ABSTRACT
MOTIVATION: The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations of 50 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to the absence of clear reference genomes and the presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing all metagenomic samples in a series (time or other metric) into a single co-assembly graph. The log fold change in graph coverage between successive samples is then calculated to call SVs that are thriving or declining. RESULTS: We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, particularly as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between successive time and temperature samples, suggesting host advantage. Our approach leverages previous work in assembly graph structural and coverage patterns to provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial gene flux. AVAILABILITY AND IMPLEMENTATION: rhea is open source and available at: https://github.com/treangenlab/rhea.
Subject(s)
Genome, Bacterial , Metagenome , Microbiota , Microbiota/genetics , Metagenomics/methods , Gene Transfer, Horizontal , Bacteria/genetics , AlgorithmsABSTRACT
Bacterial genome dynamics are vital for understanding the mechanisms underlying microbial adaptation, growth, and their broader impact on host phenotype. Structural variants (SVs), genomic alterations of 10 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to absence of clear reference genomes and presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing a single metagenome coassembly graph constructed from all samples in a series. The log fold change in graph coverage between subsequent samples is then calculated to call SVs that are thriving or declining throughout the series. We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, which is particularly noticeable as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between subsequent time and temperature samples, suggesting host advantage. Our innovative approach leverages raw read patterns rather than references or MAGs to include all sequencing reads in analysis, and thus provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial genome dynamics.
ABSTRACT
Chloroflexus sp. MS-CIW-1 was isolated from a phototrophic mat in Mushroom Spring, an alkaline hot spring in Yellowstone National Park, WY, USA. We report the draft genome of 4.8 Mb consisting of 6 contigs with 3755 protein-coding genes and a GC content of 54.45%.
ABSTRACT
Bacterial species often undergo rampant recombination yet maintain cohesive genomic identity. Ecological differences can generate recombination barriers between species and sustain genomic clusters in the short term. But can these forces prevent genomic mixing during long-term coevolution? Cyanobacteria in Yellowstone hot springs comprise several diverse species that have coevolved for hundreds of thousands of years, providing a rare natural experiment. By analyzing more than 300 single-cell genomes, we show that despite each species forming a distinct genomic cluster, much of the diversity within species is the result of hybridization driven by selection, which has mixed their ancestral genotypes. This widespread mixing is contrary to the prevailing view that ecological barriers can maintain cohesive bacterial species and highlights the importance of hybridization as a source of genomic diversity.
ABSTRACT
High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.
Subject(s)
Bacteriophages , RNA Viruses , Bacteriophages/genetics , DNA-Directed RNA Polymerases/genetics , Genome, Viral , Phylogeny , RNA , RNA Viruses/genetics , RNA-Dependent RNA Polymerase/genetics , ViromeABSTRACT
The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the host cell and the engulfed prokaryote became genetically codependent, with the integration of genes from the endosymbiont into the host nuclear genome and subsequent gene loss from the endosymbiont. This process required that horizontally transferred genes become active and properly regulated despite inherent differences in genetic features between donor (endosymbiont) and recipient (host). Although this genetic reorganization is considered critical for early stages of organellogenesis, we have little knowledge about the mechanisms governing this process. The photosynthetic amoeba Paulinella micropora offers a unique opportunity to study early evolutionary events associated with organellogenesis and primary endosymbiosis. This amoeba harbors a "chromatophore," a nascent photosynthetic organelle derived from a relatively recent cyanobacterial association (â¼120 million years ago) that is independent of the evolution of primary plastids in plants (initiated â¼1.5 billion years ago). Analysis of the genome and transcriptome of Paulinella revealed that retrotransposition of endosymbiont-derived nuclear genes was critical for their domestication in the host. These retrocopied genes involved in photoprotection in cyanobacteria became expanded gene families and were "rewired," acquiring light-responsive regulatory elements that function in the host. The establishment of host control of endosymbiont-derived genes likely enabled the cell to withstand photo-oxidative stress generated by oxygenic photosynthesis in the nascent organelle. These results provide insights into the genetic mechanisms and evolutionary pressures that facilitated the metabolic integration of the hostendosymbiont association and sustained the evolution of a photosynthetic organelle.
Subject(s)
Amoeba , Biological Evolution , Rhizaria , Symbiosis , Amoeba/genetics , Eukaryota/genetics , Plastids/genetics , Symbiosis/geneticsABSTRACT
Phototrophic biofilms in most environments experience major changes in light levels throughout a diel cycle. Phototaxis can be a useful strategy for optimizing light exposure under these conditions, but little is known about its role in cyanobacteria from thermal springs. We examined two closely related Synechococcus isolates (Synechococcus OS-A dominates at 60 to 65°C and OS-B' at 50 to 55°C) from outflows of Octopus Spring in Yellowstone National Park. Both isolates exhibited phototaxis and photokinesis in white light, but with differences in speed and motility bias. OS-B' exhibited phototaxis toward UVA, blue, green, and red wavelengths, while OS-A primarily exhibited phototaxis toward red and green. OS-A also exhibited negative phototaxis under certain conditions. The repertoires of photoreceptors and signal transduction elements in both isolates were quite different from those characterized in other unicellular cyanobacteria. These differences in the photoresponses between OS-A and OS-B' in conjunction with in situ observations indicate that phototactic strategies may be quite versatile and finely tuned to the light and local environment. IMPORTANCE Optimizing light absorption is of paramount importance to photosynthetic organisms. Some photosynthetic microbes have evolved a sophisticated process called phototaxis to move toward or away from a light source. In many hot springs in Yellowstone National Park, cyanobacteria thrive in thick, laminated biofilms or microbial mats, where small movements can result in large changes in light exposure. We quantified the light-dependent motility behaviors in isolates representing two of the most abundant and closely related cyanobacterial species from these springs. We found that they exhibited unexpected differences in their speed, directionality, and responses to different intensities or qualities of light. An examination of their genomes revealed several variations from well-studied phototaxis-related genes. Studying these recently isolated cyanobacteria reveals that diverse phototactic strategies can exist even among close relatives in the same environment. It also provides insights into the importance of phototaxis for growth and survival in microbial biofilm communities.
Subject(s)
Hot Springs , Synechococcus , Biofilms , Hot Springs/microbiology , Photosynthesis , Phototaxis/physiology , Synechococcus/geneticsABSTRACT
Cyanobacteria rely on photosynthesis, and thus have evolved complex responses to light. These include phototaxis, the ability of cells to sense light direction and move towards or away from it. Analysis of mutants has demonstrated that phototaxis requires the coordination of multiple photoreceptors and signal transduction networks. The output of these networks is relayed to type IV pili (T4P) that attach to and exert forces on surfaces or other neighboring cells to drive "twitching" or "gliding" motility. This, along with the extrusion of polysaccharides or "slime" by cells, facilitates the emergence of group behavior. We evaluate recent models that describe the emergence of collective colony-scale behavior from the responses of individual, interacting cells. We highlight the advantages of "active matter" approaches in the study of bacterial communities, discussing key differences between emergent behavior in cyanobacterial phototaxis and similar behavior in chemotaxis or quorum sensing.
Subject(s)
Phototaxis , Synechocystis/physiology , Synechocystis/radiation effects , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chemotaxis , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/physiology , Fimbriae, Bacterial/radiation effects , Light , Mutation , Quorum Sensing , Synechocystis/geneticsABSTRACT
Cyanobacteria have an inherited advantage in phosphonate phytoremediation. However, studies on phosphonate metabolism in cyanobacteria are rare and mostly focus on physiology and ecology. Here, C-P lyase gene cluster regulation in an undomesticated thermophilic Synechococcus OS-B' was examined in Synechocystis sp. PCC6803, a unicellular cyanobacterial model. Phylogenetic and cluster synteny analysis of C-P lyase genes revealed a closer relationship between Syn OS-B' and Thermus thermophilus, than with other cyanobacteria. Pho boxes were identified in the 5'-end-flanking region of the C-P lyase gene cluster, through which the downstream gene expression was regulated in a phosphate concentration-dependent manner. Unexpectedly, the phosphate concentration that thoroughly inhibited Pho boxes was almost two orders of magnitude higher than that of any natural or anthropogenic wastewater reported so far. The Pho boxes mediated regulation was achieved through the Pho regulon two-component system, and the absence of either SphS or SphR ablated the cell's ability to sense ambient phosphate changes. The three tandems of Pho boxes maintained inequivalent roles, of which the third tandem was not essential; however, it played a role in adjusting Pho boxes response in both positive and negative manner under phosphorus limitation.
Subject(s)
Operon , Synechocystis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Lyases , Operon/genetics , Phosphates/metabolism , Phylogeny , Regulon/genetics , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
Protein quality control pathways require AAA+ proteases, such as Clp and Lon. Lon protease maintains UmuD, an important component of the error-prone DNA repair polymerase (Pol V), at very low levels in E. coli. Most members of the phylum Cyanobacteria lack Lon (including the model cyanobacterium, Synechocystis sp. PCC6803), so maintenance of UmuD at low levels must employ different proteases. We demonstrate that the first 19 residues from the N-terminus of UmuD (Sug1-19 ) fused to a reporter protein are adequate to trigger complete proteolysis and that mutation of a single leucine residue (L6) to aspartic acid inhibits proteolysis. This process appears to follow the N-end rule and is mediated by ClpA/P protease and the ClpS adaptor. Additionally, mutations of arginine residues in the Sug1-19 tag suggest that the ClpX/P pathway also plays a role in proteolysis. We propose that there is a dual degron at the N-terminus of the UmuD protein in Synechocystis sp. PCC6803, which is distinct from the degron required for degradation of UmuD in E. coli. The use of two proteolysis pathways to tune levels of UmuD might reflect how a photosynthetic organism responds to multiple environmental stressors.
Subject(s)
Bacterial Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Synechocystis/enzymology , Amino Acid Motifs , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA Repair , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Mutation , Proteolysis , Synechocystis/chemistry , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
Polyphosphate (polyP), a polymer of orthophosphate (PO4 3-) of varying lengths, has been identified in all kingdoms of life. It can serve as a source of chemical bond energy (phosphoanhydride bond) that may have been used by biological systems prior to the evolution of ATP. Intracellular polyP is mainly stored as granules in specific vacuoles called acidocalcisomes, and its synthesis and accumulation appear to impact a myriad of cellular functions. It serves as a reservoir for inorganic PO4 3- and an energy source for fueling cellular metabolism, participates in maintaining adenylate and metal cation homeostasis, functions as a scaffold for sequestering cations, exhibits chaperone function, covalently binds to proteins to modify their activity, and enables normal acclimation of cells to stress conditions. PolyP also appears to have a role in symbiotic and parasitic associations, and in higher eukaryotes, low polyP levels seem to impact cancerous proliferation, apoptosis, procoagulant and proinflammatory responses and cause defects in TOR signaling. In this review, we discuss the metabolism, storage, and function of polyP in photosynthetic microbes, which mostly includes research on green algae and cyanobacteria. We focus on factors that impact polyP synthesis, specific enzymes required for its synthesis and degradation, sequestration of polyP in acidocalcisomes, its role in cellular energetics, acclimation processes, and metal homeostasis, and then transition to its potential applications for bioremediation and medical purposes.
ABSTRACT
To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc1O and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.
Subject(s)
Bacteriophage T7/genetics , DNA-Directed RNA Polymerases/genetics , Promoter Regions, Genetic/genetics , Synechocystis/genetics , Viral Proteins/genetics , Escherichia coli/geneticsABSTRACT
Despite extensive DNA sequencing data derived from natural microbial communities, it remains a major challenge to identify the key evolutionary and ecological forces that shape microbial populations. We have focused on the extensive microdiversity of the cyanobacterium Synechococcus sp., which is a dominant member of the dense phototrophic biofilms in the hot springs of Yellowstone National Park. From deep amplicon sequencing of many loci and statistical analyses of these data, we showed previously that the population has undergone an unexpectedly high degree of homologous recombination, unlinking synonymous SNP-pair correlations even on intragenic length scales. Here, we analyze the genic amino acid diversity, which provides new evidence of selection and insights into the evolutionary history of the population. Surprisingly, some features of the data, including the spectrum of distances between genic-alleles, appear consistent with primarily asexual neutral drift. Yet the non-synonymous site frequency spectrum has too large an excess of low-frequency polymorphisms to result from negative selection on deleterious mutations given the distribution of coalescent times that we infer. And our previous analyses showed that the population is not asexual. Taken together, these apparently contradictory data suggest that selection, epistasis, and hitchhiking all play essential roles in generating and stabilizing the diversity. We discuss these as well as potential roles of ecological niches at genomic and genic levels. From quantitative properties of the diversity and comparative genomic data, we infer aspects of the history and inter-spring dispersal of the meta-population since it was established in the Yellowstone Caldera. Our investigations illustrate the need for combining multiple types of sequencing data and quantitative statistical analyses to develop an understanding of microdiversity in natural microbial populations.
Subject(s)
Biological Evolution , Cyanobacteria/genetics , Epistasis, Genetic/genetics , Selection, Genetic/genetics , Alleles , Ecology , Ecosystem , Genetic Variation/genetics , Phylogeny , Polymorphism, Single Nucleotide/genetics , Recombination, GeneticABSTRACT
To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.
ABSTRACT
Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium-Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.
Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , RNA-Directed DNA Polymerase/genetics , Spirulina/genetics , CRISPR-Associated Proteins/genetics , Gene Transfer, Horizontal , Genes, Bacterial , Phylogeny , RNAABSTRACT
Environmental cues can stimulate a variety of single-cell responses, as well as collective behaviors that emerge within a bacterial community. These responses require signal integration and transduction, which can occur on a variety of time scales and often involve feedback between processes, for example, between growth and motility. Here, we investigate the dynamics of responses of the phototactic, unicellular cyanobacterium Synechocystis sp. PCC6803 to complex light inputs that simulate the natural environments that cells typically encounter. We quantified single-cell motility characteristics in response to light of different wavelengths and intensities. We found that red and green light primarily affected motility bias rather than speed, while blue light inhibited motility altogether. When light signals were simultaneously presented from different directions, cells exhibited phototaxis along the vector sum of the light directions, indicating that cells can sense and combine multiple signals into an integrated motility response. Under a combination of antagonistic light signal regimes (phototaxis-promoting green light and phototaxis-inhibiting blue light), the ensuing bias was continuously tuned by competition between the wavelengths, and the community response was dependent on both bias and cell growth. The phototactic dynamics upon a rapid light shift revealed a wavelength dependence on the time scales of photoreceptor activation/deactivation. Thus, Synechocystis cells achieve exquisite integration of light inputs at the cellular scale through continuous tuning of motility, and the pattern of collective behavior depends on single-cell motility and population growth.IMPORTANCE The photosynthetic cyanobacterium Synechocystis sp. exhibits phototaxis that is dependent on the incident light wavelength through the action of various photoreceptors. In natural environments, cells experience a set of highly dynamic and complex light inputs, yet how cells transduce multiple or dynamic inputs into motion is unknown. In this study, we measured the phototactic behaviors of single cells and communities as a function of light intensity or when illuminated by combinations of lights of different wavelengths or incidence directions. Responses to a spectrum of light regimes revealed that Synechocystis sp. integrates information about the light environment to tune its phototactic response, which is likely generated by competition among photoreceptors and the degree of wavelength-regulated growth to sensitively control the direction and degree of movement.
Subject(s)
Light , Phototaxis , Synechocystis/physiology , Synechocystis/radiation effects , Photoreceptors, Microbial , Signal TransductionABSTRACT
BACKGROUND: Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics. RESULTS: To overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the "sizer" rule, where division timing is based on cell size, or the "timer" rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the "adder" rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control. CONCLUSIONS: Our analyses revealed that the "adder" model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth kinetics of individual cells we can build testable models of circadian control of the cell cycle in cyanobacteria.
Subject(s)
Cell Division , Cell Tracking/methods , Microfluidics/methods , Synechocystis/cytology , Cell Division/radiation effects , Cell Proliferation , Image Processing, Computer-Assisted , Light , Models, Biological , Probability , Synechocystis/growth & development , Synechocystis/radiation effects , Time FactorsABSTRACT
The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, Synechococcus sp. JA-2-3B'a(2-13), Synechococcus sp. JA-3-3Ab, Chloroflexus sp. Y-400-fl, and Roseiflexus sp. RS-1, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Endopeptidases/genetics , Metagenome , Metagenomics , Microbiota , Bacteriophages/classification , Bacteriophages/genetics , Biofilms , Catalytic Domain/genetics , Computational Biology/methods , Environmental Microbiology , Gene Targeting , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions , Metagenomics/methods , Molecular Sequence Annotation , Open Reading Frames , Phylogeny , Sequence Analysis, DNAABSTRACT
Certain cyanobacteria look green if grown in red light and vice versa. This dramatic color change, called complementary chromatic adaptation (CCA), is caused by alterations of the major colored light-harvesting proteins. A major controller of CCA is the cyanobacteriochrome (CBCR) RcaE, a red-green reversible photoreceptor that triggers a complex signal transduction pathway. Now, a new study demonstrates that CCA is also modulated by DpxA, a CBCR that senses yellow and teal (greenish blue) light. DpxA acts to expand the range of wavelengths that can impact CCA, by fine-tuning the process. This dual control of CCA might positively impact the fitness of cells growing in the shade of competing algae or in a water column where light levels and spectral quality change gradually with depth. This discovery adds to the growing number of light-responsive phenomena controlled by multiple CBCRs. Furthermore, the diverse CBCRs which are exclusively found in cyanobacteria have significant biotechnological potential.
Subject(s)
Bacterial Proteins/metabolism , Cyanobacteria/cytology , Light , Photoreceptors, Microbial , Bacterial Proteins/genetics , Color , Cyanobacteria/genetics , Cyanobacteria/metabolism , Signal Transduction/radiation effectsABSTRACT
CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.