sxtA4+ and sxtA4- Genotypes Occur Together within Natural Pyrodinium bahamense Sub-Populations from the Western Atlantic

Saxitoxin (STX) is a secondary metabolite and potent neurotoxin produced by several genera of harmful algal bloom (HAB) marine dinoflagellates. The basis for variability in STX production within natural bloom populations is undefined as both toxic and non-toxic strains (of the same species) have been isolated from the same geographic locations. Pyrodinium bahamense is a STX-producing bioluminescent dinoflagellate that blooms along the east coast of Florida as well as the bioluminescent bays in Puerto Rico (PR), though no toxicity reports exist for PR populations. The core genes in the dinoflagellate STX biosynthetic pathway have been identified, and the sxtA4 gene is essential for toxin production. Using sxtA4 as a molecular proxy for the genetic capacity of STX production, we examined sxtA4+ and sxtA4- genotype frequency at the single cell level in P. bahamense populations from different locations in the Indian River Lagoon (IRL), FL, and Mosquito Bay (MB), a bioluminescent bay in PR. Multiplex PCR was performed on individual cells with Pyrodinium-specific primers targeting the 18S rRNA gene and sxtA4. The results reveal that within discrete natural populations of P. bahamense, both sxtA4+ and sxtA4- genotypes occur, and the sxtA4+ genotype dominates. In the IRL, the frequency of the sxtA4+ genotype ranged from ca. 80–100%. In MB, sxtA4+ genotype frequency ranged from ca 40–66%. To assess the extent of sxtA4 variation within individual cells, sxtA4 amplicons from single cells representative of the different sampling sites were cloned and sequenced. Overall, two variants were consistently obtained, one of which is likely a pseudogene based on alignment with cDNA sequences. These are the first data demonstrating the existence of both genotypes in natural P. bahamense sub-populations, as well as sxtA4 presence in P. bahamense from PR. These results provide insights on underlying genetic factors influencing the potential for toxin variability among natural sub-populations of HAB species and highlight the need to study the genetic diversity within HAB sub-populations at a fine level in order to identify the molecular mechanisms driving HAB evolution.

Download Full-text

Toxic and Nontoxic Microcystis Colonies in Natural Populations Can Be Differentiated on the Basis of rRNA Gene Internal Transcribed Spacer Diversity

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.3979-3987.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 3979-3987 ◽

Cited By ~ 92

Author(s):

Ingmar Janse ◽

W. Edwin A. Kardinaal ◽

Marion Meima ◽

Jutta Fastner ◽

Petra M. Visser ◽

...

Keyword(s):

Internal Transcribed Spacer ◽

Denaturing Gradient Gel Electrophoresis ◽

Natural Populations ◽

Genotypic Diversity ◽

Toxin Production ◽

Its Sequences ◽

Rrna Gene ◽

Flight Mass Spectrometry ◽

Gradient Gel Electrophoresis ◽

Natural Communities

ABSTRACT Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

Download Full-text

Mechanistic strategies of microbial communities regulating lignocellulose deconstruction in a UK salt marsh

Microbiome ◽

10.1186/s40168-020-00964-0 ◽

2021 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Daniel R. Leadbeater ◽

Nicola C. Oates ◽

Joseph P. Bennett ◽

Yi Li ◽

Adam A. Dowle ◽

...

Keyword(s):

Salt Marsh ◽

Salt Marshes ◽

Molecular Mechanisms ◽

Surface Sediments ◽

Gene Profiling ◽

Oxidative Enzymes ◽

Lignocellulose Degradation ◽

Rrna Gene ◽

Enzymatic Mechanisms

Abstract Background Salt marshes are major natural repositories of sequestered organic carbon with high burial rates of organic matter, produced by highly productive native flora. Accumulated carbon predominantly exists as lignocellulose which is metabolised by communities of functionally diverse microbes. However, the organisms that orchestrate this process and the enzymatic mechanisms employed that regulate the accumulation, composition and permanence of this carbon stock are not yet known. We applied meta-exo-proteome proteomics and 16S rRNA gene profiling to study lignocellulose decomposition in situ within the surface level sediments of a natural established UK salt marsh. Results Our studies revealed a community dominated by Gammaproteobacteria, Bacteroidetes and Deltaproteobacteria that drive lignocellulose degradation in the salt marsh. We identify 42 families of lignocellulolytic bacteria of which the most active secretors of carbohydrate-active enzymes were observed to be Prolixibacteracea, Flavobacteriaceae, Cellvibrionaceae, Saccharospirillaceae, Alteromonadaceae, Vibrionaceae and Cytophagaceae. These families secreted lignocellulose-active glycoside hydrolase (GH) family enzymes GH3, GH5, GH6, GH9, GH10, GH11, GH13 and GH43 that were associated with degrading Spartina biomass. While fungi were present, we did not detect a lignocellulolytic contribution from fungi which are major contributors to terrestrial lignocellulose deconstruction. Oxidative enzymes such as laccases, peroxidases and lytic polysaccharide monooxygenases that are important for lignocellulose degradation in the terrestrial environment were present but not abundant, while a notable abundance of putative esterases (such as carbohydrate esterase family 1) associated with decoupling lignin from polysaccharides in lignocellulose was observed. Conclusions Here, we identify a diverse cohort of previously undefined bacteria that drive lignocellulose degradation in the surface sediments of the salt marsh environment and describe the enzymatic mechanisms they employ to facilitate this process. Our results increase the understanding of the microbial and molecular mechanisms that underpin carbon sequestration from lignocellulose within salt marsh surface sediments in situ and provide insights into the potential enzymatic mechanisms regulating the enrichment of polyphenolics in salt marsh sediments.

Download Full-text

Molecular Basis of Macrolide Resistance inCampylobacterStrains Isolated from Poultry in South Korea

BioMed Research International ◽

10.1155/2018/4526576 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Bai Wei ◽

Min Kang

Keyword(s):

Molecular Mechanisms ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Erythromycin Resistance ◽

23S Rrna Gene ◽

Ribosomal Protein L22 ◽

Resistant Strains ◽

Azithromycin Resistance

We investigated the molecular mechanisms underlying macrolide resistance in 38 strains ofCampylobacterisolated from poultry. Twenty-seven strains were resistant to azithromycin and erythromycin, five showed intermediate azithromycin resistance and erythromycin susceptibility, and six showed azithromycin resistance and erythromycin susceptibility. FourCampylobacter jejuniand sixCampylobacter colistrains had azithromycin MICs which were 8–16 and 2–8-fold greater than those of erythromycin, respectively. The A2075G mutation in the 23S rRNA gene was detected in 11 resistant strains with MICs ranging from 64 to ≥ 512μg/mL. Mutations including V137A, V137S, and a six-amino acid insertion (114-VAKKAP-115) in ribosomal protein L22 were detected in theC. jejunistrains. Erythromycin ribosome methylase B-erm(B) was not detected in any strain. All strains except three showed increased susceptibility to erythromycin with twofold to 256-fold MIC change in the presence of phenylalanine arginine ß-naphthylamide (PAßN); the effects of PAßN on azithromycin MICs were limited in comparison to those on erythromycin MICs, and 13 strains showed no azithromycin MIC change in the presence of PAßN. Differences between azithromycin and erythromycin resistance and macrolide resistance phenotypes and genotypes were observed even in highly resistant strains. Further studies are required to better understand macrolide resistance inCampylobacter.

Download Full-text

Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽

10.1099/mic.0.038224-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2080-2091 ◽

Cited By ~ 28

Author(s):

Anne-Laure Michon ◽

Fabien Aujoulat ◽

Laurent Roudière ◽

Olivier Soulier ◽

Isabelle Zorgniotti ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Diversity ◽

Natural Populations ◽

Variable Region ◽

Housekeeping Gene ◽

Single Copy ◽

Population Based ◽

Rrna Gene ◽

Gene Copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

Download Full-text

Methanobacterium movens sp. nov. and Methanobacterium flexile sp. nov., isolated from lake sediment

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.027540-0 ◽

2011 ◽

Vol 61 (12) ◽

pp. 2974-2978 ◽

Cited By ~ 34

Author(s):

Jinxing Zhu ◽

Xiaoli Liu ◽

Xiuzhu Dong

Keyword(s):

Phylogenetic Analysis ◽

16S Rrna ◽

16S Rrna Gene ◽

Type Strain ◽

Qaidam Basin ◽

Sequence Similarity ◽

Single Cells ◽

Rrna Gene ◽

Qinghai Province ◽

Gram Staining

Two mesophilic methanogenic strains, designated TS-2T and GHT, were isolated from sediments of Tuosu lake and Gahai lake, respectively, in the Qaidam basin, Qinghai province, China. Cells of both isolates were rods (about 0.3–0.5×2–5 µm) with blunt rounded ends and Gram-staining-positive. Strain TS-2T was motile with one or two polar flagella and used only H2/CO2 for growth and methanogenesis. Strain GHT was non-motile, used both H2/CO2 and formate and displayed a variable cell arrangement depending on the substrate: long chains when growing in formate (50 mM) or under high pressure H2 and single cells under low pressure H2. Phylogenetic analysis based on 16S rRNA gene sequences placed the two isolates in the genus Methanobacterium. Strain TS-2T was most closely related to Methanobacterium alcaliphilum NBRC 105226T (96 % 16S rRNA gene sequence similarity). Phylogenetic analysis based on the alpha subunit of methyl-coenzyme M reductase also supported the affiliation of the two isolates with the genus Methanobacterium. DNA–DNA relatedness between the isolates and M. alcaliphilum DSM 3387T was 39–53 %. Hence we propose two novel species, Methanobacterium movens sp. nov. (type strain TS-2T = AS 1.5093T = JCM 15415T) and Methanobacterium flexile sp. nov. (type strain GHT = AS 1.5092T = JCM 15416T).

Download Full-text

Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer

Genes & Development ◽

10.1101/gad.348621.121 ◽

2021 ◽

Vol 35 (19-20) ◽

pp. 1383-1394

Author(s):

Yuxiao Zhou ◽

Siyuan Xu ◽

Mo Zhang ◽

Qiang Wu

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Single Cells ◽

Functional Characterization ◽

Three Dimensional ◽

Elongation Factor ◽

Locked Nucleic Acid ◽

Loop Formation ◽

Long Distance ◽

Enhancer Region

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5′ capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA–DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

Download Full-text

641 Spatially organized multicellular immune hubs in MMRd and MMRp colorectal cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.641 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A670-A670

Author(s):

Jonathan Chen ◽

Karin Pelka ◽

Matan Hofree ◽

Marios Giannakis ◽

Genevieve Boland ◽

...

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Mismatch Repair ◽

Molecular Mechanisms ◽

Immune Cell ◽

Single Cells ◽

Large Set ◽

Colon Tissue

BackgroundImmune responses to cancer are highly variable, with DNA mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. Almost all tumors are infiltrated with immune cells, but the types of immune responses and their effects on tumor growth, metastasis and death, vary greatly between different cancers and individual tumors. Which of the numerous cell subsets in a tumor contribute to the response, how their interactions are regulated, and how they are spatially organized within tumors remains poorly understood.MethodsTo understand the rules governing these varied responses, we transcriptionally profiled 371,223 single cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd treatment-naive patients. We developed a systematic approach to discover cell types, their underlying gene programs, and cellular communities based on single cell RNA-seq (scRNAseq) profiles and applied it to study the distinguishing features of human MMRd and MMRp colorectal cancer. Cellular communities discovered from this analysis were spatially mapped in tissue sections using multiplex RNA in situ hybridization microscopy.ResultsTo understand the basis for differential immune responses in CRC, we first determined and compared the immune cell composition of MMRd and MMRp CRC and normal colon tissue, finding dramatic remodeling between tumor and normal tissue and between MMRd and MMRp tumors, particularly within the myeloid, T cell, and stromal compartments. Among the clusters enriched in MMRd tumors were activated CXCL13+ CD8 T cells. Importantly, gene program co-variation analysis revealed multicellular networks. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated IFNG+ and CXCL13+ T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines (figure 1).ConclusionsOur study provides a rich dataset of cellular states, gene programs and their transformations in tumors across a relatively large cohort of patients with colorectal cancer. Our predictions of several multicellular hubs based on co-variation of gene expression programs, and subsequent spatial localization of two major immune-malignant hubs, organizes a large set of cell states and programs into a smaller number of coordinated networks of cells and processes. Understanding the molecular mechanisms underlying these hubs, and studying their temporal and spatial regulation upon treatment will be critical for advancing cancer therapy.Ethics ApprovalThis study was approved by the DF-HCC institutional review board (protocols 03-189 and 02-240).Abstract 641 Figure 1A coordinated network of CXCL13+ T cells with myeloid and malignant cells expressing ISGs. Image shows a portion of formalin-fixed paraffin-embedded tissue from an MMRd CRC specimen stained with multiplex RNA ISH / IF for PanCK-IF, CD3E-ISH, CXCL10/CXCL11-ISH, CXCL13-ISH, and IFNG-ISH. Note IFNG+ and CXCL13+ cells in proximity to cells expressing the chemokines CXCL10/CXCL11

Download Full-text

CAMITAX: Taxon labels for microbial genomes

10.1101/532473 ◽

2019 ◽

Author(s):

Andreas Bremges ◽

Adrian Fritz ◽

Alice C. McHardy

Keyword(s):

Single Cells ◽

Ensemble Classification ◽

Rrna Gene ◽

Genome Sequences ◽

Microbial Genomes ◽

Phylogenetic Placement ◽

Gene Homology ◽

Reference Databases ◽

Taxonomic Assignments ◽

Genome Distance

The number of microbial genome sequences is growing exponentially, also thanks to recent advances in recovering complete or near-complete genomes from metagenomes and single cells. Assigning reliable taxon labels to genomes is key and often a prerequisite for downstream analyses. We introduce CAMITAX, a scalable and reproducible workflow for the taxonomic labelling of microbial genomes recovered from isolates, single cells, and metagenomes. CAMI-TAX combines genome distance-, 16S rRNA gene-, and gene homology-based taxonomic assignments with phylogenetic placement. It uses Nextflow to orchestrate reference databases and software containers, and thus combines ease of installation and use with computational re-producibility. We evaluated the method on several hundred metagenome-assembled genomes with high-quality taxonomic annotations from the TARA Oceans project, and show that the ensemble classification method in CAMITAX improved on all individual methods across tested ranks. While we initially developed CAMITAX to aid the Critical Assessment of Metagenome Interpretation (CAMI) initiative, it evolved into a comprehensive software to reliably assign taxon labels to microbial genomes. CAMITAX is available under the Apache License 2.0 at: https://github.com/CAMI-challenge/CAMITAX

Download Full-text

A new western Atlantic snapping shrimp of the Alpheus macrocheles group (Caridea, Alpheidae) revealed by morphological, molecular and color data

European Journal of Taxonomy ◽

10.5852/ejt.2019.581 ◽

2019 ◽

Author(s):

Guidomar O. Soledade ◽

Mariana Terossi ◽

Justin A. Scioli ◽

Fernando Luis Mantelatto ◽

Alexandre O. Almeida

Keyword(s):

Color Pattern ◽

Rrna Gene ◽

Undescribed Species ◽

16S Rrna Gene Sequences ◽

Morphological Comparison ◽

Western Atlantic ◽

The North ◽

Color Data ◽

Morphological Differences ◽

Northeastern Atlantic

Alpheus macrocheles (Hailstone, 1835), a species originally described from the northeastern Atlantic, has been reported from Brazil based on material from the north and northeast coasts and Espírito Santo. However, a thorough morphological comparison between Brazilian material reported as A. macrocheles and eastern Atlantic material of A. macrocheles revealed consistent differences, suggesting that the Brazilian specimens belong to an undescribed species. Alpheus ramosportoae sp. nov. is therefore now described based on material from Amapá to Pernambuco, Brazil. Morphological differences between the new species and A. macrocheles s. str. were supported by the clear divergence of 16S rRNA gene sequences (18% of genetic distance), separating the species in two distinct clades. Differences in the color pattern also were observed and illustrated.

Download Full-text

Real-time dynamics of mutagenesis reveal the chronology of DNA repair and damage tolerance responses in single cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801101115 ◽

2018 ◽

Vol 115 (28) ◽

pp. E6516-E6525 ◽

Cited By ~ 23

Author(s):

Stephan Uphoff

Keyword(s):

Dna Repair ◽

Real Time ◽

Molecular Mechanisms ◽

Damage Tolerance ◽

Single Cells ◽

Dna Alkylation ◽

Alkylation Damage ◽

Time Dynamics ◽

Dna Mismatches ◽

The Creation

Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. Toward this goal, I detected nascent DNA mismatches as a proxy for mutagenesis and simultaneously followed gene expression dynamics in single Escherichia coli cells using microfluidics. This general microscopy-based approach revealed the real-time dynamics of mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating the creation of DNA mismatches to the chronology of the underlying molecular processes. By avoiding population averaging, I discovered cell-to-cell variation in mutagenesis that correlated with heterogeneity in the expression of alternative responses to DNA damage. Pulses of mutagenesis are shown to arise from transient DNA repair deficiency. Constitutive expression of DNA repair pathways and induction of damage tolerance by the SOS response compensate for delays in the activation of inducible DNA repair mechanisms, together providing robustness against the toxic and mutagenic effects of DNA alkylation damage.

Download Full-text