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Heliyon May 2024Controlling the microbial load in the environment is crucial to prevent the spread of organisms. The continuous spread of nosocomial infections in hospital facilities...
Controlling the microbial load in the environment is crucial to prevent the spread of organisms. The continuous spread of nosocomial infections in hospital facilities and the emergence of the coronavirus (COVID-19) highlighted the importance of disinfection processes in health safety. This work aimed to evaluate the effectiveness of LED-based disinfection lamps on bacteria from the ESKAPEE group and virus phage in vitro inactivation to be applied in hospital environments and health facilities disinfection. This study evaluated the effect of different UV wavelengths (275 nm, 280 nm (UVC), 310 nm (UVB) and 340 nm (UVA)) on the disinfection process of various microbial indicators including , , , and Bacteriophage lambda DSM 4499. Exposure time (5 min-30 min), exposure distance (0.25 m and 0.5 m) and surface materials (glass, steel, and polished wood) were evaluated on the disinfection efficiency. Furthermore, the study determined the recovery capacity of each species after UV damage. UVC-LED lamps could inactivate 99.99 % of microbial indicators after 20 min exposures at a 0.5 m distance. The exposure time needed to completely inactivate , , , and Bacteriophage lambda DSM 4499 can be decreased by reducing the exposure distance. UVB-LED and UVA-LED lamps were not able to promote a log reduction of 4 and were not effective on or bacteriophage lambda DSM 4499 inactivation. Thus, only UVC-LED lamps were tested on the decontamination of different surface materials, which was successful. showed the ability to recover from UV damage, but its inactivation rate remains 99.99 %, and spores from were not completely inactivated. Nevertheless, the inactivation rate of these indicators remained at 99.99 % with 24 h incubation after UVC irradiation. UVC-LED lamps emitting 280 nm were the most indicated to disinfect surfaces from microorganisms usually found in hospital environments.
PubMed: 38765034
DOI: 10.1016/j.heliyon.2024.e30738 -
Nature Communications May 2024Bacteriophage infection, a pivotal process in microbiology, initiates with the phage's tail recognizing and binding to the bacterial cell surface, which then mediates...
Bacteriophage infection, a pivotal process in microbiology, initiates with the phage's tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system's biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development.
Topics: Bacteriophage lambda; Cryoelectron Microscopy; Shigella sonnei; Viral Tail Proteins; Porins; Bacterial Outer Membrane Proteins; Protein Binding; Models, Molecular; Protein Conformation; Receptors, Virus
PubMed: 38760367
DOI: 10.1038/s41467-024-48686-3 -
Frontiers in Microbiology 2024In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising...
In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising method to elucidate the functions of targeted bacteria in microbiomes. However, few methods to selectively kill target microorganisms in the microbiome without affecting the growth of nontarget microorganisms are available. In this study, we focused on the host-specific lytic ability of virulent phages and validated their potency for precise microbiome engineering. In an artificial microbiome consisting of , , , and , the addition of bacteriophages infecting their respective host strains specifically reduced the number of these bacteria more than 10 orders. Remarkably, the reduction in target bacteria did not affect the growth of nontarget bacteria, indicating that bacteriophages were effective tools for precise microbiome engineering. Moreover, a virulent derivative of the λ phage was synthesized from prophage DNA in the genome of λ lysogen by DNA assembly and phage-rebooting techniques, and -targeted microbiome engineering was achieved. These results propose a novel approach for precise microbiome engineering using bacteriophages, in which virulent phages are synthesized from prophage DNA in lysogenic strains without isolating phages from environmental samples.
PubMed: 38756723
DOI: 10.3389/fmicb.2024.1403903 -
Journal of Molecular Biology Jun 2024Redβ is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease...
Redβ is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redβ is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redβ can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redβ with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redβ and SSB involves the C-terminal domain (CTD) of Redβ and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redβ CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redβ and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redβ-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.
Topics: DNA-Binding Proteins; Escherichia coli Proteins; Escherichia coli; Crystallography, X-Ray; Protein Binding; DNA, Single-Stranded; Models, Molecular; Bacteriophage lambda; Viral Proteins; Protein Conformation; Binding Sites
PubMed: 38663547
DOI: 10.1016/j.jmb.2024.168590 -
BioRxiv : the Preprint Server For... Apr 2024A major challenge faced by is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, has...
A major challenge faced by is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, has evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized more were encoded in given the relative paucity of phage that have been isolated which infect this species. Using a genomic library, we identified a Type IV restriction system consisting of two genes within a 16kB region of the pathogenicity island-2 that we name TgvA and TgvB (ype I-embedded mrSD-like system of PI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modification are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We show that additional phage defense genes are encoded in VPI-2 that protect against other phage like T3, secΦ18, secΦ27 and λ. Our study uncovers a novel Type IV restriction system in , increasing our understanding of the evolution and ecology of while highlighting the evolutionary interplay between restriction systems and phage genome modification.
PubMed: 38617239
DOI: 10.1101/2024.04.05.588314 -
Molecular Biology and Evolution Apr 2024When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold....
When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.
Topics: Evolution, Molecular; Mutation; Receptors, Virus; Protein Binding
PubMed: 38586942
DOI: 10.1093/molbev/msae056 -
Nucleic Acids Research May 2024RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that...
RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.
Topics: Bacteriophage lambda; Crystallography, X-Ray; Viral Proteins; DNA-Binding Proteins; Models, Molecular; Protein Binding; Protein Multimerization; DNA, Viral; Mutation; Lysogeny; Escherichia coli; DNA
PubMed: 38554102
DOI: 10.1093/nar/gkae212 -
Molecular Microbiology May 2024The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that... (Review)
Review
The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.
Topics: Bacteriophage lambda; Recombination, Genetic; DNA, Viral; Viral Proteins; DNA, Bacterial; Binding Sites; Integration Host Factors
PubMed: 38372210
DOI: 10.1111/mmi.15241 -
Scientific Reports Feb 2024The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In...
The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic Escherichia coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lytic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63 with micromolar affinity. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.
Topics: Bacteriophage lambda; Enterohemorrhagic Escherichia coli; Shiga Toxin; Viral Proteins
PubMed: 38360900
DOI: 10.1038/s41598-024-54508-9 -
Scientific Reports Feb 2024The ea22 gene resides in a relatively uncharacterized region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed...
The ea22 gene resides in a relatively uncharacterized region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed upon infection. In lambda and Shiga toxin-producing phages found in enterohemorrhagic E. coli (EHEC) associated with food poisoning, Ea22 favors a lysogenic over lytic developmental state. The Ea22 protein may be considered in terms of three domains: a short amino-terminal domain, a coiled-coiled domain, and a carboxy-terminal domain (CTD). While the full-length protein is tetrameric, the CTD is dimeric when expressed individually. Here, we report the NMR solution structure of the Ea22 CTD that is described by a mixed alpha-beta fold with a dimer interface reinforced by salt bridges. A conserved mobile loop may serve as a ligand for an unknown host protein that works with Ea22 to promote bacterial survival and the formation of new lysogens. From sequence and structural comparisons, the CTD distinguishes lambda Ea22 from homologs encoded by Shiga toxin-producing bacteriophages.
Topics: Humans; Bacteriophage lambda; Lysogeny; Bacteriophages; Enterohemorrhagic Escherichia coli; Shiga Toxin; Escherichia coli Infections
PubMed: 38302537
DOI: 10.1038/s41598-024-52996-3