scholarly journals Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

2016 ◽  
Vol 199 (3) ◽  
Author(s):  
Annum Munir ◽  
Stewart Shuman
Keyword(s):  
Nucleic Acid ◽  
Kinase Domain ◽  
Nucleoside Triphosphate ◽  
Content Type ◽  
Bacterial Phyla ◽  
Domain Composition ◽  
Modular Units ◽  
Polynucleotide Kinase ◽  
Key Steps

ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla.

scholarly journals Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Annum Munir ◽  
Stewart Shuman
Keyword(s):  
Nucleic Acid ◽  
Gene Cluster ◽  
Mutational Analysis ◽  
Function Analysis ◽  
Repair Pathway ◽  
Content Type ◽  
Dna And Rna ◽  
Polynucleotide Kinase ◽  
Metal Cofactor ◽  
Key Steps

ABSTRACT Runella slithyformis HD-Pnk is the prototype of a family of dual 5′ and 3′ nucleic acid end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. HD-Pnk is composed of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Here, we probed the phosphoesterase activity of HD-Pnk by querying its ability to hydrolyze non-nucleic acid phosphoester substrates and by conducting a mutational analysis of conserved amino acid constituents of the HD domain. We report that HD-Pnk catalyzes vigorous hydrolysis of p-nitrophenylphosphate (Km = 3.13 mM; kcat = 27.8 s−1) using copper as its metal cofactor. Mutagenesis identified Gln28, His33, His73, Asp74, Lys77, His94, His127, Asp162, and Arg166 as essential for p-nitrophenylphosphatase and DNA 3′ phosphatase activities. Structural modeling places these residues at the active site, wherein His33, His73, Asp74, His94, and His127 are predicted to coordinate a binuclear metal complex and Lys77 and Arg166 engage the scissile phosphate. HD-Pnk homologs are distributed broadly (and exclusively) in bacteria, usually in a two-gene cluster with a putative ATP-dependent polynucleotide ligase (LIG). We speculate that HD-Pnk and LIG comprise the end-healing and end-sealing components of a bacterial nucleic acid repair pathway. IMPORTANCE 5′-end healing and 3′-end healing are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase, and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate 5′-PO4 and 3′-OH termini needed for joining by DNA and RNA ligases. This study interrogates, biochemically and via mutagenesis, the phosphoesterase activity of Runella slithyformis HD-Pnk, a bifunctional bacterial 5′- and 3′-end-healing enzyme composed of HD phosphoesterase and P-loop kinase modules. HD-Pnk homologs are found in 129 bacterial genera from 11 phyla. In 123/129 instances, HD-Pnk is encoded in an operon-like gene cluster with a putative ATP-dependent polynucleotide ligase (LIG), suggesting that HD-Pnk and LIG are agents of a conserved bacterial nucleic acid repair pathway.

scholarly journals Release of Nonstop Ribosomes Is Essential

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Heather A. Feaga ◽  
Patrick H. Viollier ◽  
Kenneth C. Keiler
Keyword(s):  
Messenger Rna ◽  
Caulobacter Crescentus ◽  
Stop Codon ◽  
Selective Advantage ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Phyla ◽  
Almost All

ABSTRACTBacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-basedtrans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity.trans-translation is essential in some species, but in others, such asCaulobacter crescentus,trans-translation can be inactivated. To determine whytrans-translation is dispensable inC. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lackingssrA, the gene encoding tmRNA. One of these genes,CC1214, was essential in ΔssrAcells. Purified CC1214 protein could release nonstop ribosomesin vitro. CC1214 is a homolog of theEscherichia coliArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in whichssrAhas been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria.IMPORTANCEGenes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required fortrans-translation,ssrAandsmpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization ofC. crescentusArfB reveals whytrans-translation is not essential inC. crescentusand suggests that many other bacteria are likely to use ArfB to survive whentrans-translation is compromised.

scholarly journals Regulatory Properties of the ADP-Glucose Pyrophosphorylase from the ClostridialFirmicutesMemberRuminococcus albus

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Antonela E. Cereijo ◽  
Matías D. Asencion Diez ◽  
Miguel A. Ballicora ◽  
Alberto A. Iglesias
Keyword(s):  
Glycogen Synthesis ◽  
Evolutionary Relationship ◽  
Catalytic Efficiency ◽  
Ruminococcus Albus ◽  
Content Type ◽  
Bacterial Phyla ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

ABSTRACTADP-glucose pyrophosphorylase fromFirmicutesis encoded by two genes (glgCandglgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups ofFirmicutes,BacillalesandLactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase fromClostridiales, the third group inFirmicutes, has been characterized. For this reason, we cloned theglgC andglgD genes fromRuminococcus albus. Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of theBacillalesenzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases fromFirmicuteswere unregulated. Our results, together with data fromLactobacillales, indicate that heterotetramericFirmicutesenzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase fromBacillalesseems to have distinctive insensitivity to regulation.IMPORTANCEThe enzymes involved in glycogen synthesis fromFirmicuteshave been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase fromRuminococcus albus. Our results showed that this protein that belongs to different groups fromFirmicutes(Bacillales,Lactobacillales, andClostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in theFirmicutesgroup, whereby we propose that these heterotetrameric enzymes, with the exception ofBacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family inFirmicutes.

scholarly journals Deinococcus radioduransHD-Pnk, a Nucleic Acid End-Healing Enzyme, Abets Resistance to Killing by Ionizing Radiation and Mitomycin C

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Brad J. Schmier ◽  
Stewart Shuman
Keyword(s):  
Nucleic Acid ◽  
Ionizing Radiation ◽  
Mitomycin C ◽  
Deinococcus Radiodurans ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Key Steps

ABSTRACT5′- and 3′-end healing are key steps in nucleic acid break repair in which 5′-OH and 3′-PO4or 2′,3′-cyclic-PO4ends are converted to 5′-PO4and 3′-OH termini suitable for sealing by polynucleotide ligases. Here, we characterizeDeinococcus radioduransHD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5′-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3′-phosphatase and 2′,3′-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis ofp-nitrophenylphosphate. HD-Pnk is encoded by the LIG–PARG–HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential forDeinococcusgrowth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitizeDeinococcusto killing by mitomycin C.IMPORTANCEEnd healing is a process whereby nucleic acid breaks with “dirty” 3′-PO4or 2′,3′-cyclic-PO4and 5′-OH ends are converted to 3′-OH and 5′-PO4termini that are amenable to downstream repair reactions.Deinococcus radioduransis resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlightsDeinococcusHD-Pnk as a bifunctional 3′- and 5′-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

scholarly journals Longitudinal Characterization of the Gut Bacterial and Fungal Communities in Yaks

2021 ◽  
Vol 7 (7) ◽  
pp. 559
Author(s):  
Yaping Wang ◽  
Yuhang Fu ◽  
Yuanyuan He ◽  
Muhammad Fakhar-e-Alam Kulyar ◽  
Mudassar Iqbal ◽  
...  
Keyword(s):  
Natural Aging ◽  
Fungal Communities ◽  
Significant Shift ◽  
Mature Adult ◽  
Bacterial Phyla ◽  
Development Stages ◽  
Composition And Structure ◽  
Intestinal Functions ◽  
Development Phases

Development phases are important in maturing immune systems, intestinal functions, and metabolism for the construction, structure, and diversity of microbiome in the intestine during the entire life. Characterizing the gut microbiota colonization and succession based on age-dependent effects might be crucial if a microbiota-based therapeutic or disease prevention strategy is adopted. The purpose of this study was to reveal the dynamic distribution of intestinal bacterial and fungal communities across all development stages in yaks. Dynamic changes (a substantial difference) in the structure and composition ratio of the microbial community were observed in yaks that matched the natural aging process from juvenile to natural aging. This study included a significant shift in the abundance and proportion of bacterial phyla (Planctomycetes, Firmicutes, Bacteroidetes, Spirochaetes, Tenericutes, Proteobacteria, and Cyanobacteria) and fungal phyla (Chytridiomycota, Mortierellomycota, Neocallimastigomycota, Ascomycota, and Basidiomycota) across all development stages in yaks. As yaks grew older, variation reduced, and diversity increased as compared to young yaks. In addition, the intestine was colonized by a succession of microbiomes that coalesced into a more mature adult, including Ruminococcaceae_UCG-005, Romboutsia, Prevotellaceae_UCG-004, Blautia, Clostridium_sensu_stricto_1, Ruminococcus_1, Ruminiclostridium_5, Rikenellaceae_RC9_gut_group, Alloprevotella, Acetitomaculum, Lachnospiraceae_NK3A20_group, Bacteroides, Treponema_2, Olsenella, Escherichia-Shigella, Candidatus_Saccharimonas, and fungal communities Mortierella, Lomentospora, Orpinomyces, and Saccharomyces. In addition, microorganisms that threaten health, such as Escherichia-Shigella, Mortierella, Lomentospora and Hydrogenoanaerobacterium, Corynebacterium_1, Trichosporon, and Coprinellus, were enriched in young and old yaks, respectively, although all yaks were healthy. The significant shifts in microflora composition and structure might reflect adaptation of gut microbiome, which is associated with physicochemical conditions changes and substrate availability in the gut across all development periods of yaks.

scholarly journals Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 396
Author(s):  
Ewa Sajnaga ◽  
Marcin Skowronek ◽  
Agnieszka Kalwasińska ◽  
Waldemar Kazimierczak ◽  
Karolina Ferenc ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Entomopathogenic Nematodes ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Bacterial Phyla ◽  
Melolontha Melolontha

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

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