconazole, we performed a large-scale analysis of gene expression in Aspergillus fumigatus using a microarray hybridization approach. By comparing the expression of genes between the reference time and after addition of voriconazole (30, 60, 120, and 240 min), we identified 2,271 genes differentially expressed in the wild-type strain. To validate the expression of some of these genes during exposure to voriconazole, we analyzed 13 genes showing higher expression in the presence of voriconazole by real-time RT-PCR. Although the magnitudes of induction differed between the two experimental systems, in about 85% of the cases they were in good agreement with the microarray data. To our knowledge this is the first study of microarray hybridization analysis for a filamentous fungus exposed to an antifungal agent. In our study, we have observed: (i) a decreased mRNA expression of various ergosterol biosynthesis genes; (ii) increased mRNA levels of genes involved in a variety of cell functions, such as transporters, transcription factors, proteins involved in cell metabolism, and hypothetical proteins; and (iii) the involvement of the cyclic AMP-protein kinase signaling pathway in the increased mRNA expression of several of these genes.

Aflatoxin contamination of food and feed is a major concern due to the carcinogenic properties of this mycotoxin. Previous studies using classical approaches have identified a cluster of genes responsible for aflatoxin production under the control of the pathway-specific transcriptional regulator aflR, but it is unknown whether aflR controls expression of other genes within the genome. Transcription profiling comparing wild type and DeltaaflR strains of Aspergillus parasiticus grown under conditions conducive for aflatoxin production identified only 23 upregulated genes in the wild type. These included 20 genes in the aflatoxin biosynthetic cluster, and three additional genes outside the aflatoxin biosynthetic cluster (nadA, hlyC, and niiA), all with AflR binding sites. This report is the first to demonstrate genes outside the biosynthetic cluster as being associated with aflR expression.

International audience; The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

We report the genome sequence of Theileria parva , an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum , and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.

The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

The thrombin receptor (TR) and proteinase activated receptor-2 (PAR-2) may represent the prototypes of an emerging family of cell-surface receptors that effect cell activation events mediated by serine proteases generated during inflammatory, fibrinolytic or haemostatic-regulated pathways. To further characterize the molecular genetics of these receptors, we have refined the genetic and physical mapping of both PAR-2 and TR. Utilization of two distinct radiation hybrid mapping panels with different levels of resolution demonstrated that both genes are tightly linked to the microsatellite markers D5S424, D5S1977, D5S2529 and D5S2596 (in order of decreasing LOD scores, from 13.7 for D5S424 to 7.7 for D5S2596). Physical mapping using yeast artificial chromosomes (YACs) and inversion field gel electrophoresis demonstrated that they are maximally separate by 90 kb. If the association of TR and PAR-2 genes resulted from a relatively recent gene duplication event from a common ancestral gene, these observations provide a general framework for the identification of gene transcripts representing alternative proteolytically activated receptors which may be clustered within this region of the human genome. These observations are especially relevant given recent evidence that murine and human platelets express alternative signalling mechanisms or receptors for thrombin.

The sequencing and annotation of the genomes of the first strains of Aspergillus nidulans, Aspergillus oryzae, and Aspergillus fumigatus will be seen in retrospect as a transformational event in Aspergillus biology. With this event the entire genetic composition of A. nidulans, the sexual experimental model organism of the genus Aspergillus, A. oryzae, the food biotechnology organism which is the product of centuries of cultivation, and A. fumigatus, the most common causative agent of invasive aspergillosis is now revealed to the extent that we are at present able to understand. Each genome exhibits a large set of genes common to the three as well as a much smaller set of genes unique to each. Moreover, these sequences serve as resources providing the major tool to expanding our understanding of the biology of each. Transcription profiling of A. fumigatus at high temperatures and comparative genomic hybridization between A. fumigatus and a closely related Aspergillus species provides microarray based examples of the beginning of functional analysis of the genomes of these organisms going forward from the genome sequence.

To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the view that high temperature negatively affects aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster.

Relatively few transcription factors that govern the virulence of Aspergillus fumigatus are known. We constructed 11 A. fumigatus transcription factor mutants and screened them for altered virulence in Galleria mellonella larvae. We discovered that the zinc cluster transcription factor, AcuM, is essential for maximal virulence in this model, as well as in murine models of hematogenously disseminated and invasive pulmonary aspergillosis. Transcriptional profiling experiments suggested that AcuM suppresses sreA and induces hapX to stimulate expression of genes involved in both reductive iron assimilation and siderophore-mediated iron uptake. Consistent with these results, a ΔacuM mutant had reduced iron incorporation, decreased extracellular siderophore production, and impaired capacity to grow under iron-limited conditions. Interestingly, an Aspergillus nidulans ΔacuM mutant had normal extracellular siderophore production and growth under iron-limited conditions, indicating that AcuM does not govern iron acquisition in this organism. A. fumigatus AcuM also regulated genes involved in gluconeogenesis, and the ΔacuM mutant had impaired growth on gluconeogenic carbon sources. Deletion of sreA in the ΔacuM mutant restored iron uptake, extracellular siderophore production, and virulence, but not the defect in gluconeogenesis. Thus, AcuM represses SreA and thereby induces iron acquisition, a process that is essential for the maximal virulence of A. fumigatus.

Secondary metabolites are of intense interest to humans due to their pharmaceutical and/or toxic properties. Also, these metabolites are clinically relevant because of their importance in fungal pathogenesis. Aspergillus species secrete secondary metabolites when grown individually and in the presence of other fungal species. However, it is not known whether secreted secondary metabolites provide a competitive advantage over other fungal species, or whether competition has any effect on the production of those metabolites. Here, we have performed co-cultivation competition assays among different species of Aspergillus to determine relative species fitness in culture, and to analyze the presence of possible antifungal activity of secondary metabolites in extracts. The results show that, for the most part, at 30 degrees C only one species is able to survive direct competition with a second species. In contrast, survival of both competitors was often observed at 37 degrees C. Consistent with these observations, antifungal activity of extracts from cultures grown at 30 degrees C was greater than that of extract from cultures at 37 degrees C. Interestingly, culture extracts from all species studied had some degree of antifungal activity, but in general, the extracts had greater antifungal activity when species were grown in the presence of a competitor. Using gas chromatography it was determined that the composition of extracts changed due to competition and a shift in temperature. These findings indicate that co-cultivation could be a very promising method for inducing and characterizing novel antifungal compounds produced by species of Aspergillus.

Background Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species. Results We found that genomic islands (GIs) vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described. Conclusion Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.

Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of approximately 60 genes, including some involved in capsule production, actin-based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up-expressed2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up-expressed as much as 30-fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS-PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His-tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp-family protein is produced in vivo during infection.

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system1 have facilitated the use of A. oryzae in modern biotechnology2-4. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans5 and Aspergillus fumigatus6. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation. © 2005 Nature Publishing Group.

Previous studies have indicated that Arabidopsis thaliana experienced a genome-wide duplication event shortly before its divergence from Brassica followed by extensive chromosomal rearrangements and deletions. While a large number of the duplicated genes have significantly diverged or lost their sister genes, we found 4222 pairs that are still highly conserved, and as a result had similar functional assignments during the annotation of the genome sequence. Using whole-genome DNA microarrays, we identified 906 duplicated gene pairs in which at least one member exhibited a significant response to oxidative stress. Among these, only 117 pairs were up- or down-regulated in both pairs and many of these exhibited dissimilar patterns of expression. Examination of the expression patterns of PAL1 and PAL2, ACD1 and ACD2, genes coding for two Hsp20s, various P450s, and electron transfer flavoproteins suggests Arabidopsis evolved a number of distinct oxidative stress response mechanisms using similar gene sets following the duplication of its genome.

References 1. Bishop, M.D., Kappes, S.M., Keele, J.W., Stone, R.T., Sunden, S.L.F., Hawkins, G.A., Toldo, S.S., Fries, R., Grosz, M.D., Yoo, J., Beattie, C.W. (1994). Genetics 136, 619-639. 2. Murakami, T., Shima, K. (1995). Biochem. Biophys. Res. Commun. 209, 944-952. 3. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). 4. Georges, M., Massey, J. (1992). Polymorphic DNA Markers in Bovidae (World Intellectual Property Org., Geneva) WO Publ. No. 92/ 13102. 5. Crawford, A.M., Dodds, K.G., Pierson, C.A., Ede, A.J., Montgomery, G.W., Garmonsway, H.G., Beattie, A.E., Davies, K., Maddox, J.F., Kappes, S.M., Stone, R.T., Nguyen, T.C., Penty, J.M., Lord, E.A., Broom, J.E., Buitkamp, J., Schwaiger, W., Epplen, J.T., Matthew, P., Matthews, M.E., Hulme, D.J., Beh, K.J., McGraw, R.A., Beattie, C.W. (1995). Genetics 140, 703-724. 6. Stone, R.T., Pulido, J.C., Duyk, G.M., Kappes, S.M., Keele, J.W., Beattie, C.W. (1995). Mamm. Genome 6, 714-724. 7. Kennedy, G.C. (1953). Proc. R. Soc. London 140, 578-592. 8. Coleman, D.L. (1978). Diabetologia 14, 141-148. 9. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M. (1994) Nature 372, 425-432. 10. Pelleymounter, M.A., Cullen, M.J., Baker, M.B., Hecht, R., Winters, D., Boone, T., Collins, F. (1995). Science 269, 540-543. 11. Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, S.L., Chait, B.T., Rabinowitz, D., Lallone, R.L., Burley, S.K., Friedman, J.M. (1995). Science 269, 543-546. 12. Rentsch, J., Levens, N., Chiesi, M. (1995). Biochem. Biophys. Res. Commun. 214, 131-136. 13. Campfield, L.A., Smith, F.J., Guisez, Y., Devos, R., Bum, P. (1995). Science 269, 546-549. 14. Stephens, T.W., Basinski, M., Bristow, P.K., Bue-Valleskey, J.M., Burgett, S.G., Craft, L., Hale, J., Hoffmann, J. Hsiung, H.M., Kriauciunas, A., MacKellar, W., Rosteck Jr., P.R., Schoner, B., Smith, D., Tinsley, F.C., Zhang, X.-Y., Heiman, M. (1995). Nature 377,530-532. 15. Isse, N. Ogawa, Y., Tamura, N., Masuzaki, H., Moil, K., Okazaki, T., Satoh, N., Shigemoto, M., Yoshimasa, Y., Nishi, S., Hosoda, K., Inazawa, J., Nakao, K. (1995). J. Biol. Chem. 270, 27728-27733. 16. O'Brien, S.J., Womack, J.E., Lyons, L.A., Moore, K.J., Jenkins, N.A., Copeland, N.G. (1993). Nature Genet. 3, 103-112.

Background The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain a balance between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence indicates that several pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential loading of ribosomes onto mRNAs. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress. Results ER stress was induced by treating the fungus with dithiothreitol, tunicamycin, or a thermal up-shift. The mRNAs were then fractionated on the basis of ribosome occupancy into an under-translated pool (U) and a well-translated pool (W). The mRNAs were used to interrogate microarrays and the ratio of the hybridization signal (W/U) was used as an indicator of the relative translational efficiency of a mRNA under each condition. The largest category of translationally upregulated mRNAs during ER stress encoded proteins involved in translation. Components of the ergosterol and GPI anchor biosynthetic pathways also showed increased polysome association, suggesting an important role for translational regulation in membrane and cell wall homeostasis. ER stress induced limited remodeling of the secretory pathway translatome. However, a select group of transcription factors was translationally upregulated, providing a link to subsequent modification of the transcriptome. Finally, we provide evidence that one component of the ER stress translatome is a novel mRNA isoform from the yvc1 gene that is induced by ER stress in a UPR-dependent manner. Conclusions Together, these findings define a core set of mRNAs subject to translational control during the adaptive response to acute ER stress in A. fumigatus and reveal a remarkable breadth of functions that are needed to resolve ER stress in this organism.

Background The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Results Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Conclusion Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

Background The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. Results To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle. Conclusions The observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile environments.