BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and euk... more BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens.FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes.ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind...
We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacter... more We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.
The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean betwee... more The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean between April and July 2010, stimulating microbial blooms of petroleum-degrading bacteria. To understand the metabolic response of marine microorganisms, we sequenced ≈ 66 million community transcripts that revealed the identity of metabolically active microbes and their roles in petroleum consumption. Reads were assigned to reference genes from ≈ 2700 bacterial and archaeal taxa, but most assignments (39%) were to just six genomes representing predominantly methane- and petroleum-degrading Gammaproteobacteria. Specific pathways for the degradation of alkanes, aromatic compounds and methane emerged from the metatranscriptomes, with some transcripts assigned to methane monooxygenases representing highly divergent homologs that may degrade either methane or short alkanes. The microbial community in the plume was less taxonomically and functionally diverse than the unexposed community below the plume; this was due primarily to decreased species evenness resulting from Gammaproteobacteria blooms. Surprisingly, a number of taxa (related to SAR11, Nitrosopumilus and Bacteroides, among others) contributed equal numbers of transcripts per liter in both the unexposed and plume samples, suggesting that some groups were unaffected by the petroleum inputs and blooms of degrader taxa, and may be important for re-establishing the pre-spill microbial community structure.
A typical marine bacterial cell in coastal seawater contains only B200 molecules of mRNA, each of... more A typical marine bacterial cell in coastal seawater contains only B200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium's responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.
Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient.... more Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient. To understand their capacity to respond to Fe stress, we screened picoplankton genomes and the Global Ocean Survey metagenome for known Fe stress genes. Many open ocean strains of Synechococcus lack most known genes for Fe stress, while coastal and upwelling strains contain many, suggesting that maintaining multiple Fe limitation compensation strategies is not a selective advantage in the open ocean. All genomes contained iron deficiency-induced protein A (IdiA) and its complementary Fe3+ transport proteins. The ubiquity of IdiA was exploited to develop an in situ Fe stress bioassay based on immunolabelling and flow cytometry. As a test of field applicability, we used the assay on natural Synechococcus populations from one station in the Costa Rica Upwelling Dome where total Fe ranged from <0.08 to 0.14 nM in the upper water column. The bioassay found Fe stress in 5–54% of the population. Based on our findings, we believe that when reactive strains are present this assay can reveal environmental and clade-specific differences in the response of Synechococcus to Fe stress.
Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collec... more Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.
Human impacts on aquatic biodiversity are often measured at the assemblage or community level, al... more Human impacts on aquatic biodiversity are often measured at the assemblage or community level, although it has been suggested that individual-level measures are more sensitive. We evaluated the effects of anthropogenic sedimentation on endemic snails in Lake Tanganyika, East Africa, by comparing assemblagelevel (i.e., species richness, evenness, and abundance) and individual-level (i.e., frequencies of predation and parasitism, fecal organic content, life history) data between sediment-disturbed and reference sites. Previous studies have indicated that sedimentation kills snails and reduces mollusc diversity in this system, but we found little evidence of changes in species richness, evenness, or snail abundance at the levels of sedimentation recorded. In contrast, individual-level data revealed a variety of differences associated with sedimentation. Frequencies of shell scarring by predatory crabs and castration by parasitic trematodes were significantly lower at disturbed sites, indicating shifts in interspecific interactions. Snails ingested large amounts of inorganic sediments at disturbed sites, suggesting a reduction in food quality. In addition, sedimentation was associated with a large downward shift in size distribution within some species and reproduction at smaller size. These strong patterns in individual-level data contrast with the lack of effects at the assemblage level. We argue that incorporating individual-level measures will often enhance the sensitivity of impact surveys and may reveal effects of disturbance on important interspecific interactions.
Page 1. IRON LIMITATION AND THE ROLE OF SIDEROPHORES IN MARINE SYNECHOCOCCUS By Adam R. Rivers BA... more Page 1. IRON LIMITATION AND THE ROLE OF SIDEROPHORES IN MARINE SYNECHOCOCCUS By Adam R. Rivers BA, New College of Florida 2001 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy ...
BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and euk... more BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens.FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes.ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind...
We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacter... more We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.
The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean betwee... more The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean between April and July 2010, stimulating microbial blooms of petroleum-degrading bacteria. To understand the metabolic response of marine microorganisms, we sequenced ≈ 66 million community transcripts that revealed the identity of metabolically active microbes and their roles in petroleum consumption. Reads were assigned to reference genes from ≈ 2700 bacterial and archaeal taxa, but most assignments (39%) were to just six genomes representing predominantly methane- and petroleum-degrading Gammaproteobacteria. Specific pathways for the degradation of alkanes, aromatic compounds and methane emerged from the metatranscriptomes, with some transcripts assigned to methane monooxygenases representing highly divergent homologs that may degrade either methane or short alkanes. The microbial community in the plume was less taxonomically and functionally diverse than the unexposed community below the plume; this was due primarily to decreased species evenness resulting from Gammaproteobacteria blooms. Surprisingly, a number of taxa (related to SAR11, Nitrosopumilus and Bacteroides, among others) contributed equal numbers of transcripts per liter in both the unexposed and plume samples, suggesting that some groups were unaffected by the petroleum inputs and blooms of degrader taxa, and may be important for re-establishing the pre-spill microbial community structure.
A typical marine bacterial cell in coastal seawater contains only B200 molecules of mRNA, each of... more A typical marine bacterial cell in coastal seawater contains only B200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium's responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.
Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient.... more Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient. To understand their capacity to respond to Fe stress, we screened picoplankton genomes and the Global Ocean Survey metagenome for known Fe stress genes. Many open ocean strains of Synechococcus lack most known genes for Fe stress, while coastal and upwelling strains contain many, suggesting that maintaining multiple Fe limitation compensation strategies is not a selective advantage in the open ocean. All genomes contained iron deficiency-induced protein A (IdiA) and its complementary Fe3+ transport proteins. The ubiquity of IdiA was exploited to develop an in situ Fe stress bioassay based on immunolabelling and flow cytometry. As a test of field applicability, we used the assay on natural Synechococcus populations from one station in the Costa Rica Upwelling Dome where total Fe ranged from <0.08 to 0.14 nM in the upper water column. The bioassay found Fe stress in 5–54% of the population. Based on our findings, we believe that when reactive strains are present this assay can reveal environmental and clade-specific differences in the response of Synechococcus to Fe stress.
Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collec... more Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.
Human impacts on aquatic biodiversity are often measured at the assemblage or community level, al... more Human impacts on aquatic biodiversity are often measured at the assemblage or community level, although it has been suggested that individual-level measures are more sensitive. We evaluated the effects of anthropogenic sedimentation on endemic snails in Lake Tanganyika, East Africa, by comparing assemblagelevel (i.e., species richness, evenness, and abundance) and individual-level (i.e., frequencies of predation and parasitism, fecal organic content, life history) data between sediment-disturbed and reference sites. Previous studies have indicated that sedimentation kills snails and reduces mollusc diversity in this system, but we found little evidence of changes in species richness, evenness, or snail abundance at the levels of sedimentation recorded. In contrast, individual-level data revealed a variety of differences associated with sedimentation. Frequencies of shell scarring by predatory crabs and castration by parasitic trematodes were significantly lower at disturbed sites, indicating shifts in interspecific interactions. Snails ingested large amounts of inorganic sediments at disturbed sites, suggesting a reduction in food quality. In addition, sedimentation was associated with a large downward shift in size distribution within some species and reproduction at smaller size. These strong patterns in individual-level data contrast with the lack of effects at the assemblage level. We argue that incorporating individual-level measures will often enhance the sensitivity of impact surveys and may reveal effects of disturbance on important interspecific interactions.
Page 1. IRON LIMITATION AND THE ROLE OF SIDEROPHORES IN MARINE SYNECHOCOCCUS By Adam R. Rivers BA... more Page 1. IRON LIMITATION AND THE ROLE OF SIDEROPHORES IN MARINE SYNECHOCOCCUS By Adam R. Rivers BA, New College of Florida 2001 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy ...
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