Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteria.

DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood. Here, we discover a highly conserved but previously uncharacterized transcription factor (Cada2), which orchestrates a methylation-dependent adaptive response in Caulobacter. This response operates independently of the SOS response, governs the expression of genes crucial for direct repair, and is essential for surviving methylation-induced damage. Our molecular investigation of Cada2 reveals a cysteine methylation-dependent posttranslational modification (PTM) and mode of action distinct from its Escherichia coli counterpart, a trait conserved across all bacteria harboring a Cada2-like homolog instead. Extending across the bacterial kingdom, our findings support the notion of divergence and coevolution of adaptive response transcription factors and their corresponding sequence-specific DNA motifs. Despite this diversity, the ubiquitous prevalence of adaptive response regulators underscores the significance of a transcriptional switch, mediated by methylation PTM, in driving a specific and essential bacterial DNA damage response.

Chronic hypoxia is associated with transcriptomic reprogramming and increased genomic instability in cancer cells.

Hypoxia afflicts the microenvironment of solid tumors fueling malignancy. We investigated the impact of long hypoxia exposure on transcriptional remodeling, tumor mutational burden (TMB), and genomic instability of cancer cells that were grouped based on their inherent sensitivity or resistance to hypoxia. A hypoxia score was used as a metric to distinguish between the most hypoxia-sensitive (hypoxia high (HH)), and most resistant (hypoxia low (HL)) cancer cells. By applying whole exome sequencing and microarray analysis, we showed that the HH group was indeed more sensitive to hypoxia, having significantly higher TMB (p = 0.03) and copy number losses (p = 0.03), as well as a trend of higher transcriptional response. Globally cells adapted by decreasing expression of genes involved in metabolism, proliferation, and protein maturation, and increasing alternative splicing. They accumulated mutations, especially frameshift insertions, and harbored increased copy number alterations, indicating increased genomic instability. Cells showing highest TMB simultaneously experienced a significant downregulation of DNA replication and repair and chromosomal maintenance pathways. A sixteen-gene common response to chronic hypoxia was put forth, including genes regulating angiogenesis and proliferation. Our findings show that chronic hypoxia enables survival of tumor cells by metabolic reprogramming, modulating proliferation, and increasing genomic instability. They additionally highlight key adaptive pathways that can potentially be targeted to prevent cancer cells residing in chronically hypoxic tumor areas from thriving.

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of 'N' (0-10) spacer-linked target specific primers without PhiX spike-in.

BACKGROUND: Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing 'N' (0-10) spacers in target gene amplification primers that are pooled for simple handling. RESULT: We evaluated the efficiency of 'N' (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of 'N' (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,"MetReTrim", to trim the 'N' (0-10) spacers from the raw reads ( https://github.com/Mohak91/MetReTrim ). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method. CONCLUSION: This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using 'N' (0-10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

Genomic Analysis of Waterpipe Smoke-Induced Lung Tumor Autophagy and Plasticity.

The role of autophagy in lung cancer cells exposed to waterpipe smoke (WPS) is not known. Because of the important role of autophagy in tumor resistance and progression, we investigated its relationship with WP smoking. We first showed that WPS activated autophagy, as reflected by LC3 processing, in lung cancer cell lines. The autophagy response in smokers with lung adenocarcinoma, as compared to non-smokers with lung adenocarcinoma, was investigated further using the TCGA lung adenocarcinoma bulk RNA-seq dataset with the available patient metadata on smoking status. The results, based on a machine learning classification model using Random Forest, indicate that smokers have an increase in autophagy-activating genes. Comparative analysis of lung adenocarcinoma molecular signatures in affected patients with a long-term active exposure to smoke compared to non-smoker patients indicates a higher tumor mutational burden, a higher CD8+ T-cell level and a lower dysfunction level in smokers. While the expression of the checkpoint genes tested-PD-1, PD-L1, PD-L2 and CTLA-4-remains unchanged between smokers and non-smokers, B7-1, B7-2, IDO1 and CD200R1 were found to be higher in non-smokers than smokers. Because multiple factors in the tumor microenvironment dictate the success of immunotherapy, in addition to the expression of immune checkpoint genes, our analysis explains why patients who are smokers with lung adenocarcinoma respond better to immunotherapy, even though there are no relative differences in immune checkpoint genes in the two groups. Therefore, targeting autophagy in lung adenocarcinoma patients, in combination with checkpoint inhibitor-targeted therapies or chemotherapy, should be considered in smoker patients with lung adenocarcinoma.

Evolutionary and Comparative Analysis of Bacterial Nonhomologous End Joining Repair.

DNA double-strand breaks (DSBs) are a threat to genome stability. In all domains of life, DSBs are faithfully fixed via homologous recombination. Recombination requires the presence of an uncut copy of duplex DNA which is used as a template for repair. Alternatively, in the absence of a template, cells utilize error-prone nonhomologous end joining (NHEJ). Although ubiquitously found in eukaryotes, NHEJ is not universally present in bacteria. It is unclear as to why many prokaryotes lack this pathway. Toward understanding what could have led to the current distribution of bacterial NHEJ, we carried out comparative genomics and phylogenetic analysis across ∼6,000 genomes. Our results show that this pathway is sporadically distributed across the phylogeny. Ancestral reconstruction further suggests that NHEJ was absent in the eubacterial ancestor and can be acquired via specific routes. Integrating NHEJ occurrence data for archaea, we also find evidence for extensive horizontal exchange of NHEJ genes between the two kingdoms as well as across bacterial clades. The pattern of occurrence in bacteria is consistent with correlated evolution of NHEJ with key genome characteristics of genome size and growth rate; NHEJ presence is associated with large genome sizes and/or slow growth rates, with the former being the dominant correlate. Given the central role these traits play in determining the ability to carry out recombination, it is possible that the evolutionary history of bacterial NHEJ may have been shaped by requirement for efficient DSB repair.

Whole-Genome Sequencing of Sphingobium sp. Strain RSMS, a Highly Efficient Tributyl Phosphate-Degrading Bacterium.

Sphingobium sp. strain RSMS was described earlier as an efficient degrader of tributyl phosphate, an organic pollutant. This report describes the generation and annotation of the genome sequence of Sphingobium sp. strain RSMS, which will facilitate future studies to identify genetic elements responsible for the degradation of tributyl phosphate.