Insights into the Fundamental Physiology of the Uncultured Fe-Oxidizing Bacterium Leptothrix ochracea.

Leptothrix ochracea is known for producing large volumes of iron oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiology. In microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, the genomic capacity for iron oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidation. Additionally, its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiology is a hybrid of those of the chemolithotrophic Gallionella-type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps.IMPORTANCELeptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that its true physiology remains unknown. Further, L. ochracea is an ecological engineer; a few L. ochracea cells can generate prodigious volumes of iron oxyhydroxides, changing the ecosystem's geochemistry and ecology. Therefore, to determine L. ochracea's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands.

Homology-based annotation yields 1,042 new candidate genes in the Drosophila melanogaster genome.

The approach to annotating a genome critically affects the number and accuracy of genes identified in the genome sequence. Genome annotation based on stringent gene identification is prone to underestimate the complement of genes encoded in a genome. In contrast, over-prediction of putative genes followed by exhaustive computational sequence, motif and structural homology search will find rarely expressed, possibly unique, new genes at the risk of including non-functional genes. We developed a two-stage approach that combines the merits of stringent genome annotation with the benefits of over-prediction. First we identify plausible genes regardless of matches with EST, cDNA or protein sequences from the organism (stage 1). In the second stage, proteins predicted from the plausible genes are compared at the protein level with EST, cDNA and protein sequences, and protein structures from other organisms (stage 2). Remote but biologically meaningful protein sequence or structure homologies provide supporting evidence for genuine genes. The method, applied to the Drosophila melanogaster genome, validated 1,042 novel candidate genes after filtering 19,410 plausible genes, of which 12,124 matched the original 13,601 annotated genes. This annotation strategy is applicable to genomes of all organisms, including human.

Haploflow: Strain-resolved de novo assembly of viral genomes.

In viral infections often multiple related viral strains are present, due to coinfection or within-host evolution. We describe Haploflow, a de Bruijn graph-based assembler for de novo genome assembly of viral strains from mixed sequence samples using a novel flow algorithm. We assessed Haploflow across multiple benchmark data sets of increasing complexity, showing that Haploflow is faster and more accurate than viral haplotype assemblers and generic metagenome assemblers not aiming to reconstruct strains. Haplotype reconstructed high-quality strain-resolved assemblies from clinical HCMV samples and SARS-CoV-2 genomes from wastewater metagenomes identical to genomes from clinical isolates.

MAGPIE/EGRET annotation of the 2.9-Mb Drosophila melanogaster Adh region.

Our challenge in annotating the 2.91-Mb Adh region of the Drosophila melanogaster genome was to identify genetic and genomic features automatically, completely, and precisely within a 6-week period. To do so, we augmented the MAGPIE microbial genome annotation system to handle eukaryotic genomic sequence data. The new configuration required the integration of eukaryotic gene-finding tools and DNA repeat tools into the automatic data collection module. It also required us to define in MAGPIE new strategies to combine data about eukaryotic exon predictions with functional data to refine the exon predictions. At the heart of the resulting new eukaryotic genome annotation system is a reverse comparison of public protein and complementary DNA sequences against the input genome to identify missing exons and to refine exon boundaries. The software modules that add eukaryotic genome annotation capability to MAGPIE are available as EGRET (Eukaryotic Genome Rapid Evaluation Tool).

Microarray-based analysis of early development in Xenopus laevis.

In order to examine transcriptional regulation globally, during early vertebrate embryonic development, we have prepared Xenopus laevis cDNA microarrays. These prototype embryonic arrays contain 864 sequenced gastrula cDNA. In order to analyze and store array data, a microarray analysis pipeline was developed and integrated with sequence analysis and annotation tools. In three independent experimental settings, we demonstrate the power of these global approaches and provide optimized protocols for their application to molecular embryology. In the first set, by comparing maternal versus zygotic transcription, we document groups of genes that are temporally regulated. This analytical approach resulted in the discovery of novel temporally regulated genes. In the second, we examine changes in gene expression spatially during development by comparing dorsal and ventral mesoderm dissected from early gastrula embryos. We have discovered novel genes with spatial enrichment from these experiments. Finally, we use the prototype microarray to examine transcriptional responses from embryonic explants treated with activin. We selected genes (two of which are novel) regulated by activin for further characterization. All results obtained by the arrays were independently tested by RT-PCR or by in situ hybridization to provide a direct assessment of the accuracy and reproducibility of these approaches in the context of molecular embryology.