Genome-wide transposon insertion sequencing (Tn-seq) is a powerful tool to measure the importance of genes for growth. In this study, we applied Tn-seq to the Gram-positive model system Bacillus subtilis, and found that after growth in liquid medium the transposon library lacked transposon insertions in several genes related to lipoteichoic acid biosynthesis and cell wall teichoic acid modification. This was unexpected since these genes are not essential for normal growth. By growing the transposon library as a confluent layer of cells, and as discrete colonies, we found that these genes are only important when the transposon library is grown as a confluent layer. Apparently, growing the transposon library as a mixed population reduces the fitness of teichoic acid mutants, which was confirmed by coculturing experiments. This phenomenon can be explained when lipoteichoic acid and teichoic acid D-alanylation mutants become sensitive to secreted autologous antimicrobials and/or toxins. Extensive mutant analyses suggested that multiple autologous antimicrobials are involved. Finally, we show that the reduced fitness of teichoic acid mutants can be countered by the addition of divalent cations. These data raise several questions concerning the evolution of kin discrimination, and show that growing genome-wide mutant libraries as mixed cultures can influence library composition. AUTHOR SUMMARY In this study, we have investigated an unexpected finding from a genome-wide genetic screen in the bacterium Bacillus subtilis. We discovered that mutants lacking certain cell wall components, known as lipoteichoic acids, were strongly disadvantaged when grown together with other bacterial cells, even though they grew normally on their own. Further experiments showed that these mutants are unusually sensitive to antimicrobial compounds and toxins produced by their own species. This suggests that teichoic acids help protect cells from attacks by closely related bacteria and may play an important role in kin discrimination, the ability of bacteria to distinguish between close relatives and non-relatives. We also found that this vulnerability can be reduced by adding divalent metal ions such as magnesium or calcium. Our findings provide new insights into bacterial social interactions and reveal an important consideration for large-scale genetic screening approaches.
Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.
Steven J. Hancock, M. Phan, Jie-Lian Zheng· Microbiology Australia· 0 citations
Transposons are fundamental genetic elements that have profoundly shaped the architecture of eukaryotic genomes. Yeasts and filamentous fungi have emerged as important chassis organisms for bioingredient production in synthetic biology and metabolic engineering. In this review, we summarise the current understanding and future opportunities in the development of transposon-based tools for genome engineering in these fungal systems. Fungal inverted terminal repeat (ITR) DNA transposons, as well as long terminal repeat (LTR) and non-LTR retrotransposons, can accelerate genomic mutagenesis, facilitating the screening of superior genotypes and phenotypes. CRISPR-associated transposons (CASTs) hold considerable potential for site-specific integration of large transgenes, bypassing the limitations imposed by low homologous recombination (HR) efficiency in non-Saccharomyces hosts. Overall, transposon-based tools represent a valuable and underexplored avenue to accelerate genome engineering and strain development in yeasts and filamentous fungi.
Bingyin Peng, Masahiro Tominaga, Chengqiang Wang et al.· Yeast· 1 citation
Summary Bacteriophage genomes are densely packed with coding sequences and frequently encode genes of unknown function. Unbiased phage functional genomics approaches are therefore needed, particularly for large lytic phages. Here, we harness the mariner transposase to develop phage transposon mutagenesis and sequencing (TnSeq), enabling pooled sequencing to identify both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage ΦKZ (280,334 bp; 371 predicted genes), we show that ~110 genes are fitness-conferring via phage TnSeq, identifying many known and previously unknown essential genes. Moreover, this phage carries ~261 non-essential genes, including some capsid and tail proteins, many of which are important for fitness across different clinical isolates or conditions. Phage TnSeq was also extended to a base-modified phage. Together, phage TnSeq is a scalable technology that can identify essential phage genes, generate knockouts in all non-essential genes, and sensitively assign the quantitative fitness contributions of every gene in parallel.
A. Chan, Wearn-Xin Yee, Deepto Mozumdar et al.· Cell· 0 citations
Klebsiella pneumoniae is a common cause of sepsis in adults and children. Given the emergence of multidrug-resistant isolates, there is an urgent need to develop novel therapeutic strategies. Essential bacterial genes and their products, which are critical to survival and replication, represent intrinsic vulnerabilities that can be leveraged as novel drug targets. A comprehensive, empirically derived essential genome required for growth under conditions relevant to human infection biology and conserved across a majority of clinically relevant strains of K. pneumoniae is currently lacking.
To define the core essential genome of K. pneumoniae, libraries comprising hundreds of thousands of individual transposon-insertion mutants were generated across multiple strains using a transposon mutagenesis vector (pSC189). To determine the K. pneumoniae strain MGH_66 genes required for growth in laboratory media, approximately 150,000 unique mutants were grown on lysogeny broth agar (LBA), and their genomic DNA (gDNA) was extracted. The gDNA was fragmented, adapter-ligated, and PCR-amplified using custom primers targeting transposon–genome junctions. Libraries were sequenced on an Illumina MiSeq platform, and insertion sites were mapped to the K. pneumoniae MGH_66 genome using Bowtie2 software. Gene essentiality was analyzed using the statistical analysis software FiTnEss.
The library of 150,000 K. pneumoniae MGH_66 transposon-insertion mutants saturated approximately 50% of all permissive transposon-insertion sites. Based on individual p values, 544 genes were predicted to be essential for K. pneumoniae MGH_66 growth on LBA, including gyrA, fusA, and argS; which encode subunit A of DNA gyrase, elongation factor G, and arginyl-tRNA synthetase, respectively. The present dataset was not powered to detect statistically significant essential genes after correction for multiple hypothesis testing.
These data suggest 544 K. pneumoniae MGH_66 genes essential for growth on laboratory media. Future work will focus on increasing statistical power of the present dataset and using this workflow to identify genes essential for growth in host-specific environments (e.g., blood, urine) for MGH_66 and additional strains. The resulting core essential genome will comprise genes required for growth across all tested conditions and strains. These findings may inform the identification of potential therapeutic targets for multidrug-resistant K. pneumoniae.
C. Wijers, Joseph A. Bagnall, Deborah T. Hung· Journal of the Pediatric Inf...· 0 citations
Abstract Transposon-insertion sequencing (Tn-seq) couples transposon mutagenesis with next-generation sequencing to identify the transposon insertion site for thousands of mutants in parallel. It is a powerful technology with a myriad of uses beyond the identification of essential genes required for a cell to grow and divide. Tn-seq is particularly useful as a high-throughput method to assign function to function-unknown genes, which have increased steadily with the abundance of newly sequenced bacterial genomes. Tn-seq has now been adapted for use in over 100 bacterial species. Here, we summarize the applications of Tn-seq for querying bacterial physiology and discuss some of the possible applications for the future.
