scholarly journals WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

2014 ◽  
Vol 80 (22) ◽  
pp. 6879-6887 ◽  
Author(s):  
Pin Yu ◽  
Shui-Ping Liu ◽  
Qing-Ting Bu ◽  
Zhen-Xing Zhou ◽  
Zhen-Hong Zhu ◽  
...  
Keyword(s):  
Morphological Differentiation ◽  
Binding Activity ◽  
Transcriptional Analysis ◽  
Antibiotic Biosynthesis ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Dna Binding Activity ◽  
In The Wild ◽  
Natamycin Production ◽  
Streptomyces Chattanoogensis

ABSTRACTDetailed mechanisms ofWhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, aStreptomyces chattanoogensisL10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression ofwblAchwas shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAchhas specific DNA-binding activity for the promoter region ofwblAch. Gene disruption and genetic complementation revealed that WblAchacts in a positive manner to regulate natamycin production. WhenwblAchwas overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels ofwhigenes (includingwhiA,whiB,whiH, andwhiI) were severely depressed in the ΔwblAchmutant, suggesting that WblAchplays a part in morphological differentiation by influencing the expression of thewhigenes.

scholarly journals Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Yoshihiro Mouri ◽  
Kenji Konishi ◽  
Azusa Fujita ◽  
Takeaki Tezuka ◽  
Yasuo Ohnishi
Keyword(s):  
Vegetative Growth ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Transcriptional Regulator ◽  
Saccharopolyspora Erythraea ◽  
Transcriptional Analysis ◽  
Morphological Development ◽  
Sequencing Analysis ◽  
Content Type ◽  
Biological Studies

ABSTRACT The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces. AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD. 3′,5′-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5′-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3′ as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation. IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis. The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

scholarly journals Toll-Like Receptor Stimulation Induces Nondefensin Protein Expression and Reverses Antibiotic-Induced Gut Defense Impairment

2014 ◽  
Vol 82 (5) ◽  
pp. 1994-2005 ◽  
Author(s):  
Ying-Ying Wu ◽  
Ching-Mei Hsu ◽  
Pei-Hsuan Chen ◽  
Chang-Phone Fung ◽  
Lee-Wei Chen
Keyword(s):  
Dna Binding ◽  
Protein Expression ◽  
Antibiotic Treatment ◽  
Intestinal Mucosa ◽  
Pathogenic Bacteria ◽  
Binding Activity ◽  
Toll Like Receptor ◽  
Content Type ◽  
Killing Activity ◽  
Dna Binding Activity

ABSTRACTPrior antibiotic exposure is associated with increased mortality in Gram-negative bacteria-induced sepsis. However, how antibiotic-mediated changes of commensal bacteria promote the spread of enteric pathogenic bacteria in patients remains unclear. In this study, the effects of systemic antibiotic treatment with or without Toll-like receptor (TLR) stimulation on bacterium-killing activity, antibacterial protein expression in the intestinal mucosa, and bacterial translocation were examined in mice receiving antibiotics with or without oral supplementation of deadEscherichia coliorStaphylococcus aureus. We developed a systemic ampicillin, vancomycin, and metronidazole treatment protocol to simulate the clinical use of antibiotics. Antibiotic treatment decreased the total number of bacteria, including aerobic bacteria belonging to the familyEnterobacteriaceaeand the genusEnterococcusas well as organisms of the anaerobic generaLactococcusandBifidobacteriumin the intestinal mucosa and lumen. Antibiotic treatment significantly decreased the bacterium-killing activity of the intestinal mucosa and the expression of non-defensin-family proteins, such as RegIIIβ, RegIIIγ, C-reactive protein-ductin, and RELMβ, but not the defensin-family proteins, and increasedKlebsiella pneumoniaetranslocation. TLR stimulation after antibiotic treatment increased NF-κB DNA binding activity, nondefensin protein expression, and bacterium-killing activity in the intestinal mucosa and decreasedK. pneumoniaetranslocation. Moreover, germfree mice showed a significant decrease in nondefensin proteins as well as intestinal defense against pathogen translocation. Since TLR stimulation induced NF-κB DNA binding activity, TLR4 expression, and mucosal bacterium-killing activity in germfree mice, we conclude that the commensal microflora is critical in maintaining intestinal nondefensin protein expression and the intestinal barrier. In turn, we suggest that TLR stimulation induces nondefensin protein expression and reverses antibiotic-induced gut defense impairment.

scholarly journals Free Fatty Acids Interfere with the DNA Binding Activity of the Virulence Regulator PrfA of Listeria monocytogenes

2020 ◽  
Vol 202 (15) ◽  
Author(s):  
Patrícia T. dos Santos ◽  
Rikke S. S. Thomasen ◽  
Mathias S. Green ◽  
Nils J. Færgeman ◽  
Birgitte H. Kallipolitis
Keyword(s):  
Fatty Acids ◽  
Dna Binding ◽  
Free Fatty Acids ◽  
Antimicrobial Agents ◽  
Virulence Gene ◽  
Binding Activity ◽  
Content Type ◽  
Dna Binding Activity ◽  
Virulence Regulator ◽  
Long Chain

ABSTRACT Naturally occurring free fatty acids (FFAs) are recognized as potent antimicrobial agents that also affect the production of virulence factors in bacterial pathogens. In the foodborne pathogen Listeria monocytogenes, some medium- and long-chain FFAs act as antimicrobial agents as well as signaling compounds, causing a repression of transcription of virulence genes. We previously observed that the master virulence regulator PrfA is involved in both the antimicrobial and virulence-inhibitory response of L. monocytogenes to selected FFAs, but the underlying mechanisms are presently unknown. Here, we present a systematic analysis of the antimicrobial and PrfA-inhibitory activities of medium- and long-chain FFAs of various carbon chain lengths and degrees of saturation. We observed that exposure to specific antimicrobial and nonantimicrobial FFAs prevented PrfA-dependent activation of virulence gene transcription and reduced the levels of PrfA-regulated virulence factors. Thus, an antimicrobial activity was not compulsory for the PrfA-inhibitory ability of an FFA. In vitro binding experiments revealed that PrfA-inhibitory FFAs were also able to prevent the constitutively active variant PrfA* from binding to the PrfA box in the promoter region of the virulence gene hly, whereas noninhibitory FFAs did not affect its ability to bind DNA. Notably, the unsaturated FFAs inhibited the DNA binding activity of PrfA* most efficiently. Altogether, our findings support a model in which specific FFAs orchestrate a generalized reduction of the virulence potential of L. monocytogenes by directly targeting the key virulence regulator PrfA. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen able to cause foodborne infections in humans and animals. Key virulence genes in L. monocytogenes are activated by the transcription regulator PrfA, a DNA binding protein belonging to the CRP/FNR family. Various signals from the environment are known to affect the activity of PrfA, either positively or negatively. Recently, we found that specific medium- and long-chain free fatty acids act as antimicrobial agents as well as signaling compounds in L. monocytogenes. Here, we show that both antimicrobial and nonantimicrobial free fatty acids inhibit PrfA-dependent activation of virulence gene transcription by interfering with the DNA binding activity of PrfA. Our findings suggest that free fatty acids could be candidates for alternative therapies against L. monocytogenes.

scholarly journals Fitting Pieces into the Puzzle ofPseudomonas aeruginosaType III Secretion System Gene Expression

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Emily A. Williams McMackin ◽  
Louise Djapgne ◽  
Jodi M. Corley ◽  
Timothy L. Yahr
Keyword(s):  
Gene Expression ◽  
Protein Delivery ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Activity ◽  
Secretion Systems ◽  
Content Type ◽  
Global Regulators ◽  
Dna Binding Activity ◽  
Host Pathogen

ABSTRACTType III secretion systems (T3SS) are widely distributed in Gram-negative microorganisms and critical for host-pathogen and host-symbiont interactions with plants and animals. Central features of the T3SS are a highly conserved set of secretion and translocation genes and contact dependence wherein host-pathogen interactions trigger effector protein delivery and serve as an inducing signal for T3SS gene expression. In addition to these conserved features, there are pathogen-specific properties that include a unique repertoire of effector genes and mechanisms to control T3SS gene expression. ThePseudomonas aeruginosaT3SS serves as a model system to understand transcriptional and posttranscriptional mechanisms involved in the control of T3SS gene expression. The central regulatory feature is a partner-switching system that controls the DNA-binding activity of ExsA, the primary regulator of T3SS gene expression. Superimposed upon the partner-switching mechanism are cyclic AMP and cyclic di-GMP signaling systems, two-component systems, global regulators, and RNA-binding proteins that have positive and negative effects on ExsA transcription and/or synthesis. In the present review, we discuss advances in our understanding of how these regulatory systems orchestrate the activation of T3SS gene expression in the context of acute infections and repression of the T3SS asP. aeruginosaadapts to and colonizes the cystic fibrosis airways.

scholarly journals BosR Is A Novel Fur Family Member Responsive to Copper and Regulating Copper Homeostasis in Borrelia burgdorferi

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Peng Wang ◽  
Zhuoteng Yu ◽  
Thomas J. Santangelo ◽  
John Olesik ◽  
Yufeng Wang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Borrelia Burgdorferi ◽  
Metal Ions ◽  
Metal Ion ◽  
Transition Metal Ions ◽  
Binding Activity ◽  
Ferric Uptake Regulator ◽  
Content Type ◽  
Dna Binding Activity ◽  
Cu Homeostasis

ABSTRACT The ferric uptake regulator (Fur) family of DNA-binding proteins represses and/or activates gene transcription via divalent metal ion-dependent signal sensing. The Borrelia burgdorferi Fur homologue, also known as Borrelia oxidative stress regulator (BosR), promotes spirochetal adaptation to the mammalian host by directly repressing the lipoproteins required for tick colonization and indirectly activating those required for establishing infection in the mammal. Here, we examined whether the DNA-binding activity of BosR was regulated by any of the four most prevalent transition metal ions in B. burgdorferi, Mn, Fe, Cu, and Zn. Our data indicated that in addition to a structural site occupied by Zn(II), BosR had two regulatory sites that could be occupied by Zn(II), Fe(II), or Cu(II) but not by Mn(II). While Fe(II) had no effect, Cu(II) and Zn(II) had a dose-dependent inhibitory effect on the BosR DNA-binding activity. Competition experiments indicated that Cu(II) had a higher affinity for BosR than Zn(II) or Fe(II). A BosR deficiency in B. burgdorferi resulted in a significant increase in the Cu level but no significant change in the levels of Mn, Fe, or Zn. These data suggest that Cu regulates BosR activity, and BosR in turn regulates Cu homeostasis in B. burgdorferi. While this regulatory paradigm is characteristic of the Fur family, BosR is the first one shown to be responsive to Cu(II), which may be an adaptation to the potentially high level of Cu present in the Lyme disease spirochete. IMPORTANCE Transition metal ions serve an essential role in the metabolism of all living organisms. Members of the ferric uptake regulator (Fur) family play critical roles in regulating the cellular homeostasis of transition metals in diverse bacteria, and their DNA-binding activity is often regulated by coordination of the cognate divalent metal ions. To date, regulators with metal ion specificity to Fe(II), Mn(II), Zn(II), and Ni(II) have all been described. In this study, we demonstrate that BosR, the sole Fur homologue in Borrelia burgdorferi, is responsive to Cu(II) and regulates Cu homeostasis in this bacterium, which may be an adaption to potentially Cu-rich milieu in the Lyme disease spirochete. This study has expanded the repertoire of the Fur family's metal ion specificity.

scholarly journals The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

2011 ◽  
Vol 77 (21) ◽  
pp. 7586-7594 ◽  
Author(s):  
Fernando Santos-Beneit ◽  
Mónica Barriuso-Iglesias ◽  
Lorena T. Fernández-Martínez ◽  
Miriam Martínez-Castro ◽  
Alberto Sola-Landa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Response Regulator ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Luciferase Reporter ◽  
Antibiotic Biosynthesis ◽  
Binding Assays ◽  
Content Type ◽  
Phosphate Depletion

ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.

scholarly journals Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Qinli Yu ◽  
Hanlin Cai ◽  
Yanfeng Zhang ◽  
Yongzhi He ◽  
Lincai Chen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Sinorhizobium Meliloti ◽  
Binding Activity ◽  
Mobility Shift ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dna Binding Activity ◽  
Dnase I Footprinting ◽  
Gntr Family

ABSTRACT Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti. However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the −35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism. IMPORTANCE Sinorhizobium meliloti is an important soil bacterium that displays symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for S. meliloti. However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in S. meliloti. We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in S. meliloti.

scholarly journals GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

2013 ◽  
Vol 80 (2) ◽  
pp. 714-722 ◽  
Author(s):  
Junhong Wei ◽  
Yuqing Tian ◽  
Guoqing Niu ◽  
Huarong Tan
Keyword(s):  
Biological Activities ◽  
Regulatory Protein ◽  
Specific Inhibitor ◽  
Biosynthetic Gene Cluster ◽  
Binding Activity ◽  
Major Facilitator Superfamily ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Inhibitor Of Protein Synthesis

ABSTRACTGougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities.gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed thatgouRplays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of thegouL-to-gouBoperon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions ofgouL,gouM, andgouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export inStreptomyces graminearus.

scholarly journals Development of a Markerless Gene Replacement System for Acidithiobacillus ferrooxidans and Construction of apfkBMutant

2011 ◽  
Vol 78 (6) ◽  
pp. 1826-1835 ◽  
Author(s):  
Huiyan Wang ◽  
Xiangmei Liu ◽  
Shuangshuang Liu ◽  
Yangyang Yu ◽  
Jianqun Lin ◽  
...  
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Homing Endonuclease ◽  
Metallurgical Industry ◽  
Gene Replacement ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Functional Studies ◽  
In The Wild ◽  
K 12

ABSTRACTThe extremely acidophilic, chemolithoautotrophicAcidithiobacillus ferrooxidansis an important bioleaching bacterium of great value in the metallurgical industry and environmental protection. In this report, a mutagenesis system based on the homing endonuclease I-SceI was developed to produce targeted, unmarked gene deletions in the strainA. ferrooxidansATCC 23270. A targeted phosphofructokinase (PFK) gene (pfkB) mutant ofA. ferrooxidansATCC 23270 was constructed by homologous recombination and identified by PCR with specific primers as well as Southern blot analysis. This potentialpfkBgene (AFE_1807) was also characterized by expression in PFK-deficientEscherichia colicells, and heteroexpression of the PFKB protein demonstrated that it had functional PFK activity, though it was significantly lower (about 800-fold) than that of phosphofructokinase-2 (PFK-B) expressed by thepfkBgene fromE. coliK-12. The function of the potential PFKB protein inA. ferrooxidanswas demonstrated by comparing the properties of thepfkBmutant with those of the wild type. ThepfkBmutant strain displayed a relatively reduced growth capacity in S0medium (0.5% [wt/vol] elemental sulfur in 9K basal salts solution adjusted to pH 3.0 with H2SO4), but the mutation did not completely preventA. ferrooxidansfrom assimilating exogenous glucose. The transcriptional analysis of some related genes in central carbohydrate metabolism in the wild-type and mutant strains with or without supplementation of glucose was carried out by quantitative reverse transcription-PCR. This report suggests that the markerless mutagenesis strategy could serve as a model for functional studies of other genes of interest fromA. ferrooxidansand multiple mutations could be made in a singleA. ferrooxidansstrain.

scholarly journals Domain Organization of Vaccinia Virus Helicase-Primase D5

2016 ◽  
Vol 90 (9) ◽  
pp. 4604-4613 ◽  
Author(s):  
Stephanie Hutin ◽  
Wai Li Ling ◽  
Adam Round ◽  
Gregory Effantin ◽  
Stefan Reich ◽  
...  
Keyword(s):  
Dna Binding ◽  
Vaccinia Virus ◽  
Genome Structure ◽  
Antiviral Drug ◽  
Binding Activity ◽  
Structural Basis ◽  
Helicase Domain ◽  
Content Type ◽  
Dna Binding Activity ◽  
Double Stranded Dna

ABSTRACTPoxviridaeare viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in thePhycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins.IMPORTANCESince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.

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