An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

Horizontal Gene Transfer and CRISPR Targeting Drive Phage-Bacterial Host Interactions and Coevolution in "Pink Berry" Marine Microbial Aggregates.

Bacteriophages (phages), which are viruses that infect bacteria, are the most abundant components of microbial communities and play roles in community dynamics and host evolution. However, the study of phage-host interactions is hindered by a paucity of model systems from natural environments. Here, we investigate phage-host interactions in the "pink berry" consortia, which are naturally occurring, low-diversity, macroscopic bacterial aggregates that are found in the Sippewissett Salt Marsh (Falmouth, MA, USA). We leverage metagenomic sequence data and a comparative genomics approach to identify eight compete phage genomes, infer their bacterial hosts from host-encoded clustered regularly interspaced short palindromic repeats (CRISPRs), and observe the potential evolutionary consequences of these interactions. Seven of the eight phages identified infect known pink berry symbionts, namely, Desulfofustis sp. PB-SRB1, Thiohalocapsa sp. PB-PSB1, and Rhodobacteraceae sp. A2, and they are largely divergent from known viruses. In contrast to the conserved bacterial community structure of pink berries, the distribution of these phages across aggregates is highly variable. Two phages persisted over a period of seven years with high sequence conservation, allowing us to identify gene gain and loss. Increased nucleotide variation in a conserved phage capsid gene that is commonly targeted by host CRISPR systems suggests that CRISPRs may drive phage evolution in pink berries. Finally, we identified a predicted phage lysin gene that was horizontally transferred to its bacterial host, potentially via a transposon intermediary. Taken together, our results demonstrate that pink berry consortia contain diverse and variable phages as well as provide evidence for phage-host coevolution via multiple mechanisms in a natural microbial system. IMPORTANCE Phages, which are viruses that infect bacteria, are important components of all microbial systems, in which they drive the turnover of organic matter by lysing host cells, facilitate horizontal gene transfer (HGT), and coevolve with their bacterial hosts. Bacteria resist phage infection, which is often costly or lethal, through a diversity of mechanisms. One of these mechanisms is CRISPR systems, which encode arrays of phage-derived sequences from past infections to block subsequent infection with related phages. Here, we investigate the bacteria and phage populations from a simple marine microbial community, known as "pink berries", found in salt marshes of Falmouth, Massachusetts, as a model of phage-host coevolution. We identify eight novel phages and characterize a case of putative CRISPR-driven phage evolution as well as an instance of HGT between a phage and its host, together suggesting that phages have large evolutionary impacts in a naturally occurring microbial community.

Phage-Antibiotic Synergy Inhibited by Temperate and Chronic Virus Competition.

As antibiotic resistance grows more frequent for common bacterial infections, alternative treatment strategies such as phage therapy have become more widely studied in the medical field. While many studies have explored the efficacy of antibiotics, phage therapy, or synergistic combinations of phages and antibiotics, the impact of virus competition on the efficacy of antibiotic treatment has not yet been considered. Here, we model the synergy between antibiotics and two viral types, temperate and chronic, in controlling bacterial infections. We demonstrate that while combinations of antibiotic and temperate viruses exhibit synergy, competition between temperate and chronic viruses inhibits bacterial control with antibiotics. In fact, our model reveals that antibiotic treatment may counterintuitively increase the bacterial load when a large fraction of the bacteria are antibiotic resistant, and both chronic and temperate phages are present.

Duplication of leucyl-tRNA synthetase in an archaeal extremophile may play a role in adaptation to variable environmental conditions.

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that play a fundamental role in protein synthesis. They catalyze the esterification of specific amino acids to the 3'-end of their cognate tRNAs and therefore play a pivotal role in protein synthesis. Although previous studies suggest that aaRS-dependent errors in protein synthesis can be beneficial to some microbial species, evidence that reduced aaRS fidelity can be adaptive is limited. Using bioinformatics analyses, we identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of the archaeal family Sulfolobaceae. Remarkably, one copy, designated LeuRS-I, had key amino acid substitutions within its editing domain that would be expected to disrupt hydrolytic editing of mischarged tRNALeu and to result in variation within the proteome of these extremophiles. We found that another copy, LeuRS-F, contains canonical active sites for aminoacylation and editing. Biochemical and genetic analyses of the paralogs within Sulfolobus islandicus supported the hypothesis that LeuRS-F, but not LeuRS-I, functions as an essential tRNA synthetase that accurately charges leucine to tRNALeu for protein translation. Although LeuRS-I was not essential, its expression clearly supported optimal S. islandicus growth. We conclude that LeuRS-I may have evolved to confer a selective advantage under the extreme and fluctuating environmental conditions characteristic of the volcanic hot springs in which these archaeal extremophiles reside.

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus.

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus-host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus-host interactions in the archaeal domain.

Temperate and chronic virus competition leads to low lysogen frequency.

The canonical bacteriophage is obligately lytic: the virus infects a bacterium and hijacks cell functions to produce large numbers of new viruses which burst from the cell. These viruses are well-studied, but there exist a wide range of coexisting virus lifestyles that are less understood. Temperate viruses exhibit both a lytic cycle and a latent (lysogenic) cycle, in which viral genomes are integrated into the bacterial host. Meanwhile, chronic (persistent) viruses use cell functions to produce more viruses without killing the cell; chronic viruses may also exhibit a latent stage in addition to the productive stage. Here, we study the ecology of these competing viral strategies. We demonstrate the conditions under which each strategy is dominant, which aids in control of human bacterial infections using viruses. We find that low lysogen frequencies provide competitive advantages for both virus types; however, chronic viruses maximize steady state density by eliminating lysogeny entirely, while temperate viruses exhibit a non-zero 'sweet spot' lysogen frequency. Viral steady state density maximization leads to coexistence of temperate and chronic viruses, explaining the presence of multiple viral strategies in natural environments.

Microhomology-Mediated High-Throughput Gene Inactivation Strategy for the Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the last archaeal eukaryote common ancestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD+ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism.IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus, providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

Sulfolobus islandicus meta-populations in Yellowstone National Park hot springs.

Abiotic and biotic forces shape the structure and evolution of microbial populations. We investigated forces that shape the spatial and temporal population structure of Sulfolobus islandicus by comparing geochemical and molecular analysis from seven hot springs in five regions sampled over 3 years in Yellowstone National Park. Through deep amplicon sequencing, we uncovered 148 unique alleles at two loci whose relative frequency provides clear evidence for independent populations in different hot springs. Although geography controls regional geochemical composition and population differentiation, temporal changes in population were not explained by corresponding variation in geochemistry. The data suggest that the influence of extinction, bottleneck events and/or selective sweeps within a spring and low migration between springs shape these populations. We suggest that hydrologic events such as storm events and surface snowmelt runoff destabilize smaller hot spring environments with smaller populations and result in high variation in the S. islandicus population over time. Therefore, physical abiotic features such as hot spring size and position in the landscape are important factors shaping the stability and diversity of the S. islandicus meta-population within Yellowstone National Park.

The apt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus.

UNLABELLED: Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus IMPORTANCE: Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.

Inferring Speciation Processes from Patterns of Natural Variation in Microbial Genomes.

Microbial species concepts have long been the focus of contentious debate, fueled by technological limitations to the genetic resolution of species, by the daunting task of investigating phenotypic variation among individual microscopic organisms, and by a lack of understanding of gene flow in reproductively asexual organisms that are prone to promiscuous horizontal gene transfer. Population genomics, the emerging approach of analyzing the complete genomes of a multitude of closely related organisms, is poised to overcome these limitations by providing a window into patterns of genome variation revealing the evolutionary processes through which species diverge. This new approach is more than just an extension of previous multilocus sequencing technologies, in that it provides a comprehensive view of interacting evolutionary processes. Here we argue that the application of population genomic tools in a rigorous population genetic framework will help to identify the processes of microbial speciation and ultimately lead to a general species concept based on the unique biology and ecology of microorganisms.

Phytoplankton succession affects the composition of Polynucleobacter subtypes in humic lakes.

Phytoplankton influence the composition of bacterial communities, but the taxonomic specificity of algal-bacterial interactions is unclear due to the aggregation of ecologically distinct bacterial populations by community characterization methods. Here we examine whether phytoplankton seasonal succession affects the composition of subtypes within the cosmopolitan freshwater bacterial genus Polynucleobacter. Changes in the composition of Polynucleobacter subtypes were characterized in samples collected weekly from May to August in 2003 and 2008 from three humic lakes using terminal restriction fragment length polymorphism fingerprinting of the protein-encoding cytochrome c oxidase ccoN gene. Changes in phytoplankton population abundances explained, on average, 30% of temporal variation in the composition of Polynucleobacter subtypes and the interaction between phytoplankton and the environment explained an additional 18% of temporal variation. The effect of phytoplankton on specific Polynucleobacter subtypes was experimentally confirmed by changes in Polynucleobacter subtype composition following incubation with different phytoplankton assemblages or a no-phytoplankton control. Phytoplankton-associated subtypes and differentiation in substrate use among subtypes likely contribute to the effects of phytoplankton on Polynucleobacter subtype composition. Interactions between unique Polynucleobacter populations and phytoplankton highlight the ecological significance and specificity of species interactions in freshwater communities.

Patterns of gene flow define species of thermophilic Archaea.

Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

ITEP: an integrated toolkit for exploration of microbial pan-genomes.

BACKGROUND: Comparative genomics is a powerful approach for studying variation in physiological traits as well as the evolution and ecology of microorganisms. Recent technological advances have enabled sequencing large numbers of related genomes in a single project, requiring computational tools for their integrated analysis. In particular, accurate annotations and identification of gene presence and absence are critical for understanding and modeling the cellular physiology of newly sequenced genomes. Although many tools are available to compare the gene contents of related genomes, new tools are necessary to enable close examination and curation of protein families from large numbers of closely related organisms, to integrate curation with the analysis of gain and loss, and to generate metabolic networks linking the annotations to observed phenotypes. RESULTS: We have developed ITEP, an Integrated Toolkit for Exploration of microbial Pan-genomes, to curate protein families, compute similarities to externally-defined domains, analyze gene gain and loss, and generate draft metabolic networks from one or more curated reference network reconstructions in groups of related microbial species among which the combination of core and variable genes constitute the their "pan-genomes". The ITEP toolkit consists of: (1) a series of modular command-line scripts for identification, comparison, curation, and analysis of protein families and their distribution across many genomes; (2) a set of Python libraries for programmatic access to the same data; and (3) pre-packaged scripts to perform common analysis workflows on a collection of genomes. ITEP's capabilities include de novo protein family prediction, ortholog detection, analysis of functional domains, identification of core and variable genes and gene regions, sequence alignments and tree generation, annotation curation, and the integration of cross-genome analysis and metabolic networks for study of metabolic network evolution. CONCLUSIONS: ITEP is a powerful, flexible toolkit for generation and curation of protein families. ITEP's modular design allows for straightforward extension as analysis methods and tools evolve. By integrating comparative genomics with the development of draft metabolic networks, ITEP harnesses the power of comparative genomics to build confidence in links between genotype and phenotype and helps disambiguate gene annotations when they are evaluated in both evolutionary and metabolic network contexts.

Differentiation between sediment and hypolimnion methanogen communities in humic lakes.

The traditional view of carbon cycling within the pelagic zone of freshwater lakes has consisted of methane production within the anoxic sediment, followed by diffusive flux and ebullition through the water column. Methanogenic archaea have been shown to be present within the water columns of freshwater lakes; however, little is known about whether these methanogenic communities are distinct from those in the sediment or how these communities change over space and time. We used the methanogen-specific phylogenetic marker mcrA to perform a 3-year study focusing on the community structure of methanogens within the sediment and anoxic hypolimnion water layer of five humic lakes in WI, USA. The hypolimnion and sediment communities were distinct in composition, richness and phylogenetic diversity. Hypolimnion communities displayed a temporally stable biogeographical pattern among lakes, which was driven by both lake-specific environmental variables and barriers to dispersal. We conclude that the hypolimnion comprised communities of methanogens that are distinct from those in the sediment, differentiated among lakes, and likely have unique ecological roles and evolutionary trajectories in these anaerobic environments.

How Do Small Things Make a Big Difference? Activities to Teach about Human-Microbe Interactions.

Recent scientific studies are providing increasing evidence for how microbes living in and on us are essential to our good health. However, many students still think of microbes only as germs that harm us. The classroom activities presented here are designed to shift student thinking on this topic. In these guided inquiry activities, students investigate human-microbe interactions as they work together to interpret and analyze authentic data from published articles and develop scientific models. Through the activities, students learn and apply ecological concepts as they come to see the human body as a fascinatingly complex ecosystem.

Computational analysis of genes with lethal knockout phenotype and prediction of essential genes in archaea.

The identification of microbial genes essential for survival as those with lethal knockout phenotype (LKP) is a common strategy for functional interrogation of genomes. However, interpretation of the LKP is complicated because a substantial fraction of the genes with this phenotype remains poorly functionally characterized. Furthermore, many genes can exhibit LKP not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes (conditionally essential genes). We analyzed the sets of LKP genes for two archaea, Methanococcus maripaludis and Sulfolobus islandicus, using a variety of computational approaches aiming to differentiate between essential and conditionally essential genes and to predict at least a general function for as many of the proteins encoded by these genes as possible. This analysis allowed us to predict the functions of several LKP genes including previously uncharacterized subunit of the GINS protein complex with an essential function in genome replication and of the KEOPS complex that is responsible for an essential tRNA modification as well as GRP protease implicated in protein quality control. Additionally, several novel antitoxins (conditionally essential genes) were predicted, and this prediction was experimentally validated by showing that the deletion of these genes together with the adjacent genes apparently encoding the cognate toxins caused no growth defect. We applied principal component analysis based on sequence and comparative genomic features showing that this approach can separate essential genes from conditionally essential ones and used it to predict essential genes in other archaeal genomes.IMPORTANCEOnly a relatively small fraction of the genes in any bacterium or archaeon is essential for survival as demonstrated by the lethal effect of their disruption. The identification of essential genes and their functions is crucial for understanding fundamental cell biology. However, many of the genes with a lethal knockout phenotype remain poorly functionally characterized, and furthermore, many genes can exhibit this phenotype not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes. We applied state-of-the-art computational methods to predict the functions of a number of uncharacterized genes with the lethal knockout phenotype in two archaeal species and developed a computational approach to predict genes involved in essential functions. These findings advance the current understanding of key functionalities of archaeal cells.

Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus.

Virus-host interactions are a key factor shaping population dynamics of microbial species. The CRISPR-Cas adaptive immune system confers sequence-specific immunity to viral infection and has the potential to dramatically shape coevolutionary interactions between viruses and their microbial hosts. To assess evolutionary dynamics of CRISPR loci, we have sampled a population of closely related Sulfolobus islandicus strains from Kamchatka, Russia at two time points, 10 years apart. Sequence analysis of the conserved trailer sequences reveals that alleles are reassorted among three CRISPR spacer loci into combinatorial genotypes. Reassortment provides the evolutionary independence of CRISPR loci from one another as demonstrated by the differential change in allele frequencies between two time points. Genome sequences of 12 strains from this population also reveal very recent horizontal gene transfer of novel, divergent cas gene cassettes. The evolutionary independence of CRISPR loci from each other and of the cas genes that control their function are consistent with the evolutionary expectation that reassortment increases the efficiency of adaptation at these loci that are likely under strong selection by lytic viruses.

Evolutionary causes and consequences of diversified CRISPR immune profiles in natural populations.

Host-pathogen co-evolution is a significant force which shapes the ecology and evolution of all types of organisms, and such interactions are driven by resistance and immunity mechanisms of the host. Diversity of resistance and immunity can affect the co-evolutionary trajectory of both host and pathogen. The microbial CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is one host immunity mechanism which offers a tractable model for examining the dynamics of diversity in an immune system. In the present article, we review CRISPR variation observed in a variety of natural populations, examine the forces which can push CRISPRs towards high or low diversity, and investigate the consequences of various levels of diversity on microbial populations.

Sulfolobus islandicus: a model system for evolutionary genomics.

Sulfolobus islandicus has been developed as a model system for combining approaches of evolutionary and molecular biology in Archaea. We describe how the application of this interdisciplinary approach can lead to novel hypotheses derived from patterns of natural variation that can be tested in the laboratory when combined with a diversity of natural variants and versatile genetic markers. We review how this approach has highlighted the importance of recombination as an evolutionary parameter and provided insight into a molecular mechanism of recombination that may be unique in the archaeal domain. We review the development and improvement of the model system S. islandicus that will enable us to study the mechanism and genomic architecture of recombination guided by evolutionary genomic analysis of Nature's ongoing experiments in wild populations.

Augmenting the genetic toolbox for Sulfolobus islandicus with a stringent positive selectable marker for agmatine prototrophy.

Sulfolobus species have become the model organisms for studying the unique biology of the crenarchaeal division of the archaeal domain. In particular, Sulfolobus islandicus provides a powerful opportunity to explore natural variation via experimental functional genomics. To support these efforts, we further expanded genetic tools for S. islandicus by developing a stringent positive selection for agmatine prototrophs in strains in which the argD gene, encoding arginine decarboxylase, has been deleted. Strains with deletions in argD were shown to be auxotrophic for agmatine even in nutrient-rich medium, but growth could be restored by either supplementation of exogenous agmatine or reintroduction of a functional copy of the argD gene from S. solfataricus P2 into the ΔargD host. Using this stringent selection, a robust targeted gene knockout system was established via an improved next generation of the MID (marker insertion and unmarked target gene deletion) method. Application of this novel system was validated by targeted knockout of the upsEF genes involved in UV-inducible cell aggregation formation.