Biology subjects

Botka, T.

Publications and source records attributed to Botka, T..

3 recordsLinked to original sources

bioRxiv2024

Cell attachment and tail contraction of S. aureus phage phi812

Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of the Kayvirus genus, which infects Gram-positive Staphylococcus strains, is formed of a core, wedge modules, and baseplate arms carrying receptor-binding proteins 1 and 2 and tripod complexes. Upon binding to a host cell, the receptor-binding proteins of phage 812 baseplate reorient and undergo conformational changes. The changes to the tripod complexes trigger the release of the central spike and weld proteins, which expose peptidoglycan-degrading domains of the hub proteins. Changes in the positions of baseplate arms are transmitted through wedge modules to tail sheath initiator proteins. The ring of the tail sheath initiator proteins expands and triggers the contraction of the tail sheath, which shortens to 50% and pushes the tail tube 10-30 nm into the bacterial cytoplasm. Homologous molecular mechanisms are probably shared by phages of the Herelleviridae family with contractile tails to infect Gram-positive bacteria.

microbiology↗

bioRxiv2024

A stargate mechanism of Microviridae genome delivery unveiled by cryogenic electron tomography

Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.

microbiology↗

bioRxiv2023

Genomic and metabolic plasticity drive alternative scenarios for adapting Pseudomonas putida to non-native substrate D-xylose.

D-Xylose, a major constituent of plant biomass and second most abundant sugar on Earth, holds a considerable potential as a substrate for sustainable bio-production. Pseudomonas putida KT2440 is an attractive bacterial host for valorizing biogenic feedstocks but lacks a xylose utilization pathway. While several attempts to engineer P. putida for growth on xylose have been reported, a comprehensive understanding of xylose metabolism in this bacterium is lacking, hindering its further improvement and rational tailoring for specific biotechnological purposes. In this study, we elucidated the xylose metabolism in the genome-reduced P. putida strain, EM42, endowed with xylose isomerase pathway (xylAB) and transporter (xylE) from Escherichia coli and used the obtained knowledge in combination with adaptive laboratory evolution to accelerate the bacteriums growth on the pentose sugar. Carbon flux analyses, targeted gene knock-outs, and in vitro enzyme assays portrayed xylose assimilation in P. putida and confirmed a partially cyclic upper xylose metabolism. Deletion of the local transcriptional regulator gene hexR de-repressed genes of several key catabolic enzymes and reduced the lag phase on xylose. Guided by metabolic modeling, we augmented P. putida with additional heterologous pentose phosphate pathway genes and subjected rationally prepared strains to adaptive laboratory evolution (ALE) on xylose. The descendants showed accelerated growth and reduced growth lag. Genomic and proteomic analysis of engineered and evolved mutants revealed the importance of a large genomic re-arrangement, transaldolase overexpression, and balancing gene expression in the synthetic xylABE operon. Importantly, omics analyses found that similar growth characteristics of two superior mutants were achieved through distinct evolutionary paths. This work provides a unique insight into how cell metabolism adjusts to a non-native substrate; it highlights the remarkable genomic and metabolic plasticity of P. putida and demonstrates the power of combining knowledge-driven engineering with ALE in generating desirable microbial phenotypes. HighlightsO_LIElucidated xylose catabolism via exogenous isomerase pathway in P. putida EM42. C_LIO_LIDeletion of transcriptional regulator HexR improved growth on xylose. C_LIO_LIKnowledge-guided interventions and adaptive evolution accelerated growth. C_LIO_LIOmics analyses of selected mutants highlighted the genomic and metabolic plasticity of P. putida. C_LIO_LITwo mutants with superior characteristics emerged from distinct evolutionary paths. C_LI

bioengineering↗