scholarly journals VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile

2021 ◽  
Author(s):  
Julia Sorokina ◽  
Irina Sokolova ◽  
Ivan Rybolovlev ◽  
Natalya Shevlyagina ◽  
Vasiliy Troitskiy ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Gene Clusters ◽  
Binding Activity ◽  
Secretion System ◽  
Amino Acid Residues ◽  
Conjugative Transposons ◽  
Dna Binding Activity ◽  
Dna And Rna ◽  
Clostridioides Difficile ◽  
Activity Form

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and which has attracted intense interest due to its roles in the pathogenesis of infectious diseases. During the current investigation we uncovered three distinct gene clusters in Clostridioides difficile strain 630 coding for proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis shows that VirB4- and VirD4-like proteins of CTn4 locus, on one hand, and those of CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in physiology of C. difficile. Our further study on VirB4- and VirD4-like products coded by CTn4 revealed that both proteins possess Mg2+-dependent ATPase activity, form oligomers (most probably, hexamers) in water solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. Its DNA binding activity strongly decreased with the W241A variant. Mutations in the nucleotide sequences coding for presumable Walker A and Walker B motifs decreased stability of the oligomers and significantly but not completely attenuated enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs resulted neither in attenuation of enzymatic activity of the protein nor influenced the oligomerization state of the ATPase.

scholarly journals VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile

2021 ◽  
Author(s):  
Julya Sorokina ◽  
Irina Sokolova ◽  
Ivan Rybolovlev ◽  
Natalya Shevlyagina ◽  
Vasiliy Troitskiy ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Virulence Factors ◽  
Gene Clusters ◽  
Binding Activity ◽  
Secretion System ◽  
Dna Binding Activity ◽  
Dna And Rna ◽  
Clostridioides Difficile ◽  
The Stability ◽  
The One

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and has attracted intense interest due to its roles in the pathogenesis of infectious diseases. In the current investigation, we uncovered three distinct gene clusters in Clostridioides difficile strain 630 encoding proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis revealed that VirB4- and VirD4-like proteins of the CTn4 locus, on the one hand, and those of the CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in the physiology of C. difficile . Our further study on VirB4- and VirD4-like products encoded by CTn4 revealed that both proteins possess Mg 2+ -dependent ATPase activity, form oligomers (most likely hexamers) in aqueous solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. The DNA-binding activity of VirD4 strongly decreased with the W241A variant. Mutations in the nucleotide sequences encoding presumable Walker A and Walker B motifs decreased the stability of the oligomers and significantly but not completely attenuated the enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs neither attenuated the enzymatic activity of the protein nor influenced the oligomerization state of the ATPase. Importance C. difficile is a gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include the toxins TcdA, TcdB and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered possible virulence factors of C. difficile . In the current paper, we describe the structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to its significance for basic science, can potentially aid the development of antivirulence drugs suitable for the treatment of C. difficile infection.

scholarly journals Role of Single-Stranded DNA Binding Activity of T Antigen in Simian Virus 40 DNA Replication

2001 ◽  
Vol 75 (6) ◽  
pp. 2839-2847 ◽  
Author(s):  
Chunxiao Wu ◽  
Rupa Roy ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Single Point ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Amino Acid Residues ◽  
Single Stranded Dna ◽  
Dna Binding Activity

ABSTRACT We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication.

scholarly journals Genomic study of the Type IVC secretion system in Clostridium difficile: understanding C. difficile evolution via horizontal gene transfer

Genome ◽  
2017 ◽  
Vol 60 (1) ◽  
pp. 8-16 ◽  
Author(s):  
Wen Zhang ◽  
Ying Cheng ◽  
Pengcheng Du ◽  
Yuanyuan Zhang ◽  
Hongbing Jia ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Gene Clusters ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Genomic Islands ◽  
Conjugative Transposons ◽  
Type Iv ◽  
Tandem Repeat Sequences ◽  
Genomic Study ◽  
Short Tandem

Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.

Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a

2011 ◽  
Vol 437 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Isao Suetake ◽  
Yuichi Mishima ◽  
Hironobu Kimura ◽  
Young-Ho Lee ◽  
Yuji Goto ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Amino Acid ◽  
Dna Binding ◽  
Dna Methyltransferase ◽  
Basic Amino Acid ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Terminal Domain ◽  
Dna Binding Activity

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1–211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.

scholarly journals ExsD Inhibits Expression of the Pseudomonas aeruginosa Type III Secretion System by Disrupting ExsA Self-Association and DNA Binding Activity

2009 ◽  
Vol 192 (6) ◽  
pp. 1479-1486 ◽  
Author(s):  
Evan D. Brutinel ◽  
Christopher A. Vakulskas ◽  
Timothy L. Yahr
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Binding ◽  
Type Iii Secretion ◽  
Genetic Data ◽  
Binding Activity ◽  
Secretion System ◽  
Host Cells ◽  
Type Iii ◽  
Dna Binding Activity ◽  
Self Association

ABSTRACT Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to damage eukaryotic host cells and evade phagocytosis. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. ExsA-dependent transcription is coupled to type III secretory activity through a cascade of three interacting proteins (ExsC, ExsD, and ExsE). Genetic data suggest that ExsD functions as an antiactivator by preventing ExsA-dependent transcription, ExsC functions as an anti-antiactivator by binding to and inhibiting ExsD, and ExsE binds to and inhibits ExsC. T3SS gene expression is activated in response to low-calcium growth conditions or contact with host cells, both of which trigger secretion of ExsE. In the present study we reconstitute the T3SS regulatory cascade in vitro using purified components and find that the ExsD·ExsA complex lacks DNA binding activity. As predicted by the genetic data, ExsC addition dissociates the ExsD·ExsA complex through formation of an ExsD·ExsC complex, thereby releasing ExsA to bind T3SS promoters and activate transcription. Addition of ExsE to the purified system results in formation of the ExsE·ExsC complex and prevents ExsC from dissociating the ExsD·ExsA complex. Although purified ExsA is monomeric in solution, bacterial two-hybrid analyses demonstrate that ExsA can self-associate and that ExsD inhibits self-association of ExsA. Based on these data we propose a model in which ExsD regulates ExsA-dependent transcription by inhibiting the DNA-binding and self-association properties of ExsA.

Characterization of Putative Type IV Secretion System of Bacillus anthracis

2020 ◽  
Author(s):  
Gopal Jee Gopal ◽  
Awanish Kumar
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Binding Activity ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Dna Binding Activity ◽  
Type Iv ◽  
Toxic Proteins

Background: Bacillus anthracis causes anthrax disease in livestock and occasionally in humans mainly through three toxic proteins: protective antigen, lethal factor and edema factor. Synthesis of the toxic proteins in bacteria and its pathogenic role in the human host is well known. But, how these toxins are secreted from bacterium to the host or in the culture medium is not known. Methods: A group of researchers have shown (through in silico prediction) that several genes of B. anthracis have similarity with the genes which codes the subunit of the Type IV Secretion System (T4SS) known in other bacteria. In this study, we have tried to explore the existence of T4SS in B. anthracis using molecular biology tools. Result: As an initial lead, three genes (GBAA-pXO1-0064, GBAA-pXO1-0085VirB11 and Bas3783) of B. anthracis T4SS (out of more than 10) were cloned and expressed in E. coli. Among them, gene Bas3783 (VirD4 homolog) is genome encoded therefore; it is sequenced and further characterized for its DNA binding activity and oligomerization. Results obtained from this study indicated the existence of T4SS in B. anthracis.

scholarly journals DNA-Binding Activity of Amino-Terminal Domains of the Bacillus subtilis AbrB Protein

2001 ◽  
Vol 183 (13) ◽  
pp. 4094-4098 ◽  
Author(s):  
Ke Xu ◽  
Mark A. Strauch
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Disulfide Bonding ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Binding Affinity ◽  
Truncated Variants

ABSTRACT Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified. Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity. Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB. These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.

Sign in / Sign up

Export Citation Format

Share Document