LoxTnSeq: Random Transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

The removal of unwanted genetic material is a key aspect in many synthetic biology efforts, and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deep-sequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.

Escape mutations circumvent a tradeoff between resistance to a beta-lactam and a beta-lactamase inhibitor

Beta-lactamase inhibitors are increasingly used to counteract microbial resistance to beta-lactam antibiotics mediated by beta-lactamase enzymes. These inhibitors compete with the beta-lactam drug for the same binding site of the beta-lactamase, thereby generating an inherent evolutionary tradeoff: enzyme mutations that increase its activity against the beta-lactam drug also increase its susceptibility towards the inhibitor. It is unclear how common and accessible are mutants that escape this adaptive tradeoff. Here, systematically constructing and phenotyping a deep mutant library of the ampC beta-lactamase gene of Escherichia coli, we identified escape mutations, which even in the presence of the enzyme inhibitor allow growth at beta-lactam concentrations far exceeding the native inhibitory levels of the wildtype strain. Importantly, while such escape mutations appear for combinations of avibactam with some beta-lactam drugs, for other drugs escape phenotypes are completely restricted. Amplicon sequencing of the selected mutant pool identified these escape mutations and showed that they are rare and drug specific. For the combination of avibactam with aztreonam, an escape phenotype was conferred via multiple substitutions in a single conserved amino acid (Tyr 150). In contrast, a different set of mutations showed an escape phenotype for cefepime, and no escape mutants appeared for piperacillin. The differential adaptive potential of ampC to combinations of avibactam and different beta-lactam drugs can help guide drug treatments that are more resilient to evolution of resistance.

LMM24 Encodes Receptor-Like Cytoplasmic Kinase 109, Which Regulates Cell Death and Defense Responses in Rice

Lesion mimic mutants are excellent models for research on molecular mechanisms of cell death and defense responses in rice. We identified a new rice lesion mimic mutant lmm24 from a mutant pool of indica rice cultivar “ZhongHui8015”. The LMM24 gene was identified by MutMap, and LMM24 was confirmed as a receptor-like cytoplasmic kinase 109 by amino acid sequence analysis. The lmm24 mutant displayed dark brown lesions in leaves and growth retardation that were not observed in wild-type ZH8015. The results of histochemical staining and TUNEL assays showed enhanced ROS accumulation and cell death in lmm24. Chloroplast degradation was observed in lmm24 leaves, with decreased expression of photosynthesis-related genes and increased expression of the senescence-induced STAYGREEN (SGR) gene and other senescence-associated genes. Furthermore, lmm24 exhibited enhanced resistance to rice blast fungus Magnaporthe oryzae (M. oryzae) and up-regulation of defense response genes. Our data demonstrate that LMM24 regulates cell death and defense responses in rice.

Use of a Yeast TRNase Killer Toxin to Diagnose Kti12 Motifs Required for TRNA Modification by Elongator

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g. nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

ABSTRACTWhole-genome fitness analysis in microbes that uses saturating transposon mutagenesis combined with massively parallel sequencing (Tn-seq) is providing a measure of the contribution of each gene to a given growth condition. With this technique, gene fitness profiles and essential genes are discovered by simultaneous analyses of whether the absence of each gene product alters the growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure to simplify and shorten the process by including delivery of the transposon through conjugation and liquid culture enrichment of the mutant pool, creating transposon liquid enrichment sequencing (TnLE-seq). To illustrate the success of these modifications and the robustness of the procedure, analyses of gene fitness of two cultures of the strictly anaerobic bacteriumDesulfovibrio vulgarisHildenborough were performed, with growth on lactate as the electron donor and sulfate as the electron acceptor. These data demonstrate reproducibility and provide a base condition for analysis of fitness changes in deletion mutants and in various growth conditions. The procedural modifications will facilitate the application of this powerful genetic analysis to microbes lacking a facile genetic system. Pilot studies produced 2.5 × 105and 3.4 × 105unique insertion mutants in the anaerobeDesulfovibrio vulgarisHildenborough grown under typical laboratory conditions in rich medium. These analyses provided two similar high-resolution maps of gene fitness across the genome, and the method was also applied to growth in minimal medium. These results were also compared to the coverage obtained with a ca. 13,000-member cataloged transposon library constructed by sequencing transposon insertion sites in individual mutants.

Novel Determinants of Intestinal Colonization of Salmonella enterica Serotype Typhimurium Identified in Bovine Enteric Infection

ABSTRACTCattle are naturally infected withSalmonella entericaserotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidalSalmonellainfection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes ofSalmonellaTyphimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants,STM3602andSTM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains.STM3602encodes a putative transcriptional regulator that may be involved in phosphonate utilization, andSTM3846encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed bySalmonellato survive within the small intestines of cattle.

Exploring the Mode of Action of Antimicrobial Peptide MUC7 12-Mer by Fitness Profiling of Saccharomyces cerevisiae Genomewide Mutant Collection

ABSTRACT The MUC7 12-mer (RKSYKCLHKRCR) is a cationic antimicrobial peptide derived from the human salivary mucin MUC7. To study its effect/mechanism of action on fungi, we performed a fitness screen of a tagged, diploid, homozygous gene deletion mutant pool of the yeast Saccharomyces cerevisiae grown in the presence of the MUC7 peptide. Forty-five strains exhibiting reduced fitness and 13 strains exhibiting increased fitness (sensitivity or resistance, respectively) were identified by hybridization intensities to tag arrays. The strongest fitness defects were observed with deletions in genes encoding elements of the RIM101 signaling pathway (regulating response to alkaline and neutral pH and other environmental conditions) and of the endosomal sorting complex required for transport (ESCRT; functioning mainly in protein sorting for degradation, but also required for activation of the RIM101 pathway). Other deletions identified as conferring fitness defect or gain are in genes associated with a variety of functions, including transcription regulation, protein trafficking, transport, metabolism, and others. The results of the pool fitness screen were validated by a set of mutant strains tested individually in the presence of the MUC7 12-mer. All tested RIM101-related deletion strains showing fitness defects confirmed their sensitivities. Taken together, the results led us to conclude that deletions of genes associated with the RIM101 pathway confer sensitivity to the peptide by preventing activation of this pathway and that this stress response plays a major role in the protection of S. cerevisiae against damage inflicted by the MUC7 12-mer peptide.