Methods for determining the genetic affinity of microorganisms and viruses

Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism

The Reverse Transcription Signature of N-\u3csub\u3e1\u3c/sub\u3e-Methyladenosine in RNA-Seq is Sequence Dependent

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m1A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3’ of m1A in the template RNA, meaning it is sequence dependent. The RT-signature ofm1Awas used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA

Microbial identification by mass cataloging

BACKGROUND: The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using hybridization and other molecular approaches. In their usual format, such assays are based on the presence of unique subsequences in the target RNA and require a prior knowledge of what organisms are likely to be in a sample. They are thus limited in generality when analyzing an unknown sample. Herein, we demonstrate the utility of catalogs of masses to characterize the bacterial 16S rRNA(s) in any sample. Sample nucleic acids are digested with a nuclease of known specificity and the products characterized using mass spectrometry. The resulting catalogs of masses can subsequently be compared to the masses known to occur in previously-sequenced 16S rRNAs allowing organism identification. Alternatively, if the organism is not in the existing database, it will still be possible to determine its genetic affinity relative to the known organisms. RESULTS: Ribonuclease T(1 )and ribonuclease A digestion patterns were calculated for 1,921 complete 16S rRNAs. Oligoribonucleotides generated by RNase T(1 )of length 9 and longer produce sufficient diversity of masses to be informative. In addition, individual fragments or combinations thereof can be used to recognize the presence of specific organisms in a complex sample. In this regard, 140 strains out of 1,921 organisms (7.3%) could be identified by the presence of a unique RNase T(1)-generated oligoribonucleotide mass. Combinations of just two and three oligoribonucleotide masses allowed 54% and 72% of the specific strains to be identified, respectively. An initial algorithm for recovering likely organisms present in complex samples is also described. CONCLUSION: The use of catalogs of compositions (masses) of characteristic oligoribonucleotides for microbial identification appears extremely promising. RNase T(1 )is more useful than ribonuclease A in generating characteristic masses, though RNase A produces oligomers which are more readily distinguished due to the large mass difference between A and G. Identification of multiple species in mixtures is also feasible. Practical applicability of the method depends on high performance mass spectrometric determination, and/or use of methods that increase the one dalton (Da) mass difference between uracil and cytosine

Bacterial species identification getting easier

The traditional methods of bacterial identification are based on observation of either the morphology of single cells or colony characteristics. However, the adoption of newer and automated methods offers advantage in terms of rapid and reliable identification of bacterial species. The review provides a comprehensive appreciation of new and improved technologies such fatty acid profiling, sequence analysis of the 16S rRNA gene, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), metabolic finger profiling using BIOLOG, ribotyping, together with the computational tools employed for querying the databases that are associated with these identification tools and high-throughput genomic sequencing in bacterial identification. It is evident that with the increase in the adoption of new technologies bacterial identification is becoming easier.Keywords: Bacteria, Biolog, computational tools, fatty acids, Gram staining, identification, metagenomics, morphology, MALDI-TOF MS, RiboPrinter, 16S rRNA gene.African Journal of Biotechnology Vol. 12(41), pp. 5975-598

Insights into the biology of Candidate Division OP3 LiM populations

The candidate division OP3, recently entitled candidate phylum Omnitrophica, is characterized by 16S rRNA gene sequences from a broad range of anoxic habitats with a broad phylogeny of up to 26% sequence dissimilarity. The 16S rRNA phylotype OP3 LiM had previously been detected in limonene-degrading, methanogenic enrichment cultures and represented small coccoid cells. Neither isolation experiments nor physiological experiments had provided insights into the metabolism of this bacterium within the complex methanogenic community. This doctoral thesis aimed at the characterization of populations of the phylotype OP3 LiM to discover its biology. Metagenomes usually yield draft population genomes. To obtain the complete closed OP3 LiM genome, in silico methods were explored to improve draft assemblies. Large genomes of planctomycete strains were assembled with a variety of methods. A taxonomic classification of contig sequences was used to differentiate and separate contigs of draft assemblies into taxon-specific groups. Reassemblies of reads obtaining from mapping onto taxon-specific contigs yielded improved draft assemblies. This knowledge was used to obtain a closed genome of OP3 LiM from a metagenome of physically enriched OP3 LiM cells. Finishing the OP3 LiM genome required the combination of data of different sequencing technologies, a variety of assembly and mapping software, over 15 reassemblies with intensive manual quality controls by read and contig mapping and, finally, laboratory work with combinatorial PCR to solve the genome puzzle. The population genome of OP3 LiM is the first closed genome of a member of candidate phylum Omnitrophica and comprises 1,974,501 bp with a GC content of 52.9%. Its 23S rRNA contains a group I intron. The genome offers a syntrophic life on hydrogen or formate, however, the metaproteome indicated that OP3 LiM uses glycolysis together with pyruvate oxidation as major catabolic pathway. The metaproteome also identified high levels of proteins potentially involved in the degradation of polymers as well as in the uptake of foreign nucleic acids. The genomic information was combined with observations of cells of the methanogenic community by different visualization methods. Images of OP3 LiM required electron microscopy due to the small cell size of 0.2a 0.3 AAmicrometre in diameter. In situ hybridizations revealed two physiological stages, free-living OP3 LiM cells with low ribosome content and OP3 LiM cells attached to either bacteria or archaea, which showed strong signals. This observation indicated a higher metabolic activity of OP3 LiM cells during the attachment and, likewise, that the bacterium utilizes surface polysaccharides as preferred substrate. In situ hybridizations revealed that the methanogen Methanosaeta in the enrichment culture contained cells in the filaments that lacked DNA and rRNA suggesting that these cells lost their cellular content. We also observed faint signals of the OP3 LiM 16S rRNA in Methanosaeta cells. The presence of the intron RNA of the 23S rRNA of OP3 LiM was visualized in Methanosaeta cells devoid of DNA and rRNA. This first direct observation of an intron transfer from a bacterium to an archaeon together with metaproteomic observations indicate the lifestyle of an epibiotic bacterium for OP3 LiM. OP3 LiM is the first predatory bacterium that preys on Archaea. We propose to name OP3 LiM a Candidatus Vampirococcus archaeovorusa

Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics

Metatranscriptomes generated by pyrosequencing hold significant potential for describing functional processes in complex microbial communities. Meeting this potential requires protocols that maximize mRNA recovery by reducing the relative abundance of ribosomal RNA, as well as systematic comparisons to identify methodological artifacts and test for reproducibility across data sets. Here, we implement a protocol for subtractive hybridization of bacterial rRNA (16S and 23S) that uses sample-specific probes and is applicable across diverse environmental samples. To test this method, rRNA-subtracted and unsubtracted transcriptomes were sequenced (454 FLX technology) from bacterioplankton communities at two depths in the oligotrophic open ocean, yielding 10 data sets representing ~350 Mbp. Subtractive hybridization reduced bacterial rRNA transcript abundance by 40–58%, increasing recovery of non-rRNA sequences up to fourfold (from 12% to 20% of total sequences to 40–49%). In testing this method, we established criteria for detecting sequences replicated artificially via pyrosequencing errors and identified such replicates as a significant component (6–39%) of total pyrosequencing reads. Following replicate removal, statistical comparisons of reference genes (identified via BLASTX to NCBI-nr) between technical replicates and between rRNA-subtracted and unsubtracted samples showed low levels of differential transcript abundance (<0.2% of reference genes). However, gene overlap between data sets was remarkably low, with no two data sets (including duplicate runs from the same pyrosequencing library template) sharing greater than 17% of unique reference genes. These results indicate that pyrosequencing captures a small subset of total mRNA diversity and underscores the importance of reliable rRNA subtraction procedures to enhance sequencing coverage across the functional transcript pool.Agouron InstituteGordon and Betty Moore FoundationUnited States. Dept. of Energy. Office of ScienceNational Science Foundation (U.S.) (NSF Science and Technology Center Award EF0424599

The role of unicellular cyanobacteria in nitrogen fixation and assimilation in subtropical marine waters

Biological N2 fixation constitutes the major source of nitrogen in open ocean systems, regulating the marine nitrogen inventory and primary productivity. Symbiotic relationships between phytoplankton and N2 fixing microorganisms (diazotrophs) have been suggested to play a significant role in the ecology and biogeochemistry in these oceanic regions. The widely distributed, uncultured N2 fixing cyanobacterium UCYN A was suggested to live in symbiosis since it has unprecedented genome reduction, including the lack of genes encoding for oxygen evolving photosystem II and the tricarboxylic acid cycle. This thesis aims to study carbon and nitrogen metabolism on field populations of UCYN A using molecular biology, as well as mass spectrometry tools to visualize metabolic activity on a single cell scale. The development of a 16S rRNA oligonucleotide probe specifically targeting UCYN A cells and its successful application on environmental samples (Manuscript I and II) revealed a symbiotic partnership with a unicellular prymnesiophyte. We demonstrated a nutrient transfer in carbon and nitrogen compounds between these two partner cells, providing an explanation how these diazotrophs thrive in open ocean systems. Further, UCYN A can also associate with globally abundant calcifying prymnesiophyte members, e.g. Braarudosphaera bigelowii, indicating that this symbiosis might impact the efficiency of the biological carbon pump. In manuscript III, we provided quantitative information on the cellular abundance and distribution of UCYN A cells in the North Atlantic Ocean and identified the eukaryotic partner cell as Haptophyta (including prymnesiophyte) via double Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD FISH). The UCYN A Haptophyta association was the dominant form (87.0±6.1%) over free living UCYN A cells. Interestingly, we also detected UCYN A cells living in association with unknown eukaryotes and non calcifying Haptophyta cells, raising questions about the host specificity. During a follow up study (Manuscript IV), we conducted various nutrient amendment experiments (including iron, phosphorus, ammonium nitrate and Saharan Dust) in order to examine physiological interactions between individual UCYN A and Haptophyta cells. Single cell measurements using nanometer scale secondary ion mass spectrometry (nanoSIMS) revealed a tight physiological coupling in the transfer of carbon (R2 = 0.6232; n = 44) and nitrogen (R2 = 0.9659; n = 44) between host and symbiont. N2 fixation was mainly stimulated when iron rich Saharan Dust was added, emphasizing on aeolian dust deposition in seawater as a major parameter in constraining N2 fixation of UCYN A. Moreover, when fixed nitrogen species (ammonium and nitrate) were added, a third unknown microbial partner cell was observed within individual UCYN A Haptophyta associations, but their menaing is unclear. Based on this thesis work we revealed how UCYN A cells thrive in the environment and established a culture independent technique to assess the in situ activity in respect to CO2 and N2 fixation of this ecological relevant group of microorganisms. Furthermore, this unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N2 fixation

The role of unicellular cyanobacteria in nitrogen fixation and assimilation in subtropical marine waters

Biological N2 fixation constitutes the major source of nitrogen in open ocean systems, regulating the marine nitrogen inventory and primary productivity. Symbiotic relationships between phytoplankton and N2 fixing microorganisms (diazotrophs) have been suggested to play a significant role in the ecology and biogeochemistry in these oceanic regions. The widely distributed, uncultured N2 fixing cyanobacterium UCYN–A was suggested to live in symbiosis since it has unprecedented genome reduction, including the lack of genes encoding for oxygen–evolving photosystem II and the tricarboxylic acid cycle. This thesis aims to study carbon and nitrogen metabolism on field populations of UCYN–A using molecular biology, as well as mass spectrometry tools to visualize metabolic activity on a single cell scale. The development of a 16S rRNA oligonucleotide probe specifically targeting UCYN– A cells and its successful application on environmental samples (Manuscript I and II) revealed a symbiotic partnership with a unicellular prymnesiophyte. We demonstrated a nutrient transfer in carbon and nitrogen compounds between these two partner cells, providing an explanation how these diazotrophs thrive in open ocean systems. Further, UCYN–A can also associate with globally abundant calcifying prymnesiophyte members, e.g. Braarudosphaera bigelowii, indicating that this symbiosis might impact the efficiency of the biological carbon pump. In manuscript III, we provided quantitative information on the cellular abundance and distribution of UCYN–A cells in the North Atlantic Ocean and identified the eukaryotic partner cell as Haptophyta (including prymnesiophyte) via double Catalyzed Reporter Deposition–Fluorescence In Situ Hybridization (CARD–FISH). The UCYN–A–Haptophyta association was the dominant form (87.0±6.1%) over free–living UCYN–A cells. Interestingly, we also detected UCYN–A cells living in association with unknown eukaryotes and non–calcifying Haptophyta cells, raising questions about the host specificity. During a follow up study (Manuscript IV), we conducted various nutrient amendment experiments (including iron, phosphorus, ammonium–nitrate and Saharan Dust) in order to examine physiological interactions between individual UCYN–A and Haptophyta cells. Single cell measurements using nanometer scale secondary ion mass spectrometry (nanoSIMS) revealed a tight physiological coupling in the transfer of carbon (R2 = 0.6232; n = 44) and nitrogen (R2 = 0.9659; n = 44) between host and symbiont. N2 fixation was mainly stimulated when iron–rich Saharan Dust was added, emphasizing on aeolian dust deposition in seawater as a major parameter in constraining N2 fixation of UCYN–A. Moreover, when fixed nitrogen species (ammonium and nitrate) were added, a third unknown microbial partner II cell was observed within individual UCYN–A–Haptophyta associations, but their menaing is unclear. Based on this thesis work we revealed how UCYN–A cells thrive in the environment and established a culture–independent technique to assess the in situ activity in respect to CO2 and N2 fixation of this ecological relevant group of microorganisms. Furthermore, this unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N2 fixation

Interactions between the Translation Machinery and a Translational preQ1 Riboswitch.

Gene expression is highly regulated with a diversity of regulation at the RNA level. In bacteria, regulation of mRNA translation into protein often occurs through RNA sequence features such as the Shine-Dalgarno (SD) sequence and local structural features. Translational riboswitches in bacteria exemplify such cis-acting regulation. This work look at how structural features of a preQ1 riboswitch effect regulation through interactions with the translation machinery. Broader questions about how individual translational machinery components, such as ribosomal protein S1 and the 30S ribosomal subunit, interact with structured RNAs are also addressed. We sought a more detailed mechanistic view of the interplay between the translational preQ1 riboswitch found in the 5′ UTR of an mRNA from T. tengcongensis, its ligand preQ1, and the SD sequence accessibility. To this end, we developed SiM-KARTS, a generalized strategy to interrogate site-specific structural dynamics of RNA molecules based on probe hybridization kinetics. Intriguingly, we found that the riboswitch expression platform alternates between conformations with differing SD accessibility, which are distinguished by “bursts” of probe binding, the pattern of which is modulated by ligand. This challenges the assumption that riboswitches behave in simple ON/OFF fashion and thus has broader implications for how we think about translational riboswitch regulation. The folding and unfolding of RNA structure influences other cellular processes besides translation. Ribosomal protein S1 performs other roles outside of the context of translation, which are related to its RNA binding or unfolding capacity. We used the well-characterized preQ1 riboswitch as a model pseudoknot to study how S1 interacts with defined, stable tertiary structure. S1 is able to bind and at least partially unfold this pseudoknot in a manner that is limited by RNA structural stability. Lastly, we investigated the influence of S1 on translation of preQ1 riboswitch-containing mRNAs and found that the effects of ligand on translation are not potentiated by the loss of S1. There is, however, a dramatic effect on translational coupling, invoking a role for S1 in polycistronic mRNA translation. These results highlight the need for additional techniques, such as assays at the single molecule level, to monitor early 30S-mRNA interactions during translation.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116677/1/palund_1.pd