Biology subjects

Sharda, M.

Publications and source records attributed to Sharda, M..

5 recordsLinked to original sources

bioRxiv2024

The evolution of sequence specificity in a DNA binding protein family

Transcriptional regulation enables bacteria to adjust to its environment. This is driven by transcription factors (TFs), which display DNA site recognition specificity with some flexibility built in. TFs, however, are not considered essential to a minimal cellular life. How do they evolve? It has been hypothesized that TFs evolve by gaining specificity (and other functions) on a background of non-specific chromosome structuring proteins. We used the IHF/HU family of DNA binding proteins, in which IHF binds DNA in a sequence-specific manner, whereas HU binds more indiscriminately, to test this hypothesis. We show that HU{beta} has been present from the bacterial root, while both IHF subunits emerged much later and diversified in Proteobacteria, with HU having possibly arisen from transfer events in Gammaproteobacteria. By reconstructing ancestral sequences in-silico on a rooted phylogeny of IHF/HU we show that the common ancestor of this family was probably HU-like and therefore non-specific in binding DNA. IHF evolved from a branch of HU after HU had substantially diverged. Various residues characteristic of IHF and shown to be involved in specific sequence recognition (at least in E. coli) have likely been co-opted from preexisting residues in HU, while those residues of IHF{beta} have likely evolved independently, suggesting that each of the IHF subunits has undergone different trajectories to acquire their DNA binding properties.

evolutionary biology↗

bioRxiv2023

Widespread prevalence of a post-translational modification in activation of an essential bacterial DNA damage response

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 post-translational modification and mode of action distinct from its E. 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 co-evolution 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 post-translational modification, in driving a specific and essential bacterial DNA damage response.

microbiology↗

bioRxiv2020

SARS-CoV-2 sequence typing, evolution and signatures of selection using CoVa, a Python-based command-line utility

The current global pandemic COVID-19, caused by SARS-CoV-2, has resulted in millions of infections worldwide in a few months. Global efforts to tackle this situation have produced a tremendous body of genomic data, which can be used for tracing transmission routes, characterization of isolates, and monitoring variants with potential for unusual virulence. Several groups have analyzed these genomes using different approaches. However, as new data become available, the research community needs a pipeline to perform a set of routine analyses, that can quickly incorporate new genome sequences and update the analysis reports. We developed a programmatic tool, CoVa, with this objective. It is a fast, accurate and user-friendly utility to perform a variety of genome analyses on hundreds of SARS-CoV-2 sequences. Using CoVa, we define a modified sequence typing nomenclature and identify sites under positive selection. Further analysis identified some peptides and sites showing geographical patterns of selection. Specifically, we show differences in sequence type distribution between sequences from India and those from the rest of the world. We also show that several sites show signatures of positive selection uniquely in sequences from India. Preliminary evolutionary analysis, using features that will be incorporated into CoVa in the near future, show a mutation rate of 7.4 x 10-4 substitutions/site/year, confirm a temporal signal with a November 2019 origin of SARS-CoV-2, and a heterogeneity in the geographical distribution of Indian samples.

bioinformatics↗

bioRxiv2020

High quality single amplicon sequencing method for illumina platforms using N (0-10) spacer primer pool without PhiX spik-in

Illumina sequencing platform requires base diversity in initial 11 cycles for efficient cluster identification and color matrix estimation. This limitation yields low quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but overall reduces the number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms we developed high throughput single amplicon sequencing method by introducing N spacers in target gene amplification primers that are pooled for simple handling. We evaluated the efficiency of N spacer primers by targeting bacterial 16S V3-V4 region, demonstrating heterogonous base library construction. Addition of N spacer causes sequencing frame shift at every base that leads to base diversity and produces heterogenous high quality reads within single amplicon library. We have written a python script "MetReTrim" to trim the heterogenous N spacers from the pre-processed reads. This method terminates the need for PhiX spike-in and allows for multiplexing of multiple samples, greatly reducing the overall cost and yields improved sequence quality.

genomics↗

bioRxiv2019

Evolutionary analysis of bacterial Non-Homologous 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 that is used as a template for repair. Alternatively, in the absence of a template, cells utilize error-prone Non-homologous 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. To understand what could have led to the current distribution of bacterial NHEJ, we carried out comparative genomics and phylogenetic analysis across ~6000 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 rates; 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.

bioinformatics↗