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1.
Appl Environ Microbiol ; 85(13)2019 07 01.
Article in English | MEDLINE | ID: mdl-31028022

ABSTRACT

Evidence suggests many marine bacteria are cosmopolitan, with widespread but sparse strains poised to seed abundant populations under conducive growth conditions. However, studies supporting this "microbial seed bank" hypothesis have analyzed taxonomic marker genes rather than whole genomes/metagenomes, leaving open the possibility that disparate ocean regions harbor endemic gene content. The Red Sea is isolated geographically from the rest of the ocean and has a combination of high irradiance, high temperature, and high salinity that is unique among the oceans; we therefore asked whether it harbors endemic gene content. We sequenced and assembled single-cell genomes of 21 SAR11 (subclades Ia, Ib, Id, and II) and 5 Prochlorococcus (ecotype HLII) samples from the Red Sea and combined them with globally sourced reference genomes to cluster genes into ortholog groups (OGs). Ordination of OG composition could distinguish clades, including phylogenetically cryptic Prochlorococcus ecotypes LLII and LLIII. Compared with reference genomes, 1% of Prochlorococcus and 17% of SAR11 OGs were unique to the Red Sea genomes (RS-OGs). Most (83%) RS-OGs had no annotated function, but 65% of RS-OGs were expressed in diel Red Sea metatranscriptomes, suggesting they are functional. Searching Tara Oceans metagenomes, RS-OGs were as likely to be found as non-RS-OGs; nevertheless, Red Sea and other warm samples could be distinguished from cooler samples using the relative abundances of OGs. The results suggest that the prevalence of OGs in these surface ocean bacteria is largely cosmopolitan, with differences in population metagenomes manifested by differences in relative abundance rather than complete presence/absence of OGs.IMPORTANCE Studies have shown that as we sequence seawater from a selected environment deeper and deeper, we approach finding every bacterial taxon known for the ocean as a whole. However, such studies have focused on taxonomic marker genes rather than on whole genomes, raising the possibility that the lack of endemism results from the method of investigation. We took a geographically isolated water body, the Red Sea, and sequenced single cells from it. We compared those single-cell genomes to available genomes from around the ocean and to ocean-spanning metagenomes. We showed that gene ortholog groups found in Red Sea genomes but not in other genomes are nevertheless common across global ocean metagenomes. These results suggest that Baas Becking's hypothesis "everything is everywhere, but the environment selects" also applies to gene ortholog groups. This widely dispersed functional diversity may give oceanic microbial communities the functional capacity to respond rapidly to changing conditions.


Subject(s)
Alphaproteobacteria/genetics , Genome, Bacterial , Metagenome , Prochlorococcus/genetics , Seawater/microbiology , Indian Ocean , Phylogeny
2.
Nature ; 444(7115): 97-101, 2006 Nov 02.
Article in English | MEDLINE | ID: mdl-17080091

ABSTRACT

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.


Subject(s)
Genome, Fungal/genetics , Ustilago/genetics , Ustilago/pathogenicity , Zea mays/microbiology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation, Fungal , Genes, Fungal/genetics , Genomics , Multigene Family/genetics , Ustilago/growth & development , Virulence/genetics
3.
Microb Ecol ; 60(3): 505-15, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20369357

ABSTRACT

We used a microarray targeting 3,524 genes to assess the transcriptional response of the actinomycete Rhodococcus aetherivorans I24 in minimal medium supplemented with various substrates (e.g., PCBs) and in both PCB-contaminated and non-contaminated sediment slurries. Relative to the reference condition (minimal medium supplemented with glucose), 408 genes were upregulated in the various treatments. In medium and in sediment, PCBs elicited the upregulation of a common set of 100 genes, including gene-encoding chaperones (groEL), a superoxide dismutase (sodA), alkyl hydroperoxide reductase protein C (ahpC), and a catalase/peroxidase (katG). Analysis of the R. aetherivorans I24 genome sequence identified orthologs of many of the genes in the canonical biphenyl pathway, but very few of these genes were upregulated in response to PCBs or biphenyl. This study is one of the first to use microarrays to assess the transcriptional response of a soil bacterium to a pollutant under conditions that more closely resemble the natural environment. Our results indicate that the transcriptional response of R. aetherivorans I24 to PCBs, in both medium and sediment, is primarily directed towards reducing oxidative stress, rather than catabolism.


Subject(s)
Geologic Sediments/microbiology , Polychlorinated Biphenyls/metabolism , Rhodococcus/genetics , Soil Microbiology , Soil Pollutants/metabolism , Biodegradation, Environmental , Culture Media , Gene Expression Regulation, Bacterial , Genes, Bacterial , Oligonucleotide Array Sequence Analysis , Oxidative Stress , RNA, Bacterial/genetics , Rhodococcus/metabolism , Sequence Analysis, DNA , Transcription, Genetic
4.
BMC Genomics ; 8: 334, 2007 Sep 24.
Article in English | MEDLINE | ID: mdl-17892571

ABSTRACT

BACKGROUND: Ustilago maydis is the basidiomycete fungus responsible for common smut of corn and is a model organism for the study of fungal phytopathogenesis. To aid in the annotation of the genome sequence of this organism, several expressed sequence tag (EST) libraries were generated from a variety of U. maydis cell types. In addition to utility in the context of gene identification and structure annotation, the ESTs were analyzed to identify differentially abundant transcripts and to detect evidence of alternative splicing and anti-sense transcription. RESULTS: Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 x 521) and haploid cells of strain 521 grown under nutrient rich, carbon starved, and nitrogen starved conditions. Using the genome sequence as a scaffold, the 15,901 ESTs were assembled into 6,101 contiguous expressed sequences (contigs); among these, 5,482 corresponded to predicted genes in the MUMDB (MIPS Ustilago maydis database), while 619 aligned to regions of the genome not yet designated as genes in MUMDB. A comparison of EST abundance identified numerous genes that may be regulated in a cell type or starvation-specific manner. The transcriptional response to nitrogen starvation was assessed using RT-qPCR. The results of this suggest that there may be cross-talk between the nitrogen and carbon signalling pathways in U. maydis. Bioinformatic analysis identified numerous examples of alternative splicing and anti-sense transcription. While intron retention was the predominant form of alternative splicing in U. maydis, other varieties were also evident (e.g. exon skipping). Selected instances of both alternative splicing and anti-sense transcription were independently confirmed using RT-PCR. CONCLUSION: Through this work: 1) substantial sequence information has been provided for U. maydis genome annotation; 2) new genes were identified through the discovery of 619 contigs that had previously escaped annotation; 3) evidence is provided that suggests the regulation of nitrogen metabolism in U. maydis differs from that of other model fungi, and 4) Alternative splicing and anti-sense transcription were identified in U. maydis and, amid similar observations in other basidiomycetes, this suggests these phenomena may be widespread in this group of fungi. These advances emphasize the importance of EST analysis in genome annotation.


Subject(s)
Expressed Sequence Tags , Genome, Fungal , RNA, Messenger/genetics , Ustilago/genetics , Zea mays/microbiology , Alternative Splicing , DNA, Complementary , Introns , Nitrogen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ustilago/growth & development , Ustilago/metabolism
5.
PLoS One ; 5(7): e11518, 2010 Jul 12.
Article in English | MEDLINE | ID: mdl-20634954

ABSTRACT

BACKGROUND: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. METHODOLOGY/PRINCIPAL FINDINGS: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. CONCLUSIONS: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.


Subject(s)
Genome/genetics , Prokaryotic Cells/metabolism , Repetitive Sequences, Nucleic Acid/genetics , Sequence Analysis, DNA/methods , Algorithms
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