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Long tandemly repeated repetitive (LTRR) sequences in the filamentous cyanobacterium Anabaena sp PCC 7120
Abstract
Nucleotide sequence analysis of the DNA region carrying transposon Tn5-1087b from the Anabaena 7120 nitrogen fixation-deficient mutant YC16 revealed the presence of a novel repeated DNA element in cyanobacteria designated long tandemly repeated repetitive (LTRR) sequence. The LTRR element is 37 bp long and contains an inverted repeat sequence. 17 copies of the LTRR element, 13 of which were completely identical, were identified within a 1.3 kb DNA fragment, which was flanked by two divergently transcribed genes homologous to bacteriophage T4 'gene 15' and Rhizobium meliloti exoD, respectively. LTRR-like sequences occur in several DNA regions in Anabaena 7120 and in other cyanobacteria. Furthermore, the presence of an LTRR-like DNA region in mitochondrial plasmids of Vicia faba indicates strong conservation of such structures during evolution.
... Analyses of molecular polymorphisms in a set of Anabaena strains were done using STRR, LTRR and Hip1 primers (Prasanna et al., 2006;Nayak et al., 2009). The LTRR sequences were used for the specific fingerprinting of individual cyanobacterial isolates of Anabaena strains by Masepohl et al. (1996). DNA amplification finger-printing (DAF), a PCR-based method using short oligonucleotides for production of characteristic banding patterns, was used by Eskew et al. (1993) for taxonomic studies of Anabaena azollae strains. ...
... The trichome morphology (straight or coiled) of Cylindrospermopsis strains was shown to be correlated with STRR profiles in the study by Wilson et al. (2000). STRR sequences might be the target of specific DNA-binding proteins involved in replication during heterocyst differentiation (Lupski and Weinstock, 1992;Masepohl et al., 1996). Rasmussen and Svenning (1998) used STRR1 to fingerprint symbiotic cyanobacterial isolates from the angiosperm Gunnera to show the high genetic diversity and distinct clus- tering of symbiotic Nostoc isolates. ...
... Only A. fertilissima and A. ambigua were out-grouped and were not consistent with the morphological classification. Masepohl et al. (1996) identified a 37-bp LTRR sequence in Anabaena strain PCC 7120. Some differences were obtained among symbiotic cyanobacterial isolates from the angiosperm Gunnera using LTRR primers (Rasmussen and Svenning, 1998). ...
In this study, ten species of Anabaena were used to test the congruence between the traditional morphological classification system and the present molecular classification system. For morphological classification, strains were categorized into two different groups based on the whether or not the akinetes were directly adjacent to heterocysts in the trichome. Genetic diversity was assessed using the banding patterns of repetitive DNA sequences including the short tandemly repeated repetitive (STRR) sequences and long tandemly repeated repetitive (LTRR) sequences that are present in the cyanobacterial genome. The phylogenetic relationships inferred from comparison of the STRR sequences generally supported the traditional classification of cyanobacteria based on morphological criteria. The dendrograms based on the LTRR sequences did not show a clear correlation with the dendrogram based on morphology.
... The first description of CRISPR loci emerged in 1987, when Nakata et al. identified an interesting locus downstream to the IAP gene (encoding alkaline phosphatase isozyme) of E. coli that contained roughly palindromic repeated sequences (direct repeat of 29 bp) interspaced by variable sequences. 23 In subsequent years, with the rapid advancement in genome sequencing, researchers started identifying similar sequences in bacteria (Mycobacterium tuberculosis, 23,24 Sulfolobus acidocaldarius, 25 Cyanobacteria, 26,27 Clostridium difficile, 24 and Lactobacillus acidophilus 28 ) and archaea. 24 These frequent clusters of repeated sequences 29 were identified independently many times by independent lab groups and given many different names such as DVR (direct variable repeats), 23 LTRR (long tandemly repeated repetitive sequences), 26,27 SRSR (short regularly spaced repeats), 25 LCTR (large clusters of tandem repeats), 30 and SPIDR (spacer interspersed direct repeats). ...
... 23 In subsequent years, with the rapid advancement in genome sequencing, researchers started identifying similar sequences in bacteria (Mycobacterium tuberculosis, 23,24 Sulfolobus acidocaldarius, 25 Cyanobacteria, 26,27 Clostridium difficile, 24 and Lactobacillus acidophilus 28 ) and archaea. 24 These frequent clusters of repeated sequences 29 were identified independently many times by independent lab groups and given many different names such as DVR (direct variable repeats), 23 LTRR (long tandemly repeated repetitive sequences), 26,27 SRSR (short regularly spaced repeats), 25 LCTR (large clusters of tandem repeats), 30 and SPIDR (spacer interspersed direct repeats). 28 The name CRISPR as "clustered regularly interspaced short palindromic repeat'' 17 was first coined by Jansen et al. in 2002. ...
The CRISPR/Cas9 system is a popular genome-editing tool with immense therapeutic potential. It is a simple two-component system (Cas9 protein and RNA) that recognizes the DNA sequence on the basis of RNA:DNA complementarity, and the Cas9 protein catalyzes the double-stranded break in the DNA. In the past decade, near-atomic resolution structures at various stages of the CRISPR/Cas9 DNA editing pathway have been reported along with numerous experimental and computational studies. Such studies have boosted knowledge of the genome-editing mechanism. Despite such advancements, the application of CRISPR/Cas9 in therapeutics is still limited, primarily due to off-target effects. Several studies aim at engineering high-fidelity Cas9 to minimize the off-target effects. Molecular Dynamics (MD) simulations have been an excellent complement to the experimental studies for investigating the mechanism of CRISPR/Cas9 editing in terms of structure, thermodynamics, and kinetics. MD-based studies have uncovered several important molecular aspects of Cas9, such as nucleotide binding, catalytic mechanism, and off-target effects. In this Review, the contribution of MD simulation to understand the CRISPR/Cas9 mechanism has been discussed, preceded by an overview of the history, mechanism, and structural aspects of the CRISPR/Cas9 system. These studies are important for the rational design of highly specific Cas9 and will also be extremely promising for achieving more accurate genome editing in the future.
... The LTRR sequence has also been identified in some cyanobacterial species (Valerio et al. 2009), for the first time in Anabaena sp. (PCC 7120) (Masepohl et al. 1996). The size of this fragment is 37 bp and could be present in the genome in multiple copies (Rasmussen & Svenning 1998). ...
Cyanobacteria are a lineage of Eubacteria that have long captured the attention of scientists. Approximately
5310 species of cyanobacteria have been hitherto described and new species are continually
being found, named and described according to established rules. The correct determination of
cyanobacteria strains concerns new biotechnological applications as well as ecological studies. There
are many situations where it is crucial to recognise distinct algae species, however methods for doing
so vary greatly. The aim of this review is to summarize the state of the art of the main and most recent
molecular studies focusing on the phylum Cyanobacteria, with particular attention to the most frequently
used gene markers. For a long time, the classification method used for cyanobacteria as well
as traditionally described species was mainly based on morphology. Over time, integrative taxonomy,
which involves the inclusion of many characters and comprehensive taxa sampling, has become the
rule as it provides a better resolution of species relationships. For a better resolution of the phylogenies
of the phylum Cyanobacteria, it is usually necessary to focus on different genetic markers: from the
most common, like the 16S and 23S rRNA, ITS, rbcLXS and rpoC genes, to genes not so widely used,
such as hetR, psbA, tufA, gyp and cpcBA. Also, the highly repetitive sequences often used for the symbiotic
cyanobacteria represent an important factor in the inference of the phylogenetic relationships.
... CRISPR-clustered regularly interspaced short palindromic repeats-were first discovered in the 3 -end flanking region of the iap gene in E. coli in 1987 [1] and later found in many other bacteria and even archaea [2][3][4]. These repeats and their associated proteins constitute an adaptive immune system to protect host microbes from invasion by foreign genetic elements, such as viruses, through targeted cleavage of the nucleic acids with RNA-guided endonuclease [5][6][7][8]. ...
Cystic fibrosis (CF) is a monogenic recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator gene (CFTR). Remarkable progress in basic research has led to the discovery of highly effective CFTR modulators. Now ~90% of CF patients are treatable. However, these modulator therapies are not curative and do not cover the full spectrum of CFTR mutations. Thus, there is a continued need to develop a complete and durable therapy that can treat all CF patients once and for all. As CF is a genetic disease, the ultimate therapy would be in-situ repair of the genetic lesions in the genome. Within the past few years, new technologies, such as CRISPR/Cas gene editing, have emerged as an appealing platform to revise the genome, ushering in a new era of genetic therapy. This review provided an update on this rapidly evolving field and the status of adapting the technology for CF therapy.
... Over the next decade, this particular repeat sequence was detected in a variety of bacteria and archaea. [35][36][37][38][39] In 2002, Janson et al. provided a generalized summary of the specific repeats that have been identified, naming these repeats as a family and using the acronym CRISPR for clustered regularly interspaced short palindromic repeats. 40 In addition, multiple CRISPR-associated proteins (Cas)-Cas1 to Cas4-have been revealed in previous studies. ...
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications.
... In their study, a group of researchers observed that approximately 70% of CRISPR transformed cells were able to segregate without using selection markers (Behler et al. 2018). Nonetheless, the CRISPR-Cas system has substantially impacted the field of cyanobacterial biotechnology by precisely engineering its genome and improving its growth and biomass under stress conditions (Masepohl et al. 1996; Barrangou and Horvath 2017). ...
Cyanobacteria are ubiquitous microorganisms that play a significant role in the maintenance of the earth’s ecology. Owing to the smaller and completely sequenced genome, some strains have emerged as appropriate candidates for manipulating their genetic sequences to enhance growth and photosynthesis under distinct environmental fluctuations. Synthetic biology tools have arisen as an indispensable means for scaling up the natural circadian rhythm of prokaryotes and eukaryotes, thus improving the physiological and metabolic processes to promote their growth under adverse environmental conditions. Although the availability of synthetic biology tools for engineering multiple pathways in cyanobacteria is still limited, in the past few years significant progress has been made in developing genetic tools including promoters, sRNA, RBS, riboswitches and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas-9 systems for engineering cyanobacteria with improved biomass production and product development. Systematic rewiring of physiological, biochemical and molecular pathways may significantly improve the growth and production of engineered cyanobacteria under stressful environments. In this chapter, recent advancement in synthetic biology tools and their application in cyanobacteria for sustainable biotechnologies is reviewed. Furthermore, it also provides valuable insights into their future developments.KeywordsCyanobacteriaSynthetic biology toolsRBSRiboswitchesCRISPR/Cas-9
... Currently, CRISPR repeats have been found in most archaeal genomes and nearly half of the studied bacterial ones, but they have not been found in eukaryotic or viral DNA sequences. The existence of CRISPR repeats in mitochondria was suggested in one of the earliest publications on the subject (the same article described CRISPR in cyanobacteria for the first time) [19]. The authors used a set of previously published data on the sequencing of mitochondrial plasmids from Vicia faba L. beans [20], and their conclusions were further cited by Mojica et al. [21], but these observations were not confirmed in later studies [8]. ...
The development of a method for genome editing based on CRISPR-Cas9 technology was awarded The Nobel Prize in Chemistry in 2020, less than a decade after the discovery of all principal molecular components of the system. For the first time in history a Nobel prize was awarded to two women, Emmanuelle Charpentier and Jennifer Doudna, who made key discoveries in the field of DNA manipulation with the CRISPR-Cas9 system, so-called "genetic scissors". It is difficult to overestimate the importance of the technique as it enables one not only to manipulate genomes of model organisms in scientific experiments, and modify characteristics of important crops and animals, but also has the potential of introducing revolutionary changes in medicine, especially in treatment of genetic diseases. The original biological function of CRISPR-Cas9 system is the protection of prokaryotes from mobile genetic elements, in particular viruses. Currently, CRISPR-Cas9 and related technologies have been successfully used to cure life-threatening diseases, make coronavirus detection tests, and even to modify human embryo cells with the consequent birth of babies carrying the introduced modifications. This intervention with human germplasm cells resulted in wide disapproval in the scientific community due to ethical concerns, and calls for a moratorium on inheritable genomic manipulations. This review focuses on the history of the discovery of the CRISPR-Cas9 system with some aspects of its current applications, including ethical concerns about its use in humans.
... Примерно в это же время похожие повторы были описаны в геномах Mycobacterium tuberculosis (Groenen et al., 1993), стрептококка (Hoe et al., 1999), цианобактерии Anabaena sp. (Masepohl et al., 1996), Shigella dysenteriae, Salmonella typhimurium (Nakata et al., 1989) и других видов бактерий. Высказывались предположения, что данные повторы могут участвовать в хромосомных перестройках, рекомбинации или являются местами посадки белков, регулирующих соседствующие с повторами гены (Nakata et al., 1989;Groenen et al., 1993), однако экспериментально эти предположения не проверялись. ...
В настоящем обзоре обсуждаются ключевые этапы развития технологии редактирования геномов CRISPR/Cas от истории открытия до современных разработок в различных областях, включая применение данной технологии в медицине. Рассматриваются также технические и этические проблемы, связанные с использованием CRISPR/Cas для редактирования геномов эмбрионов человека.
... Aufgrund der charakteristischen Struktur wurde der Lokus als clustered regularly interspaced short palindromic repeats (CRISPR) bezeichnet. Durch weitere Forschung konnten in den darauffolgenden Jahren homologe Sequenzen in weiteren Bakterien und Archaeen entdeckt werden (Groenen et al., 1993, Mojica et al., 1995, Masepohl et al., 1996. Im Jahre 2005 kamen mehrere Forschergruppen zu der Erkenntnis, dass die Spacer-Sequenzen Homologien zu DNA-Sequenzen aus dem Genom von Viren und Plasmiden aufweisen (Bolotin et al., 2005, Mojica et al., 2005, Pourcel et al., 2005. ...
Die Entwicklung des Schädeldachs beginnt beim Menschen bereits in der frühen Embryogenese und ist erst im Erwachsenenalter abgeschlossen. Das Wachstum der Schädelknochen muss sich während der Entwicklung fortwährend dem Gehirnwachstum anpassen. An den Stellen, wo zwei Schädelknochen aufeinandertreffen, formen sich Schädelnähte, die aus mesenchymalem Bindegewebe bestehen und als Wachstumsfugen des Schädels dienen. Tritt eine frühzeitige Verknöcherung innerhalb einer oder mehrerer Schädelnähte auf, spricht man von einer Kraniosynostose. Als Konsequenz wird ein weiteres Knochenwachstum verhindert, sodass sich das Neurokranium in dieser Region nicht dem expansiven Wachstum des Gehirns anpassen kann. Dies geht in der Regel mit einem kompensatorischen Wachstum des Schädels und infolgedessen mit kraniofazialen Dysmorphien und einem erhöhten intrakraniellen Druck einher. Klinische Studien und Forschungen an Modellorganismen konnten bereits eine Vielzahl an Genen mit der Entstehung von Kraniosynostosen assoziieren, darunter die Transkriptionsfaktoren TCF12 und TWIST1. Beim Menschen sind heterozygote Mutationen in TCF12 und TWIST1 mit Kraniosynostosen der Koronarnaht assoziiert. Bei Mäusen hingegen führt eine heterozygote Tcf12 Mutation nur in Kombination mit einer heterozygoten Twist1 Mutation zu Fusionen der Koronarnaht. Der Zebrabärbling (Danio rerio, überwiegend auch Zebrafisch genannt) weist eine bemerkenswerte Ähnlichkeit bezüglich der Anatomie und Morphologie des Schädeldachs zum Menschen auf. Um die genaue Funktion von TCF12 bei der Ausbildung der Schädelnähte zu untersuchen, wurde im Rahmen dieser Arbeit der Zebrafisch als in vivo Modell für die Entstehung tcf12-induzierter Kraniosynostosen etabliert. Zu Beginn der Arbeit wurde das Expressionsmuster von tcf12 über die Entwicklung hinweg analysiert. Ein besonderer Fokus lag dabei auf einem Expressionsnachweis während der Entwicklung der Schädelplatten und der Schädelnähte. Ein erster Expressionsnachweis von tcf12 mittels PCR-Analysen und Whole-mount RNA in-situ Hybridisierungen zeigte eine breite Expression von tcf12 ab dem 1-3 Somiten Stadium an. Für tiefergehende in vivo Analysen wurden im Zuge dieser Arbeit tcf12:EGFP Reportergenlinien generiert. Mit diesen gelang ein Nachweis der tcf12 Expression entlang der Wachstumsfronten der Schädelplatten, innerhalb der Schädelnähte sowie im Periost und der Dura mater. Mit den tcf12:EGFP Fischen als Referenz wurde in weiterführenden Experimenten die Aktivität drei hochkonservierter CNEs (engl. conserved non-coding elements) in vivo im Zebrafisch untersucht. Zwei der CNEs konnten als tcf12 Enhancer verifiziert werden, die eine Genexpression während der Neurogenese des zentralen Nervensystems (ZNS) steuern. Die beiden Enhancer-Elemente zeichnen sich durch eine hohe Konservierung vom Menschen bis hin zum Zebrafisch aus. Aufgrund der unterschiedlichen Sensitivität gegenüber einem Funktionsverlust von TCF12 und TWIST1 in Mensch und Maus sollte die Auswirkung eines Knockouts der orthologen Gene auf die Entwicklung der Schädelnähte des Zebrafisches untersucht werden. Mittels CRISPR/Cas9 wurden verschiedene Knockout-Linien für die Gene tcf12, twist1a und twist1b generiert. Analysen der Knockoutmutanten zeigten, dass ein heterozygoter Verlust von tcf12 und twist1b in seltenen Fällen zu partiellen Fusionen der Koronarnähte im Zebrafisch führt. Des Weiteren konnte bei tcf12 und twist1b Einzel- und Doppelmutanten ein abnormes Wachstum der Schädelplatten im Bereich der Suturen beobachtet werden. Die Expressionsstudien und die Analysen der Knockoutmutanten deuten auf eine Regulation von TCF12 bei der Differenzierung der Stammzellen sowie der Proliferation der Osteoblasten innerhalb der Schädelnähte hin. Um die Auswirkung von TCF12 Mutationen auf funktioneller Ebene zu untersuchen wurden im Verlauf dieser Arbeit Luciferase-Reporter Assays durchgeführt. Anhand dieser konnte nachgewiesen werden, dass Mutationen, die die basic helix-loop-helix (bHLH)-Domäne beeinträchtigen, die Transaktivierungsfähigkeit von TCF12 aufheben. Co-Transfektions-Experimente mit TWIST1 offenbarten eine Regulation der Transaktivierung von TCF12 durch TWIST1, sowohl im Menschen, als auch im Zebrafisch. Im Rahmen dieser Arbeit konnten die genauen Expressionsorte von TCF12 während der Morphogenese des Schädeldachs nachgwiesen und die Funktion von TCF12 und seinem Interaktionspartner TWIST1 bei der Entstehung von Kraniosynostosen weiter aufgeklärt werden.
... The following section describes the components of CRISPR/Cas9 systems in brief. Ishino et al. (1987) Discovery of the presence of clustered repeats in E. coli 2. Nakata et al. (1989) Specifc study of the clustered repeats in enterobacteria 3. Groenen et al. (1993) Use of CRISPR for M. tuberculosis typing 4. Mojica et al. (1995) Identifcation of CRISPRs in Haloferax 5. Masepohl et al. (1996) Description of CRISPRs in cyanobacteria 6. Hoe et al. (1999) Use of CRISPR for S. pyogenes rapid genetic subtyping 7. Mojica et al. (2000) Widespread occurrence of CRISPRs in archaea and bacteria 8. Makarova et al. (2002) DNA repair hypothesis for Cas genes 9. Jansen et al. (2002a,b) Term 'CRISPR' coined; description of Cas genes 10. Pourcel et al. (2005), Mojica et al. (2005), Spacer sequences are probably snippets from foreign genome and Bolotin et al. (2005) 11. Makarova et al. (2006) Putative RNAi-based mechanism proposed 12. Barrangou et al. (2007) Establishment of CRISPR/Cas9 as adaptive immune system of S. thermophilus (a bacteria) against viruses 13. ...
... The following section describes the components of CRISPR/Cas9 systems in brief. Ishino et al. (1987) Discovery of the presence of clustered repeats in E. coli 2. Nakata et al. (1989) Specifc study of the clustered repeats in enterobacteria 3. Groenen et al. (1993) Use of CRISPR for M. tuberculosis typing 4. Mojica et al. (1995) Identifcation of CRISPRs in Haloferax 5. Masepohl et al. (1996) Description of CRISPRs in cyanobacteria 6. Hoe et al. (1999) Use of CRISPR for S. pyogenes rapid genetic subtyping 7. Mojica et al. (2000) Widespread occurrence of CRISPRs in archaea and bacteria 8. Makarova et al. (2002) DNA repair hypothesis for Cas genes 9. Jansen et al. (2002a,b) Term 'CRISPR' coined; description of Cas genes 10. Pourcel et al. (2005), Mojica et al. (2005), Spacer sequences are probably snippets from foreign genome and Bolotin et al. (2005) 11. Makarova et al. (2006) Putative RNAi-based mechanism proposed 12. Barrangou et al. (2007) Establishment of CRISPR/Cas9 as adaptive immune system of S. thermophilus (a bacteria) against viruses 13. ...
... The repeats were identical (or nearly identical) in sequence and length, while the intervening spacers had a common length but seemingly random sequence. The authors searched for and found the repeats in genomes of two other species of gramnegative bacteria and other groups found similar repeats in a range of bacteria and archaea (Groenen et al., 1993;Mojica et al., 1993;Mojica et al., 1995;Masepohl et al., 1996;Hoe et al., 1999). The broad distribution and surprisingly well-conserved layout suggested an important functional role for CRISPR arrays (Mojica et al., 2000;Jansen et al., 2002). ...
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) is a type of prokaryotic immune system that is unique in its ability to provide sequence-specific adaptive protection, which can be updated in response to new threats. CRISPR-Cas does this by storing fragments of DNA from invading genetic elements in an array interspersed with short repeats. The CRISPR array can be continuously updated through integration of new DNA fragments (termed spacers) at one end, but over time existing spacers become obsolete. To optimize immunity, spacer uptake, residency, and loss must be regulated. This mini-review summarizes what is known about how spacers are organized, maintained, and lost from CRISPR arrays.
... These DNA repeats were also studied in many bacterial species like Mycobacterium tuberculosis (Groenen et al. 1993), Streptococcus pyogenes (Hoe et al. 1999), Methanocaldococcus jannaschii (Bult et al. 1996), in archaeal species namely Haloferax mediterranei, Haloferax volcanii (Mojica et al. 1995), Thermotoga maritima (Nelson et al. 1999) and in filamentous cyanobacterium Anabaena sp. (Masepohl et al. 1996) as well as in other archaeal and bacterial species. The acronym 'CRISPR' was coined in 2002 (Jansen et al. 2002). ...
In recent years, CRISPR-Cas9 technology is widely acknowledged for having major applications in the field of biotechnology for editing genome of any organism to treat a variety of complex diseases and for other purposes. The acronym 'CRISPR-Cas' stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated genes. This genetic organization exists in prokaryotic organisms and aids in the development of adaptive immunity since a protein called Cas9 nuclease cleaves specific target nucleic acid sequences from foreign invaders and destroys them. This mode of action has gained interest of the researchers to understand the insights of CRISPR-Cas9 technology. Here, we review that CRISPR-Cas organization is restricted to two classes and possesses different protein effectors. We also review the architecture of CRISPR loci, mechanism involved in genome editing by CRISPR-Cas9 technology and pathways of repairing double-strand breaks (DSBs) generated during the process of genome editing. This review also presents the strategies to increase the Cas9 specificity and reduce off-target activity to achieve accurate genome editing. Further, this review provides information on CRISPR tools used for genome editing, databases that are required for storing data on loci, strategies for delivering CRISPR-Cas9 to cells under study and applications of CRISPR-Cas9 to various fields. Safety measures are implemented on this technology to avoid misuse or ethical issues. We also discuss about the future aspects and potential applications of CRISPR-Cas9 technology required mainly for the treatment of dreadful diseases, crop improvement as well as genetic improvement in human.
... In the year 1987 an unusual structure of arranged repeats at the 3 0 region of the Iap gene was discovered in the DNA of Escherichia coli ( Fig. 1; Ishino et al. 1987). Soon after, a similar pattern was reported in a number of other bacteria and archaea systems (Bult et al. 1996;Groenen et al. 1993;Kawarabayasi et al. 1998Kawarabayasi et al. , 1999Klenk et al. 1998;Masepohl et al. 1996;Mojica et al. 1995;Nelson et al. 1999;Sensen et al. 1998). As a result of these conserved characteristics, the complex of repeated sequences and spacers were referred to as Clustered Regularly Interspaced Short Palindromic Repeats or simply CRISPR (Lander 2016). ...
The world stands at a new threshold today. As a planet, we face various challenges, and the key one is how to continue to produce enough food, feed, fiber, and fuel to support the burgeoning population. In the past, plant breeding and the ability to genetically engineer crops contributed to increasing food production. However, both approaches rely on random mixing or integration of genes, and the process can be unpredictable and time-consuming. Given the challenge of limited availability of natural resources and changing environmental conditions, the need to rapidly and precisely improve crops has become urgent. The discovery of CRISPR-associated endonucleases offers a precise yet versatile platform for rapid crop improvement. This review summarizes a brief history of the discovery of CRISPR-associated nucleases and their application in genome editing of various plant species. Also provided is an overview of several new endonucleases reported recently, which can be utilized for editing of specific genes in plants through various forms of DNA sequence alteration. Genome editing, with its ever-expanding toolset, increased efficiency, and its potential integration with the emerging synthetic biology approaches hold promise for efficient crop improvement to meet the challenge of supporting the needs of future generations.
... Analyses of molecular polymorphisms from a set of Anabaena strains were done using repetitive DNA sequences (STRR, LTRR, and Hip1) (Prasanna et al., 2006). The LTRR sequences were used for the specific fingerprinting of individual cyanobacterial isolates of Anabaena strains (Masepohl et al., 1996;Ezhilarasi and Anand, 2009b). Muralitharan and Thajuddin (2008) identified tRNA fMet group I introns in six strains of marine Synechococcus elongatus with sizes of around 280 bp were consistently obtained in all the strains tested and compared with other unicellular cyanobacterial strains and the phylogenetic tree inferred from the intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history (Figs. ...
... Repeat structures embedded in the genome of halophilic archaea were described as early as the 1990s [1,2]. Similar alternating repeat sequences were also described in E. coli and were subsequently identified in several prokaryotic species [3][4][5][6]. Their role as key players in an adaptive and specific immune system remained elusive until the late 2000s when bioinformatics analyses confirmed that foreign genetic elements are the origin of CRISPR spacers [7][8][9]. CRISPR-Cas systems have since been identified in almost half of bacteria and most archaea [10]. ...
Invading genetic elements pose a constant threat to prokaryotic survival, requiring an effective defence. Eleven years ago, the arsenal of known defence mechanisms was expanded by the discovery of the CRISPR-Cas system. Although CRISPR-Cas is present in the majority of archaea, research often focuses on bacterial models. Here, we provide a perspective based on insights gained studying CRISPR-Cas system I-B of the archaeon Haloferax volcanii. The system relies on more than 50 different crRNAs, whose stability and maintenance critically depend on the proteins Cas5 and Cas7, which bind the crRNA and form the Cascade complex. The interference activity requires a seed sequence and can interact with multiple PAM sequences. H. volcanii stands out as the first example of an organism that can tolerate autoimmunity via the CRISPR-Cas system while maintaining a constitutively active system. In addition, the H. volcanii system was successfully developed into a tool for gene regulation.
... Similarly, repeat arrays were soon found in other archaeal genomes, notably Archaeoglobus fulgidus (Klenk et al. 1997), Methanothermobacter thermoautotrophicum (Smith et al. 1997), Sulfolobus solfataricus (Sensen et al. 1998), Pyrococcus horikoshii (Kawarabayasi et al. 1998), Aeropyrum pernix (Kawarabayasi et al. 1999) and the Sulfolobus conjugative plasmid pNOB8 (She et al. 1998). Repeat arrays were also detected in the bacterium Thermotoga maritima (Nelson et al. 1999), and partial sequences were found in Streptococcus pyogenes (Hoe et al. 1999) and in cyanobacterial strains (Masepohl et al. 1996). ...
In the late 1980s and early 1990s, arrays of regularly spaced repeats were detected in both bacterial and archaeal genomes. They are currently known as Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR. Advances in our understanding of their biological significance and potential applications for biotechnology have followed a two-phased development. Initial studies were few and mainly descriptive of arrays of interspaced repeats in bacteria and archaea and of physically linked conserved genes that were inferred to be co-functional. Moreover, before their function was revealed, repeat-spacer arrays of Mycobacterium spp were employed as novel markers for bacterial genotyping. The second phase began in 2005, with the discovery of a link between CRISPR arrays and host protection against invading genetic elements. This finding fuelled a plethora of biochemical and genetic studies directed at characterizing the mechanistic details of this novel and complex genetic barrier. First, this led to the finding that the repeats, spacers, CRISPR-associated (Cas) proteins and partially conserved leader regions flanking one end of the CRISPR array, constitute the essential functional components. Subsequently, three primary functional steps were defined: (1) acquisition (also termed adaptation): uptake of new spacers at or near the leader sequence, (2) expression: generation of CRISPR transcripts from within the leader region and their processing into small mature CRISPR RNAs (crRNAs) carrying all or most of the spacer sequence and (3) interference: involving protein-crRNA complexes targeting and cleaving foreign genetic elements. Only now can we begin to comprehend the complex functional interactions and diversity of CRISPR-based systems, and the implications of their adaptive nature. Here, we describe the early developments in the CRISPR field and relate them to our current understanding of how these novel, complex and diverse systems function.
... These repeat sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. In addition, a 37 bp long tandemly repeated repetitive (LTRR) sequence has been identified in Anabaena strain PCC 7120, and also in non-heterocystous cyanobacteria [66]. Rasmussen and Svenning [67] developed a fingerprinting technique based on STRR and LTRR sequences which was useful in revealing heterogeneity among symbiotic isolates from Gunnera species besides distinguishing them from free living isolates. ...
Cyanobacteria (blue-green algae) are Gram-negative oxygenic photosynthetic prokaryotes with a long evolutionary history. They have potential applications in diverse areas, especially in agriculture, as nutrient supplements in agriculture and industry (as biofertilizer, plant growth promoting rhizobacteria and as biocontrol agents). Their role as food supplements/nutraceuticals and in bioremediation and wastewater treatment is an emerging area of interest. In addition, they are known to produce wide array of bioactive compounds (secondary metabolites) with diverse biological activities — including antiviral, antibacterial, antifungal, antimalarial, antitumoral and anti-inflammatory properties, having therapeutic, industrial and agricultural significance. One of the major problems has been regarding their classification being incongruent with the phylogeny, because the phenotype of cyanobacterial strains is known to be altered under different environmental/nutritional conditions. However, because of their simple growth needs, they are the favourite model organisms for deeper understanding of several metabolic processes and for the production of recombinant compounds of medicinal and commercial value. In recent years, cyanobacteria have gained interest for producing third generation biofuels (both biomass and H2 production). With the recent advances in metabolic engineering techniques and availability of genome sequences, novel approaches are being explored for realising the potential of cyanobacteria. Our review provides an overview of the polyphasic approaches used in the analyses of cyanobacterial biodiversity and the potential of these organisms in providing viable solutions to global problems of food, energy and environmental degradation, which need further impetus through adoption of multidisciplinary collaborative programs.
... The first description of CRISPR elements dates back to 1987 when Ishino et al. (1987) discovered a series of short palindromic sequences regularly repeated and separated by short unique sequences on the genome of Escherichia coli. Later, CRISPR arrays were also detected in archaea (Groenen et al., 1993;Masepohl et al., 1996;Mojica et al., 2005) and three independent studies identified the viral and plasmid source of spacer sequences (Bolotin et al., 2005;Mojica et al., 2005;Pourcel et al., 2005). The observation of spacers matching foreign genetic elements, combined with a detailed bioinformatic analysis of the Cas proteins revealing putative nuclease and helicase domains, led to the proposal that CRISPR-Cas functions as an RNA-mediated adaptive immune system (Makarova et al., 2006). ...
Multiple organisms face the threat of viral infections. To combat phage invasion, bacteria and archaea have evolved an adaptive mechanism of protection against exogenic mobile genetic elements, called CRISPR-Cas. In this defense strategy, phage infection is memorized via acquisition of a short invader sequence, called a spacer, into the CRISPR locus of the host genome. Upon repeated infection, the 'vaccinated' host expresses the spacer as a precursor RNA, which is processed into a mature CRISPR RNA (crRNA) that guides an endonuclease to the matching invader for its ultimate destruction. Recent efforts have uncovered molecular details underlying the crRNA biogenesis and interference steps. However, until recently the step of adaptation had remained largely uninvestigated. In this minireview, we focus on recent publications that have begun to reveal molecular insights into the adaptive step of CRISPR-Cas immunity, which is required for the development of the heritable memory of the host against viruses.
... CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) were first found in Escherichia coli downstream from the iap gene, as a stretch of 29 nt direct repeats separated by unique sequences of 32 nt, known as spacer sequences (Ishino et al., 1987). After this, CRISPRs were also identified in other bacteria and Archaea, such as Haloferax mediterranei, Streptococcus pyogenes, Anabaena and Mycobacterium tuberculosis (Groenen et al., 1993;Mojica et al., 1995;Masepohl et al., 1996;Hoe et al., 1999). Transcription of these CRISPRs was first demonstrated in the archaeon Archeoglobus fulgidus (Tang et al., 2002). ...
Bacteria and Archaea are continuously exposed to mobile genetic elements (MGE), such as viruses and plasmids. MGEs may provide a selective advantage, may be neutral or may cause cell damage. To protect against invading DNA, prokaryotes utilize a number of defence systems, including the CRISPR/Cas system. CRISPR/Cas systems rely on integration of invader sequences (spacers) into CRISPR loci that act as a genetic memory of past invasions. Processed CRISPR transcripts are utilized as guides by Cas proteins to cleave complementary invader nucleic acids. In this issue, two groups report on spacer acquisition and turnover dynamics of CRISPR loci in a thermoacidophilic archeon and a pathogenic bacterium. Erdmann and Garrett (2012) demonstrate that three of the six CRISPR loci of Sulfolobus solfataricus rapidly acquire new spacer sequences from a conjugative plasmid present in a virus mixture. Intriguingly, two distinct mechanisms of spacer integration are utilized: leader adjacent and internal CRISPR spacer acquisition. Lopez-Sanchez et al. (2012) studied the type II system of Streptococcus agalactiae and observe heterogeneity in the bacterial population. A fraction of the population lost one or more anti-mobilome spacer sequences during its cultivation, allowing the transfer of a MGE in this subpopulation and a rapid response to altering selection pressures.
... The CRISPR story began with the discovery of a peculiar short repeat in the E. coli genome by Ishino and coworkers in the 1980s (Ishino et al., 1987; Nakata et al., 1989). Subsequently, similar repeats were noted in a number of bacteria and archaea (Bult et al., 1996; Groenen et al., 1993; Hermans et al., 1991; Hoe et al., 1999; Kawarabayasi et al., 1999; Kawarabayasi et al., 1998; Klenk et al., 1997; Masepohl et al., 1996; Mojica et al., 1995; Nelson et al., 1999; Sensen et al., 1998; She et al., 1998; She et al., 2001; Smith et al., 1997). Mojica and Jansen and their colleagues unified these observations, coined the CRISPR acronym, and characterized the CRISPR locus (Jansen et al., 2002; Mojica et al., 2000). ...
All cellular systems evolve ways to combat predators and genomic parasites. In bacteria and archaea, numerous resistance mechanisms have developed against phage. Our understanding of this defensive repertoire has recently been expanded to include the CRISPR system of clustered, regularly interspaced short palindromic repeats. In this remarkable pathway, short sequence tags from invading genetic elements are actively incorporated into the host's CRISPR locus to be transcribed and processed into a set of small RNAs that guide the destruction of foreign genetic material. Here we review the inner workings of this adaptable and heritable immune system and draw comparisons to small RNA-guided defense mechanisms in eukaryotic cells.
... and sequenced in a subsequent study 4 . This constituted the first report of a CRISPR locus and was followed by similar descriptions of repeats derived from gene [5][6][7][8] or wholegenome 9-14 sequencing projects in bacteria and archaea. The accumulation of available genomic sequences led mojica et al. 15 to recognize CRISPRs as a family of repeats that are present in many such species. ...
Sequence-directed genetic interference pathways control gene expression and preserve genome
integrity in all kingdoms of life. The importance of such pathways is highlighted by the
extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many
bacteria and most archaea, clustered, regularly interspaced short palindromic repeats
(CRISPRs) are involved in a more recently discovered interference pathway that protects
cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive,
heritable record of past infections and express CRISPR RNAs — small RNAs that target
invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles
in microbial physiology and evolution. We also discuss potential applications of this novel
interference pathway.
... These heptanucleotide sequences have been identified in several cyanobacterial genera and species, so far mostly in heterocystous cyanobacteria (Rasmussen & Svenning, 1998; Zheng et al., 1999; Nilsson et al., 2000; Wilson et al., 2000; Teaumroong et al., 2002; Lyra et al., 2005; Prasanna et al., 2006), but also in some non-heterocystous ones (Rasmussen & Svenning, 1998). Furthermore, a 37 bp long tandemly repeated repetitive sequence (LTRR) has also been identified in some cyanobacterial species (Masepohl et al., 1996; Rasmussen & Svenning, 1998; Prasanna et al., 2006). Analysis of STRRs and LTRRs has been described as a powerful tool for taxonomic studies (Mazel et al., 1990). ...
In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon-Wiener (J') indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D=0.83; J'=0.88) and Aphanizomenon flos-aquae the highest ones (D=J'=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.
Discovery of the CRISPR/Cas system revolutionized biology and biomedicine in the 21st century. Here we discuss the milestones in the development of CRISPR/Cas genome editing technology, from the history of discovery to current developments, including medical applications. Technical and ethical problems associated with the use of CRISPR/Cas for editing human embryonic genomes are also discussed.
Since 2012, the new gene editing technique called CRISPR took the world by storm because theoretically it can be used to edit any organism quickly, precisely, and at low cost. Because of these features, many fear that CRISPR could become a technology of choice for terrorists or states who wish to produce novel threat agents or bioweapons. Others fear that it could be the source of a catastrophic event caused by unsafe laboratory practices by amateur or practicing scientists. In this chapter we review the ethical, safety, and security challenges that the technology raises. We conclude that while safety concerns are founded due to the vague regulatory framework worldwide, the risks of misuse by inexperienced terrorists are limited by the fact that the technology currently has a number of limitations and still presents a number of technical challenges.
This year marks the tenth anniversary of the identification of the biological function of CRISPR-Cas as adaptive immune systems in bacteria. In just a decade, the characterization of CRISPR-Cas systems has established a novel means of adaptive immunity in bacteria and archaea and deepened our understanding of the interplay between prokaryotes and their environment, and CRISPR-based molecular machines have been repurposed to enable a genome editing revolution. Here, we look back on the historical milestones that have paved the way for the discovery of CRISPR and its function, and discuss the related technological applications that have emerged, with a focus on microbiology. Lastly, we provide a perspective on the impacts the field has had on science and beyond. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Microbes live in multi-microbial communities called microbiome. Discoveries that can help in the regulation of the composition of the microbiome are likely to impact diverse functions of microbes from health, environment, to biotechnology. Antimicrobials offer such regulatory potential and are slowly but surely evolving for the benefit of human health and biotechnology. Antibiotics are the first discovered antimicrobials which are low molecular weight natural microbial products that inhibit the growth of other microbes. However, emergence of microbial resistance to conventional antibiotics has presented an urgent need for novel antimicrobials. Here, we describe another native microbial machinery, CRISPR (“clustered regularly interspaced short palindromic repeats”)–Cas (“CRISPR associated”) system, that confers adaptive immunity to microbes by employing CRISPR RNAs to recognize and destroy complementary nucleic acids of invasive foreign genetic elements. Further, sequence-based targeting by CRISPR–Cas system has been leveraged for the development of sequence-specific novel antimicrobials, genome editing, and genome regulation tools.
Cyanobacteria enter into efficient nitrogen-fixing symbiosis with a wide range of representatives within the plant kingdom. These include plants from the divisions: Bryophyta (mosses, liverworts and hornworts), gymnosperms of the family Cycadaceae, an aquatic ferns within Pteridohyta (Azolla) and an angiosperm Gunnera, as well as diverse lichenized fungi. This host range places cyanobacteria in an exclusive position among symbiotic nitrogen-fixers. With few exceptions the cyanobacteria belongs to the filamentous genus Nostoc. Very little is known about the diversity of symbiotic cyanobacteria both within and between the different host plants.
The biodiversity of nitrogen-fixing organisms is huge. Taxonomic and phylogenetic research is needed to structure this diversity, to facilitate communication among scientists, and to increase our understanding of the evolution and biology of diazotrophs. Molecular tools for taxonomic and biodiversity studies of diazotrophic rhizobia, frankiae, cyanobacteria and bacilli are presented in sections 2 to 5. Sections 6 to 9 focus on problems with genus and species assignment.
The diversity of cyanobacteria in the North-Eastern region of India has not been studied except for a few sporadic and inconclusive
reports. Loktak Lake is a huge reservoir for various kinds of organisms, including cyanobacteria. The present study describes
the isolation and molecular diversity of 72 filamentous, heterocystous cyanobacterial strains isolated from samples collected
from Loktak Lake, its adjoining rice fields and rice fields in Indian Council of Agricultural Research (ICAR) complex, Shillong,
Meghalaya, India. The isolated strains belonged to the genera Anabaena, Nostoc, Calothrix, Cylindrospermum and Mastigocladus. The molecular analysis of isolates revealed the occurrence of certain strains being present in the sample collected from
the rice fields falling in the catchment area of Loktak Lake, Manipur and rice fields in ICAR complex, Shillong, Meghalaya
both. A polyphasic approach based on morphological features and PCR based molecular polymorphism revealed enormous level of
molecular diversity. Out of three primers targeted regions used for determining genetic polymorphism, STRR1A produced best
fingerprint profile of cyanobacterial strains. The morphological diversity of isolates was assured by light microscope whereas
PCR based multiple fingerprint profile was used for molecular characterization. Molecular typing using short tandemly repeated
repetitive STRR1A sequences as primer provided strain specific fingerprint profiles of the isolates.
We examined 32 cyanobacterial strains, representative of 11 different genera, for the presence of the highly iterated genomic palindrome, called HIP1, using HIP1 extended PCR primers. Cryolysates or cell suspensions, and even single colonies, of Microcystis aeruginosa , strain PCC 7806, yielded PCR products equivalent to those obtained with purified DNA, as judged from the banding patterns obtained by gel electrophoresis. Cryolysates were used for amplification and genotyping of all other strains. Representatives of different genera differed extensively in HIP1-based migration patterns, and the amplification products of axenic cyanobacteria were comparable to those obtained with duplicate, non-axenic strains. In addition, HIP1 repeats were present in 11 strains of the species Microcystis aeruginosa and in isolates assignable to Planktothrix agardhii and P. rubescens. Based on available 16S rDNA sequence data and DNA/DNA hybridization results for members of these genera, the differences detected by HIP1-based profiling correlate with the interspecies distinctions between Planktothrix agardhii and P. rubescens, and reflect intraspecific diversity for members of Microcystis aeruginosa. Thus, we have confirmed the value of this method for rapid genotyping and verification of presumably identical strains from different culture collections.
Leptolyngbya strains VRUC135 and CSC7-1, isolated from biofilms present in Roman hypogea, were employed as experimental strains to optimize molecular techniques to approach the taxonomy of different epilithic strains of Leptolyngbya, maintained in our laboratory. High quality genomic DNAs were obtained according to the CTAB method, while low quality DNAs were obtained with the hot phenol method. High quality DNAs were used in gradient PCRs to optimize annealing temperatures for 16S-23S rRNA operon amplification and genomic PCR fingerprinting, with primers derived from HIP1, STRR, and LTRR sequences. At the optimized conditions, no significant differences in PCR products were observed using as template low quality DNAs. The employment of both molecular approaches suggested that Leptolyngbya VRUC135 and CSC7-1 are different species: the partial sequence of 16S rRNA gene from Leptolyngbya CSC7-1 showed 91.6% nucleotide identity with the 16S rRNA gene of Leptolyngbya VRUC135, and both strains exhibited a high genetic diversity when typed with HIP1, STRR, and LTRR PCR fingerprinting.
All organisms need to continuously adapt to changes in their environment. Through horizontal gene transfer, bacteria and archaea can rapidly acquire new traits that may contribute to their survival. However, because new DNA may also cause damage, removal of imported DNA and protection against selfish invading DNA elements are also important. Hence, there should be a delicate balance between DNA uptake and DNA degradation. Here, we describe prokaryotic antiviral defense systems, such as receptor masking or mutagenesis, blocking of phage DNA injection, restriction/modification, and abortive infection. The main focus of this review is on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated), a prokaryotic adaptive immune system. Since its recent discovery, our biochemical understanding of this defense system has made a major leap forward. Three highly diverse CRISPR/Cas types exist that display major structural and functional differences in their mode of generating resistance against invading nucleic acids. Because several excellent recent reviews cover all CRISPR subtypes, we mainly focus on a detailed description of the type I-E CRISPR/Cas system of the model bacterium Escherichia coli K12.
The second edition of the highly acclaimed Bacterial Stress Responses incorporates and reviews the vast number of new findings that have greatly advanced the understanding of bacterial stress responses in the decade since the publication of the first edition. Readers will discover how this improved understanding not only enhances our knowledge of all cellular regulation at the molecular level, but also provides new ammunition in the fight against pathogens and helps optimize the use of bacteria in biotechnology.
The first section explores general regulatory principles, including the latest findings from genomics studies. In the second and third sections readers will learn how much more researchers have discovered about both specific and general stress responses. Next, the fourth section reviews how stress responses affect the interactions between bacteria and host cells. The fifth section describes bacterial stress responses in different niches and communities, with an emphasis on extreme environments. The final section examines how our growing understanding of bacterial stress responses can be used to better combat bacterial infection with antibiotics and improve biofuel production and bioremediation.
All chapters have been contributed by leaders and pioneers in their respective fields and then carefully edited to ensure conciseness and clarity. With its coverage of a broad range of model organisms as well as biotechnologically, medically, and environmentally relevant bacteria, this new edition fully encapsulates our understanding of bacterial stress responses. Moreover, it serves as a springboard for new investigations and new applications.
New in the Second Edition
· Incorporates new fields such as network analysis, metagenomics, and regulatory RNAs
· Examines new findings from genomics studies that have changed our understanding of regulation
· Explains how new findings from bacterial stress response studies facilitate the development of antibiotics
· Discusses the latest efforts to exploit bacterial stress responses for biofuel production and bioremediation
· Presents significant insights into how bacteria survive stress conditions by undergoing changes of state, morphology, or cell surface
More Key Features
· Features contributors who are leaders in the investigation of bacterial stress responses and the development of new applications based on new findings
· Serves as a gateway to the literature in the field
· Highlights important directions for future research
Cyanobacteria are the microscopic photosynthetic ‘cell factories’ that are known to produce a wide variety of bioactive compounds and have a very significant role in environment and ecology of water bodies and other ecosystems. However, the systematics and classification of this group of organism is presently in a state of disarray. These were traditionally identified on the basis of morphology, and were classified as blue-green algae (Cyanophyta) under the Botanical codes whereas; the Bergey’s Manual of Systematic Bacteriology uses Bacteriological approach for this purpose. Their greater morphological, physiological, biochemical and molecular variability; incorrect use of old or revised names; misidentification of the strains; horizontal gene transfer; and lack of appropriate genetic information complicates the process. Further, this group of microorganisms has wide adaptation qualities, show spatial and temporal variation in their genetic community structure, and respond differently to factors such as nutrient deprivation, light intensity and predation. Therefore, the application of various molecular tools, functional genomics, proteomics, metabolomics, and analysis of important bioactive compounds along with the morphological, physiological and biochemical attributes will magnify the diversity present among the members of this group and will be highly useful in correct deciphering of their phylogenetic relationships.
Species of Nostoc are found on every continent on Earth in a wide range of terrestrial and aquatic ecosystems where their growths become both
conspicuous and abundant. Environments where water is scarce, or absent for protracted periods, remain some of the most extreme
on the Earth yet they typically support populations of the terrestrial desiccation-tolerant form N. commune. The nitrogen-fixing activities of Nostoc spp. contribute to the quality of nutrient-poor soils, especially in karst regions, and several forms contribute to the productivity
of rice paddies throughout an expansive region of Asia (Chapter 8). Some Nostoc spp. enter into a range of associations with higher and lower plants and these include the only known symbiosis with a flowering
plant (Chapter 19). The only known endocyanosis of a fungus, Geosiphon pyriforme, involves N. punctijorme and may represent a type of photoautotrophic association which was one step in the evolution of all land plants. Growths
of Nostoc elaborate a range of natural products that range from the innocuous, which contribute no more than a mildly offensive odor
to potable water supplies, to potent toxins which cause sickness, cell transformation and the death of wildlife and humans
(Chapter 22). Despite the fact that Nostoc spp., collectively, represent a unique biological resource, there is no adequate species concept for the group; the origins
of Nostoc and other heterocystous cyanobacteria are unclear; and the physiological and molecular traits which could provide diagnostic
features of a Nostoc remain elusive (Chapter 1). Nostoc is collected and cultivated as a source of food by the indigenous populations of many countries and in at least one of these,
China, Nostoc occupies a position in human social behavior; it has done so for at least 1500 years. This chapter considers in a broad sense
what is known and what needs to be known about Nostoc spp.
A 2.5 kb high-copy-number plasmid, pM A4 in thermophilic cyanobacterium Synechococcus sp. M A4 was isolated and characterized to develop a genetic engineering system for thermophilic cyanobacteria. The copy
number of pM A4 was determined to be by densitometry about 350/cell. The pM A4 may be a type of rolling-circle plasmid, because
a possible rep gene encoding 34 k D-protein and a consensus sequence of a double-stranded origin nick site of rolling circle plasmids were
found in the pM A4 sequence. The pM A4 was electro-introduced into another thermophile, Synechococcus sp. MA 19, which is the strongest poly-β-hydroxybutyrate (PHB) accumulator in photoau totrophic organisms. The pM A4 was
incorporated and retained in MA 19. These results indicate that pM A4 could be developed as a useful vector for thermophilic
cyanobacteria.
The presence of repetitive DNA sequences viz., short tandemly repeated repetitive (STRR) and highly iterated palindrome (HIP), in the cyanobacterial genome were used to
generate a PCR-based fingerprint pattern of nine cyanobacterial cultures (both stress tolerant and non-tolerant), belonging
to the genus Westiellopsis. By this method it was possible to generate distinguishing fingerprint patterns for all the isolates and cluster isolates
with similar stress tolerance properties. This study reveals the utility of repetitive DNA sequences in the cyanobacterial
genome, for differentiation of Westiellopsis cultures and clustering strains that posses similar stress tolerance properties.
Significant transitions in the availability. toxicity and subsequent use of metals are predicted to have coincided with the
release of dioxygen in quantity and hence coincided with the first appearance of cyanobacteria. During the rapid adaptive
radiation of the cyanobacteria there will have been (i) selection for the evolution of new metal resistance determinants and
(ii) greater opportunity for more available metals to be recruited for use in newly evolved proteins. Today, the availability
of metals in many environments fluctuates both spatially and temporally not only as a result of ‘natural’ processes. but also
as a consequence of anthropogenic factors. The first part of this chapter considers the metabolism and toxicity of metals
in cyanobacteria. The second part of this chapter considers a separate topic, the abundance of short sequences which are now
known to be repeated throughout the DNA of many cyanobacteria. It is predicted that the study of cyanobacteria will also give
unique insights into the evolution of repetitive DNA.
Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.
Les répétitions en tandem sont constituées de successions de motifs d'ADN. Ces structures sont présentes dans tous les organismes, procaryotes comme eucaryotes et, même si leur rôle biologique est encore peu compris, elles ont des applications dans de nombreux domaines. Tout d'abord, chez les bactéries, les répétitions en tandem polymorphes, dont le nombre d'unités varie, se révèlent un outil puissant pour l'identification de souches à des fins épidémiologiques. Par ailleurs, certaines répétitions en tandem humaines ont la propriété de muter à des fréquences élevées : les minisatellites hypermutables sont les éléments les plus instables du génome humain. Ils peuvent être utilisés comme biomarqueurs d'exposition à des agents potentiellement mutagènes tels que les radiations ionisantes. D'un point de vue plus fondamental, ils sont également un modèle d'étude des mécanismes d'instabilité des génomes. Dans cette thèse, nous mettons à profit les données issues du séquençage afin d'identifier des répétitions en tandem polymorphes. Nous avons tout d'abord élaboré une base de données des répétitions en tandem accessible sur le web (http://minisatellites.u-psud.fr), qui fournit un accès aux répétitions en tandem de génomes entiers. Ensuite, dans le but de sélectionner les répétitions en tandem polymorphes, plusieurs stratégies ont été mises en oeuvre. D'une part, chez les bactéries pour lesquelles les séquences de plusieurs souches étaient disponibles, nous avons créé un utilitaire de comparaison de souches, afin d'identifier des marqueurs polymorphes utilisables en épidémiologie. D'autre part, une étude menée sur les minisatellites humains a permis de définir des critères prédictifs du polymorphisme à partir de la séquence d'un seul allèle de minisatellite, et a en outre mis en évidence un nouveau minisatellite hypermutable situé dans une séquence codante putative. Les critères prédictifs ont également été appliqués à l'identification de minisatellites codants potentiellement polymorphes dans le génome humain.
Les répétitions en tandem sont constituées de successions de motifs d'ADN. Ces structures présentes dans tous les organismes, procaryotes comme eucaryotes, ont des applications dans de nombreux domaines. Depuis quelques années seulement, les répétitions en tandem sont étudiées chez les bactéries. Le polymorphisme associé à ces séquences peut être utilisé pour le génotypage de bactéries pathogènes, permettant une identification précise au niveau de la souche. Le polymorphisme des séquences répétées est de deux types : polymorphisme de longueur et mutations internes aux motifs. Les génomes des deux bactéries pathogènes responsables d'infections nosocomiales, Staphylococcus aureus et Pseudomonas aeruginosa, ont été étudiés dans le but d'identifier des séquences répétées polymorphes. Un ensemble de marqueurs polymorphes a été validé expérimentalement pour ces deux espèces permettant un typage dit MLVA (pour « Multiple Locus VNTR Analysis »). Le travail plus classique de typage par la taille de la répétition a été complété par un travail de séquençage de certains allèles. Les résultats obtenus montrent comment le typage « MLVA » complété si nécessaire par le séquençage d'allèles, pourraient constituer de nouvelles méthodes peu coûteuses participant au contrôle des infections bactériennes.
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), the basis of spoligotyping technology, can provide prokaryotes with heritable adaptive immunity against phages' invasion. Studies on CRISPR loci and their associated elements, including various CAS (CRISPR-associated) proteins and leader sequences, are still in its infant period. We introduce the brief history', structure, function, bioinformatics research and application of this amazing immunity system in prokaryotic organism for inspiring more scientists to find their interest in this developing topic.
The direct repeat region in Mycobacterium tuberculosiscomplex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat
(DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive
polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of
tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain
differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking
DNA of M. tuberculosis complex strains. We identified 94 different spacer sequences among 26 M. tuberculosis complex strains. No sequence homology was found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence
or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that
the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous
DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling
of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined
to isolates of the rare Mycobacterium canettii taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.
Toxic and non-toxic cyanobacterial strains from Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis, Planktothrix (Oscillatoria agardhii), Oscillatoria and Synechococcus genera were examined by RFLP of PCR-amplified 16S rRNA genes and 16S rRNA gene sequencing. With both methods, high 16S rRNA gene similarity was found among planktic, anatoxin-a-producing Anabaena and non-toxic Aphanizomenon, microcystin-producing and non-toxic Microcystis, and microcystin-producing and non-toxic Planktothrix strains of different geographical origins. The respective sequence similarities were 99.9-100%, 94.2-99.9% and 99.3-100%. Thus the morphological characteristics (e.g. Anabaena and Aphanizomenon), the physiological (toxicity) characteristics or the geographical origins did not reflect the level of 16S rRNA gene relatedness of the closely related strains studied. In addition, cyanobacterial strains were fingerprinted with repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR. All the strains except two identical pairs of Microcystis strains had different band profiles. The overall grouping of the trees from the 16S rRNA gene and the REP- and ERIC-PCR analyses was similar. Based on the 16S rRNA gene sequence analysis, four major clades were formed. (i) The clade containing filamentous heterocystous cyanobacteria was divided into three discrete groups of Anabaena/Aphanizomenon, Anabaena/Cylindrospermum/ Nodularia/Nostoc and Calothrix strains. The three other clades contained (ii) filamentous non-heterocystous Planktothrix, (iii) unicellular non-heterocystous Microcystis and (iv) Synechococcus strains.
The rapid increase in genomic sequences provides new opportunities for comparative genomics. In this report, we describe a novel family of repeat sequences that is present in Bacteria and Archaea but not in Eukarya. The repeat loci typically consisted of repetitive stretches of nucleotides with a length of 25 to 37 bp alternated by nonrepetitive DNA spacers of approximately equal size as the repeats. The nucleotide sequences and the size of the repeats were highly conserved within a species, but between species the sequences showed no similarity. Due to their characteristic structure, we have designated this family of repeat loci as SPacers Interspersed Direct Repeats (SPIDR). The SPIDR loci were identified in more than forty different prokaryotic species. Individual species such as Mycobacterium tuberculosis contain one SPIDR locus, while other species such as Methanococcus jannaschii contained up to 20 different loci. The number of repeats in a locus varies greatly from two repeats to several dozens of repeats. The SPIDR loci were flanked by a common 300-500-bp leader sequence, which appeared to be conserved within a species but not between species. The SPIDR locus of M. tuberculosis is extensively used for strain typing. The finding of SPIDR loci in other prokaryotes, including the pathogens Salmonella, Campylobacter, and Pasteurella may extend this surveillance to other species.
Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.
We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.
An octameric palindrome (5'-GCGATCGC-3') is abundant in cyanobacterial sequences within databases (GenBank/EMBL) and was designated HIP1 (highly iterated palindrome). The frequency of occurrence of all 256 octameric palindromes has now been determined in sub-databases revealing large and unique over-representation of HIP1 in cyanobacterial entries. DNA sequences from other bacteria were searched for any over-represented octameric palindromes analogous to HIP1. Only two sequences were identified, in the genomes of a thermophile and halophilic archaebacteria, although these were less abundant than HIP1 in cyanobacteria and relate to codon usage. To test the proposed widespread distribution of HIP1 in DNA from the cyanobacterium Synechococcus PCC 6301, randomly selected genomic clones were partly sequenced. HIP1 constituted 2.5% of the novel sequences, equivalent to a site on average once every 320 nucleotides. An oligonucleotide including HIP1 was also tested in PCR. Multiple products were obtained using template DNA from cyanobacterial strains in which HIP1 is abundant in known sequences, and some strains generated characteristic HIP-PCR banding patterns. However, analysis of DNA from one strain (not previously represented in databases) by random sequencing, HIP-PCR and Pvul digestion, confirms that not all cyanobacterial genomes are rich in HIP1.
In bacteria, there is growing evidence that highly repeated DNA sequence families are quite widespread. The palindromic unit (PU) family may represent up to 1% of the genome of E. coli and Salmonella typhimurium. The genomic localization of PUs and their consensus sequence appear to be species-specific. At least some PUs display functions at the mRNA level such as mRNA stabilization and, occasionally, mRNA processing or transcription termination. However, some of their properties, including indications that they bind a specific protein, suggest that PUs are involved in the structure of the bacterial nucleoid.
Three mitochondrial plasmids (1704, 1695 and 1478 bp) were isolated from sterile cytoplasms of Vicia faba L. and cloned into a bacterial plasmid vector. Their nucleotide sequence was found to be 99 to 100% homologous to their counterparts isolated from a fertile cytoplasm (J.A. Wahleithner and D.R. Wolstenholme, Curr Genet 12 (1987) 55-76). Several overlapping transcripts were localized in the region which is unique to each of the three plasmids. S1 nuclease mapping indicated for all of them several 3' termini but a unique 5' boundary which was located downstream of the consensus sequence CNTAAGTGANNNNNGAA also found at the transcript 5' boundary of other plant mitochondrial plasmids. Southern blot hybridization with nuclear DNA indicated the presence of nuclear sequences homologous to each plasmid.
Rhizobium meliloti exoD mutants are deficient in invasion of alfalfa nodules and, as a consequence, the nodules that exoD strains induce fail to fix nitrogen. These nodules appear to be arrested at the same stage as nodules induced by other exo mutants, which do not make an acidic exopolysaccharide called EPS I, or by ndv mutants, which do not produce a periplasmic cyclic beta(1,2) glucan. However previous genetic and biochemical evidence suggested that the nodule invasion defect of exoD mutants arose from a biochemical deficiency distinct from those of both EPS I-deficient exo mutants and ndv mutants. In this study, we characterize mutant phenotypes of exoD strains in both free-living and symbiotic states. Nodules induced by exoD mutants are generally small and empty of bacteria, and exhibit the same structural features as nodules induced by other invasion-deficient mutants. Putative incipient infection threads were visible in outer cortical cells of these nodules but not in the plant cells in the interior of the nodule. We show that exoD mutants are sensitive to alkaline conditions, ceasing to grow at elevated pH in liquid yeast extract cultures and exhibiting decreased viability in alkaline medium. Interestingly, we find that buffering the plant growth medium at slightly acidic pH (6.0-6.5) restores the ability of exoD mutants to invade alfalfa nodules. exoD mutants are thus alkali sensitive for both free-living and symbiotic phenotypes. This result implies that the nodule invasion defect of exoD mutants arises from their sensitivity to alkaline conditions and, furthermore, that alkaline conditions may obtain in the developing infection thread. The deduced amino acid sequence of ExoD is extremely hydrophobic, suggesting that the protein is membrane associated. We propose models whereby absence of a putative membrane protein might lead to sensitivity to alkaline conditions and consequent arrest of nodule invasion.
An unusual cluster of tandemly repeated DNA sequences (TRS) was found downstream from the gene encoding DsaV methyltransferase, the DNA modification enzyme in the DsaV restriction-modification system found in a strain of Dactylococcopsis salina (Ds). The repeat unit is about 32-bp long and is present 13 times in the cluster. Each repeat unit can be divided into two distinct parts based on the level of sequence conservation and evolution. Hybridization of Ds DNA with a probe specific for this cluster revealed that there were at least two additional sites within the genome with similar TRS. The TRS units are localized in one region of the Ds genome. They do not share significant sequence similarity with other TRS found in prokaryotes.
Genomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal-tolerant eukaryotes. Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus, smt, in cells of Synechococcus PCC 6301 selected for Cd tolerance. Following the characterization of this locus, the altered smt region has now been isolated from a Cd-tolerant cell line, C3.2, and its nucleotide sequence determined. This has identified a deletion within smtB, which encodes a trans-acting repressor of smt transcription. Two identical palindromic octanucleotides (5'-GCGATC-GC-3') traverse both borders of the excised element. This palindromic sequence is highly represented in the smt locus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL/DDBJ DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences from Synechococcus strains (estimated to occur at an average frequency of once every c. 664 bp). HIP1 is also abundant in the genomes of other cyanobacteria. The functional significance of smtB deletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteria are discussed.