Hari Sharan Misra

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged genome. In this study, we investigate the role of the natural transformation-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks primarily occur through SSA repair pathway. Additionally, our findings suggest that the DprA protein may hinder both the SSA and ESDSA repair pathways, albeit in distinct manners. Overall, our results highlight the crucial function of DprA in the selection between SSA and ESDSA pathways for DNA repair in heavily irradiated D. radiodurans . IMPORTANCE: Deinococcus radiodurans exhibits an extraordinary ability to endure and thrive in extreme environments, including exposure to radiation, desiccation, and damaging chemicals, as well as intense UV radiation. The bacterium has evolved highly efficient repair mechanisms capable of rapidly mending hundreds of DNA fragments in its genome. Our research indicates that natural transformation (NT)-specific dprA genes play a pivotal role in regulating DNA repair in response to radiation. Remarkably, we found that DprA is instrumental in selecting DNA double-strand break repair pathways, a novel function that has not been reported before. This unique regulatory mechanism highlights the indispensable role of DprA beyond its native function in NT and underscores its ubiquitous presence across various bacterial species, regardless of their NT proficiency. These findings shed new light on the resilience and adaptability of Deinococcus radiodurans , opening avenues for further exploration into its exceptional survival strategies.

Deinococcus radiodurans , a radioresistant bacterium, lacks LexA/RecA-mediated DNA damage response and cell cycle regulation as known in other bacteria. However, it adjusts its transcriptome and proteome upon DNA damage.

Natural transformation enables bacteria to acquire DNA from the environment and contributes to genetic diversity, DNA repair, and nutritional requirements. DNA processing protein A (DprA) receives incoming single-stranded DNA and assists RecA loading for homology-directed natural chromosomal transformation and DNA strand annealing during plasmid transformation.

Deinococcus radiodurans show extraordinary resistance to gamma radiation. It is polyploid and harbors a multipartite genome comprised of 2 chromosomes and 2 plasmids, packaged in a doughnut-shaped toroidal nucleoid.

The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiation-resistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation-responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho-mimetic mutant lost the wild-type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post-bleaching recovery for drFtsZ and phospho-mimetic mutant were nearly similar at 2 h post-irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria.

FtsK/SpoIIIE protein family are DNA translocases known as the fastest DNA motor proteins that use ATP for their movement on DNA. Most of the studies in single chromosome-containing bacteria have established the role of FtsK in chromosome dimer resolution (CDR) connecting the bacterial chromosome segregation process with cell division. But, only limited reports are available on the interdependent regulation of genome segregation and cell division in multipartite genome harbouring (MGH) bacteria. In this study, for the first time, we report the characterization of FtsK from the radioresistant MGH bacterium Deinococcus radiodurans R1 (drFtsK). drFtsK shows the activity characteristics of a typical FtsK/SpoIIIE/Tra family. It stimulates the site-specific recombination catalyzed by Escherichia coli tyrosine recombinases. drFtsK interacts with various cell division and genome segregation proteins of D. radiodurans . Microscopic examination of different domain deletion mutants of this protein reveals alterations in cellular membrane architecture and nucleoid morphology. In-vivo localization studies of drFtsK-RFP show that it forms multiple foci on nucleoid as well as on the membrane with maximum density on the septum. drFtsK coordinates its movement with nucleoid separation. The alignment of its foci shifts from old to new septum indicating its cellular dynamics with the FtsZ ring during the cell division process. Nearly, similar positional dynamicity of FtsK was observed in cells recovering from gamma radiation exposure. These results suggest that FtsK forms a part of chromosome segregation, cell envelope, and cell division machinery in D. radiodurans . Importance: Deinococcus radiodurans show extraordinary resistance to γ-radiation. It is polyploid and harbours a multipartite genome comprised of two chromosomes and two plasmids, packaged in a doughnut-shaped toroidal nucleoid. Very little is known about how the tightly packed genome is accurately segregated and the next divisional plane is determined. FtsK, a multifunctional protein, helps in pumping the septum-trapped DNA in several bacteria. Here, we characterized FtsK of D. radiodurans R1 (drFtsK) for the first time and showed it to be an active protein. The absence of drFtsK causes many defects in morphology at both cellular and nucleoid levels. The compact packaging of the deinococcal genome and cell membrane formation is hindered in ftsK mutants. In-vivo drFtsK is dynamic, forms foci on both nucleoid and septum, and coordinates with FtsZ for the next cell division. Thus, drFtsK role in maintaining the normal genome phenotype and cell division in Deinococcus radiodurans is suggested.

The polymerization/depolymerization dynamics of FtsZ plays the pivotal role in cell division in majority bacteria. Deinococcus radiodurans , a radiation resistant bacterium, shows an arrest of growth in response to DNA damage, despite no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode homologs of E. coli ’s SulA, which attenuate FtsZ functions in response to DNA damage in different bacteria. A radiation responsive Ser/Thr quinoprotein kinase (RqkA) characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we report the detailed characterization of S235 and S335 phosphorylation effect in the regulation of drFtsZ functions, and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans . Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ while phospho-mimetic mutant showed the loss of signatures characteristics to wild type enzyme including the arrest in its dynamics under normal conditions. The post-bleaching recovery kinetics for drFtsZ and phospho-mimetic mutant was nearly similar at 2h post irradiation recovery but found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage and is first time demonstrated in this prokaryote.