Pharmacological Improvement of CFTR Function Rescues Airway Epithelial Homeostasis and Host Defense in Children with Cystic Fibrosis.

RATIONALE: Pharmacological improvement of CFTR function with elexacaftor/tezacaftor/ivacaftor (ETI) provides unprecedented improvements in lung function and other clinical outcomes to patients with cystic fibrosis (CF). However, ETI effects on impaired mucosal homeostasis and host defense at the molecular and cellular level in the airways of CF patients remain unknown. OBJECTIVES: To investigate effects of ETI on the transcriptiome of nasal epithelial and immune cells from children with CF at the single cell level. METHODS: Nasal swabs from 13 children with CF and at least one F508del allele aged 6 to 11 years were collected at baseline and three months after initiation of ETI, subjected to scRNA-seq and compared to swabs from 12 age-matched healthy children. MEASUREMENTS AND MAIN RESULTS: Proportions of CFTR-positive cells were decreased in epithelial basal, club and goblet cells, but not in ionocytes from children with CF at baseline and were restored on ETI therapy to near healthy levels. Single cell transcriptomics revealed an impaired interferon signalling and reduced expression of MHC I and II encoding genes in epithelial cells of CF children at baseline, which was partially restored by ETI. Additionally, ETI therapy markedly reduced the inflammatory phenotype of immune cells, particularly of neutrophils and macrophages. CONCLUSIONS: Pharmacological improvement of CFTR function improves innate mucosal immunity and reduces immune cell inflammatory responses in the upper airways of children with CF at the single cell level, highlighting the potential to restore epithelial homeostasis and host defense in CF airways by early initiation of ETI therapy. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Non-classical circulating monocytes expressing high levels of microsomal prostaglandin E2 synthase-1 tag an aberrant IFN-response in systemic sclerosis.

Systemic sclerosis (SSc) is a complex disease that affects the connective tissue, causing fibrosis. SSc patients show altered immune cell composition and activation in the peripheral blood (PB). PB monocytes (Mos) are recruited into tissues where they differentiate into macrophages, which are directly involved in fibrosis. To understand the role of CD14+ PB Mos in SSc, a single-cell transcriptome analysis (scRNA-seq) was conducted on 8 SSc patients and 8 controls. Using unsupervised clustering methods, CD14+ cells were assigned to 11 clusters, which added granularity to the known monocyte subsets: classical (cMos), intermediate (iMos) and non-classical Mos (ncMos) or type 2 dendritic cells. NcMos were significantly overrepresented in SSc patients and showed an active IFN-signature and increased expression levels of PTGES, in addition to monocyte motility and adhesion markers. We identified a SSc-related cluster of IRF7+ STAT1+ iMos with an aberrant IFN-response. Finally, a depletion of M2 polarised cMos in SSc was observed. Our results highlighted the potential of PB Mos as biomarkers for SSc and provided new possibilities for putative drug targets for modulating the innate immune response in SSc.

The salmon louse genome: Copepod features and parasitic adaptations.

Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.

Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy.

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in pre-neoplastic monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significantly differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and post-therapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state, and a distinct expression profile together with MBL cells compared to normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically-driven growth dynamics and together with its persistent presence suggests a central role in the normal-to-cancer transition.

An Unbiased Molecular Approach Using 3'-UTRs Resolves the Avian Family-Level Tree of Life.

Presumably, due to a rapid early diversification, major parts of the higher-level phylogeny of birds are still resolved controversially in different analyses or are considered unresolvable. To address this problem, we produced an avian tree of life, which includes molecular sequences of one or several species of ∼90% of the currently recognized family-level taxa (429 species, 379 genera) including all 106 family-level taxa of the nonpasserines and 115 of the passerines (Passeriformes). The unconstrained analyses of noncoding 3-prime untranslated region (3'-UTR) sequences and those of coding sequences yielded different trees. In contrast to the coding sequences, the 3'-UTR sequences resulted in a well-resolved and stable tree topology. The 3'-UTR contained, unexpectedly, transcription factor binding motifs that were specific for different higher-level taxa. In this tree, grebes and flamingos are the sister clade of all other Neoaves, which are subdivided into five major clades. All nonpasserine taxa were placed with robust statistical support including the long-time enigmatic hoatzin (Opisthocomiformes), which was found being the sister taxon of the Caprimulgiformes. The comparatively late radiation of family-level clades of the songbirds (oscine Passeriformes) contrasts with the attenuated diversification of nonpasseriform taxa since the early Miocene. This correlates with the evolution of vocal production learning, an important speciation factor, which is ancestral for songbirds and evolved convergent only in hummingbirds and parrots. As 3'-UTR-based phylotranscriptomics resolved the avian family-level tree of life, we suggest that this procedure will also resolve the all-species avian tree of life.

Epigenetic regulator function through mouse gastrulation.

During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type-specific transcription factors and ubiquitous epigenetic machinery, which recognizes universally available histone residues or nucleotides in a context-dependent manner1,2. The molecular functions of these regulators are generally well understood, but assigning direct developmental roles to them is hampered by complex mutant phenotypes that often emerge after gastrulation3,4. Single-cell RNA sequencing and analytical approaches have explored this highly conserved, dynamic period across numerous model organisms5-8, including mouse9-18. Here we advance these strategies using a combined zygotic perturbation and single-cell RNA-sequencing platform in which many mutant mouse embryos can be assayed simultaneously, recovering robust  morphological and transcriptional information across a panel of ten essential regulators. Deeper analysis of central Polycomb repressive complex (PRC) 1 and 2 components indicates substantial cooperativity, but distinguishes a dominant role for PRC2 in restricting the germline. Moreover, PRC mutant phenotypes emerge after gross epigenetic and transcriptional changes within the initial conceptus prior to gastrulation. Our experimental framework may eventually lead to a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.

Ice-Age Climate Adaptations Trap the Alpine Marmot in a State of Low Genetic Diversity.

Some species responded successfully to prehistoric changes in climate [1, 2], while others failed to adapt and became extinct [3]. The factors that determine successful climate adaptation remain poorly understood. We constructed a reference genome and studied physiological adaptations in the Alpine marmot (Marmota marmota), a large ground-dwelling squirrel exquisitely adapted to the "ice-age" climate of the Pleistocene steppe [4, 5]. Since the disappearance of this habitat, the rodent persists in large numbers in the high-altitude Alpine meadow [6, 7]. Genome and metabolome showed evidence of adaptation consistent with cold climate, affecting white adipose tissue. Conversely, however, we found that the Alpine marmot has levels of genetic variation that are among the lowest for mammals, such that deleterious mutations are less effectively purged. Our data rule out typical explanations for low diversity, such as high levels of consanguineous mating, or a very recent bottleneck. Instead, ancient demographic reconstruction revealed that genetic diversity was lost during the climate shifts of the Pleistocene and has not recovered, despite the current high population size. We attribute this slow recovery to the marmot's adaptive life history. The case of the Alpine marmot reveals a complicated relationship between climatic changes, genetic diversity, and conservation status. It shows that species of extremely low genetic diversity can be very successful and persist over thousands of years, but also that climate-adapted life history can trap a species in a persistent state of low genetic diversity.

Global delay in nascent strand DNA methylation.

Cytosine methylation is widespread among organisms and essential for mammalian development. In line with early postulations of an epigenetic role in gene regulation, symmetric CpG methylation can be mitotically propagated over many generations with extraordinarily high fidelity. Here, we combine BrdU labeling and immunoprecipitation with genome-wide bisulfite sequencing to explore the inheritance of cytosine methylation onto newly replicated DNA in human cells. Globally, we observe a pronounced lag between the copying of genetic and epigenetic information in embryonic stem cells that is reconsolidated within hours to accomplish faithful mitotic transmission. Populations of arrested cells show a global reduction of lag-induced intermediate CpG methylation when compared to proliferating cells, whereas sites of transcription factor engagement appear cell-cycle invariant. Alternatively, the cancer cell line HCT116 preserves global epigenetic heterogeneity independently of cell-cycle arrest. Taken together, our data suggest that heterogeneous methylation largely reflects asynchronous proliferation, but is intrinsic to actively engaged cis-regulatory elements and cancer.

Author Correction: Global delay in nascent strand DNA methylation.

Following online publication of this article, the Gene Expression Omnibus records corresponding to accession codes GSM2406773, MN-d6, and GSM2406772, MN-d14, listed in the data availability statement were deleted. The data are now available under accession codes GSM3039355, WGBS_hESC_WT_D6_R4 (MN day 6), and GSM3039351, WGBS_hESC_WT_D14_R4 (MN day 14), and the data availability statement has been updated with the new accession codes in the HTML and PDF versions of the article.

Genome-wide tracking of dCas9-methyltransferase footprints.

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

Allele frequencies of BRAFV600 mutations in primary melanomas and matched metastases and their relevance for BRAF inhibitor therapy in metastatic melanoma.

BACKGROUND: The detection of BRAFV600 mutations in patients with metastatic melanoma is important because of the availability of BRAF inhibitor therapy. However, the clinical relevance of the frequency of BRAFV600 mutant alleles is unclear. PATIENTS AND METHODS: Allele frequencies of BRAFV600 mutations were analyzed by ultra-deep next-generation sequencing in formalin-fixed, paraffin-embedded melanoma tissue (75 primary melanomas and 88 matched metastases). In a second study, pretreatment specimens from 76 patients who received BRAF inhibitors were retrospectively analyzed, and BRAFV600 allele frequencies were correlated with therapeutic results. RESULTS: Thirty-five patients had concordantly BRAF-positive and 36 (48%) patients had concordantly BRAF-negative primary melanomas and matched metastases, and four patients had discordant samples with low allele frequencies (3.4-5.2%). Twenty-six of 35 patients with concordant samples had BRAFV600E mutations, three of whom had additional mutations (V600K in two patients and V600R in one) and nine patients had exclusively non-V600E mutations (V600K in eight patients and V600E -c.1799_1800TG > AA- in one patient). The frequency of mutated BRAFV600 alleles was similar in the primary melanoma and matched metastasis in 27/35 patients, but differed by >3-fold in 8/35 of samples. BRAFV600E allele frequencies in pretreatment tumor specimens were not significantly correlated with treatment outcomes in 76 patients with metastatic melanoma who were treated with BRAF inhibitors. CONCLUSIONS: BRAFV600 mutation status and allele frequency is consistent in the majority of primary melanomas and matched metastases. A small subgroup of patients has double mutations. BRAFV600 allele frequencies are not correlated with the response to BRAF inhibitors.

Using the canary genome to decipher the evolution of hormone-sensitive gene regulation in seasonal singing birds.

BACKGROUND: While the song of all songbirds is controlled by the same neural circuit, the hormone dependence of singing behavior varies greatly between species. For this reason, songbirds are ideal organisms to study ultimate and proximate mechanisms of hormone-dependent behavior and neuronal plasticity. RESULTS: We present the high quality assembly and annotation of a female 1.2-Gbp canary genome. Whole genome alignments between the canary and 13 genomes throughout the bird taxa show a much-conserved synteny, whereas at the single-base resolution there are considerable species differences. These differences impact small sequence motifs like transcription factor binding sites such as estrogen response elements and androgen response elements. To relate these species-specific response elements to the hormone-sensitivity of the canary singing behavior, we identify seasonal testosterone-sensitive transcriptomes of major song-related brain regions, HVC and RA, and find the seasonal gene networks related to neuronal differentiation only in the HVC. Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation. Among the testosterone-regulated genes of canary HVC, 20% lack estrogen response elements and 4 to 8% lack androgen response elements in orthologous promoters in the zebra finch. CONCLUSIONS: The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior. Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation.

The European sea bass (Dicentrarchus labrax) is a temperate-zone euryhaline teleost of prime importance for aquaculture and fisheries. This species is subdivided into two naturally hybridizing lineages, one inhabiting the north-eastern Atlantic Ocean and the other the Mediterranean and Black seas. Here we provide a high-quality chromosome-scale assembly of its genome that shows a high degree of synteny with the more highly derived teleosts. We find expansions of gene families specifically associated with ion and water regulation, highlighting adaptation to variation in salinity. We further generate a genome-wide variation map through RAD-sequencing of Atlantic and Mediterranean populations. We show that variation in local recombination rates strongly influences the genomic landscape of diversity within and differentiation between lineages. Comparing predictions of alternative demographic models to the joint allele-frequency spectrum indicates that genomic islands of differentiation between sea bass lineages were generated by varying rates of introgression across the genome following a period of geographical isolation.

Transcriptome sequencing and microarray design for functional genomics in the extremophile Arabidopsis relative Thellungiella salsuginea (Eutrema salsugineum).

BACKGROUND: Most molecular studies of plant stress tolerance have been performed with Arabidopsis thaliana, although it is not particularly stress tolerant and may lack protective mechanisms required to survive extreme environmental conditions. Thellungiella salsuginea has attracted interest as an alternative plant model species with high tolerance of various abiotic stresses. While the T. salsuginea genome has recently been sequenced, its annotation is still incomplete and transcriptomic information is scarce. In addition, functional genomics investigations in this species are severely hampered by a lack of affordable tools for genome-wide gene expression studies. RESULTS: Here, we report the results of Thellungiella de novo transcriptome assembly and annotation based on 454 pyrosequencing and development and validation of a T. salsuginea microarray. ESTs were generated from a non-normalized and a normalized library synthesized from RNA pooled from samples covering different tissues and abiotic stress conditions. Both libraries yielded partially unique sequences, indicating their necessity to obtain comprehensive transcriptome coverage. More than 1 million sequence reads were assembled into 42,810 unigenes, approximately 50% of which could be functionally annotated. These unigenes were compared to all available Thellungiella genome sequence information. In addition, the groups of Late Embryogenesis Abundant (LEA) proteins, Mitogen Activated Protein (MAP) kinases and protein phosphatases were annotated in detail. We also predicted the target genes for 384 putative miRNAs. From the sequence information, we constructed a 44 k Agilent oligonucleotide microarray. Comparison of same-species and cross-species hybridization results showed superior performance of the newly designed array for T. salsuginea samples. The developed microarrays were used to investigate transcriptional responses of T. salsuginea and Arabidopsis during cold acclimation using the MapMan software. CONCLUSIONS: This study provides the first comprehensive transcriptome information for the extremophile Arabidopsis relative T. salsuginea. The data constitute a more than three-fold increase in the number of publicly available unigene sequences and will greatly facilitate genome annotation. In addition, we have designed and validated the first genome-wide microarray for T. salsuginea, which will be commercially available. Together with the publicly available MapMan software this will become an important tool for functional genomics of plant stress tolerance.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Long-term survival of hydrated resting eggs from Brachionus plicatilis.

BACKGROUND: Several organisms display dormancy and developmental arrest at embryonic stages. Long-term survival in the dormant form is usually associated with desiccation, orthodox plant seeds and Artemia cysts being well documented examples. Several aquatic invertebrates display dormancy during embryonic development and survive for tens or even hundreds of years in a hydrated form, raising the question of whether survival in the non-desiccated form of embryonic development depends on pathways similar to those occurring in desiccation tolerant forms. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, Illumina short read sequencing was used to generate transcription profiles from the resting and amictic eggs of an aquatic invertebrate, the rotifer, Brachionus plicatilis. These two types of egg have very different life histories, with the dormant or diapausing resting eggs, the result of the sexual cycle and amictic eggs, the non-dormant products of the asexual cycle. Significant transcriptional differences were found between the two types of egg, with amictic eggs rich in genes involved in the morphological development into a juvenile rotifer. In contrast, representatives of classical "stress" proteins: a small heat shock protein, ferritin and Late Embryogenesis Abundant (LEA) proteins were identified in resting eggs. More importantly however, was the identification of transcripts for messenger ribonucleoprotein particles which stabilise RNA. These inhibit translation and provide a valuable source of useful RNAs which can be rapidly activated on the exit from dormancy. Apoptotic genes were also present. Although apoptosis is inconsistent with maintenance of prolonged dormancy, an altered apoptotic pathway has been proposed for Artemia, and this may be the case with the rotifer. CONCLUSIONS: These data represent the first transcriptional profiling of molecular processes associated with dormancy in a non-desiccated form and indicate important similarities in the molecular pathways activated in resting eggs compared with desiccated dormant forms, specifically plant seeds and Artemia.

Genomic insights into the physiology and ecology of the marine filamentous cyanobacterium Lyngbya majuscula.

Filamentous cyanobacteria of the genus Lyngbya are important contributors to coral reef ecosystems, occasionally forming dominant cover and impacting the health of many other co-occurring organisms. Moreover, they are extraordinarily rich sources of bioactive secondary metabolites, with 35% of all reported cyanobacterial natural products deriving from this single pantropical genus. However, the true natural product potential and life strategies of Lyngbya strains are poorly understood because of phylogenetic ambiguity, lack of genomic information, and their close associations with heterotrophic bacteria and other cyanobacteria. To gauge the natural product potential of Lyngbya and gain insights into potential microbial interactions, we sequenced the genome of Lyngbya majuscula 3L, a Caribbean strain that produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide, using a combination of Sanger and 454 sequencing approaches. Whereas ∼ 293,000 nucleotides of the draft genome are putatively dedicated to secondary metabolism, this is far too few to encode a large suite of Lyngbya metabolites, suggesting Lyngbya metabolites are strain specific and may be useful in species delineation. Our analysis revealed a complex gene regulatory network, including a large number of sigma factors and other regulatory proteins, indicating an enhanced ability for environmental adaptation or microbial associations. Although Lyngbya species are reported to fix nitrogen, nitrogenase genes were not found in the genome or by PCR of genomic DNA. Subsequent growth experiments confirmed that L. majuscula 3L is unable to fix atmospheric nitrogen. These unanticipated life history characteristics challenge current views of the genus Lyngbya.

Deep sequencing of naupliar-, cyprid- and adult-specific normalised Expressed Sequence Tag (EST) libraries of the acorn barnacle Balanus amphitrite.

In order to improve the genetic characterisation of the barnacle Balanus amphitrite, normalised EST libraries for the developmental stages, viz. nauplius (a mix of instars I and II), cyprid and adult, were generated. The libraries were sequenced independently using 454 technologies and 575,666 reads were generated. For adults, 4843 unique isotigs were estimated and 6754 and 7506 in the cyprid and naupliar stage, respectively. It was found that some of the previously proposed cyprid-specific bcs genes were also expressed during the naupliar and adult stage. Furthermore, as lectins have been hypothesised to influence settlement cue recognition in barnacles, the database was searched for lectin-like isotigs. Two proteins, uniquely expressed in either the cyprid or the adult stage, matched a mannose receptor, and their nucleotide sequences were 33% and 31% identical to a lectin (BRA-3) isolated from Megabalanus rosa. Further characterisation of these genes may suggest their involvement in settlement.

Genome of Streptococcus oralis strain Uo5.

Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci, which includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple-antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci and their potential to engage in interspecies gene transfer.

Identification of genes essential for prey-independent growth of Bdellovibrio bacteriovorus HD100.

Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Whole-genome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.