scholarly journals Functional Analysis of PilT from the Toxic Cyanobacterium Microcystis aeruginosa PCC 7806

2006 ◽  
Vol 189 (5) ◽  
pp. 1689-1697 ◽  
Author(s):  
Kenlee Nakasugi ◽  
Ralitza Alexova ◽  
Charles J. Svenson ◽  
Brett A. Neilan
Keyword(s):  
Gene Transfer ◽  
Microcystis Aeruginosa ◽  
Structural Models ◽  
Toxin Gene ◽  
Dna Uptake ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Critical Residues ◽  
Pcc 6803

ABSTRACT The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 ± 1.8 nmol Pi min−1 mg protein−1, with a requirement for Mg2+. Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.

scholarly journals Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

2016 ◽  
Vol 113 (44) ◽  
pp. 12467-12472 ◽  
Author(s):  
Christof Hepp ◽  
Berenike Maier
Keyword(s):  
Gene Transfer ◽  
Single Molecule ◽  
Initial Step ◽  
Dna Uptake ◽  
Chromosomal Dna ◽  
Type Iv Pilus ◽  
Membrane Pore ◽  
Type Iv ◽  
Force Relation ◽  
Uptake Velocity

Horizontal gene transfer can speed up adaptive evolution and support chromosomal DNA repair. A particularly widespread mechanism of gene transfer is transformation. The initial step to transformation, namely the uptake of DNA from the environment, is supported by the type IV pilus system in most species. However, the molecular mechanism of DNA uptake remains elusive. Here, we used single-molecule techniques for characterizing the force-dependent velocity of DNA uptake by Neisseria gonorrhoeae. We found that the DNA uptake velocity depends on the concentration of the periplasmic DNA-binding protein ComE, indicating that ComE is directly involved in the uptake process. The velocity–force relation of DNA uptake is in very good agreement with a translocation ratchet model where binding of chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of uptake. The model yields a speed of DNA uptake of 900 bp⋅s−1 and a reversal force of 17 pN. Moreover, by comparing the velocity–force relation of DNA uptake and type IV pilus retraction, we can exclude pilus retraction as a mechanism for DNA uptake. In conclusion, our data strongly support the model of a translocation ratchet with ComE acting as a ratcheting chaperone.

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Marie Zachary ◽  
Susanne Bauer ◽  
Maximilian Klepsch ◽  
Katharina Wagler ◽  
Bruno Hüttel ◽  
...  
Keyword(s):  
Target Genes ◽  
Target Prediction ◽  
Type Iv Pilus ◽  
Content Type ◽  
Type Iv ◽  
Gene Expression Control ◽  
Initial Characterization ◽  
Pilus Biogenesis ◽  
Regulatory Rnas

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae , TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

scholarly journals Identification of Pilus-Like Structures and Genes in Microcystis aeruginosa PCC7806

2005 ◽  
Vol 71 (11) ◽  
pp. 7621-7625 ◽  
Author(s):  
Kenlee Nakasugi ◽  
Brett A. Neilan
Keyword(s):  
Electron Microscopy ◽  
Microcystis Aeruginosa ◽  
Type Iv Pilus ◽  
Type Iv

ABSTRACT Four putative type IV pilus genes from the toxic, naturally transformable Microcystis aeruginosa PCC7806 were identified. Three of these genes were clustered in an arrangement which is identical to that from other cyanobacterial genomes. Type IV pilus-like appendages were also observed by electron microscopy.

scholarly journals Characterization of the Francisella tularensis subsp. novicida type IV pilus

Microbiology ◽  
2008 ◽  
Vol 154 (7) ◽  
pp. 2139-2150 ◽  
Author(s):  
Xhavit Zogaj ◽  
Subhra Chakraborty ◽  
Jirong Liu ◽  
David G. Thanassi ◽  
Karl E. Klose
Keyword(s):  
Francisella Tularensis ◽  
Type Iv Pilus ◽  
Tularensis Subsp ◽  
Type Iv

scholarly journals Insertion-Duplication Mutagenesis ofNeisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island

2001 ◽  
Vol 183 (16) ◽  
pp. 4718-4726 ◽  
Author(s):  
Holly L. Hamilton ◽  
Kevin J. Schwartz ◽  
Joseph P. Dillard
Keyword(s):  
Type Iv Secretion ◽  
Phenotypic Characterization ◽  
Insertion Mutation ◽  
Dna Uptake ◽  
Chromosomal Dna ◽  
Chromosome Walking ◽  
Erythromycin Resistance ◽  
Type Iv ◽  
Transfer Genes

ABSTRACT We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization oftraG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.

scholarly journals Identification and Characterization of a Novel Competence Gene,comC, Required for DNA Binding and Uptake inAcinetobacter sp. Strain BD413

1998 ◽  
Vol 180 (6) ◽  
pp. 1592-1595 ◽  
Author(s):  
Caroline Link ◽  
Sandra Eickernjäger ◽  
Dirk Porstendörfer ◽  
Beate Averhoff
Keyword(s):  
Electron Microscopy ◽  
Dna Binding ◽  
Natural Transformation ◽  
Leader Sequence ◽  
Wild Type ◽  
Type Iv Pilus ◽  
Competence Gene ◽  
Type Iv ◽  
Identification And Characterization

ABSTRACT A gene (comC) essential for natural transformation was identified in Acinetobacter sp. strain BD413. ComC has a typical leader sequence and is similar to different type IV pilus assembly factors. A comC mutant (T308) is not able to bind or take up DNA but exhibits a piliation phenotype indistinguishable from the transformation wild type as revealed by electron microscopy.

scholarly journals Structure of the PilM-PilN Inner Membrane Type IV Pilus Biogenesis Complex from Thermus thermophilus

2011 ◽  
Vol 286 (27) ◽  
pp. 24434-24442 ◽  
Author(s):  
Vijaykumar Karuppiah ◽  
Jeremy P. Derrick
Keyword(s):  
Binding Site ◽  
Atp Hydrolysis ◽  
Thermus Thermophilus ◽  
Narrow Channel ◽  
Dna Uptake ◽  
Type Iv Pilus ◽  
Inner Membrane ◽  
Atp Binding Site ◽  
Type Iv ◽  
Pilus Biogenesis

Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other Gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.

scholarly journals Requirement of Novel Competence Genes pilT andpilU of Pseudomonas stutzeri for Natural Transformation and Suppression of pilT Deficiency by a Hexahistidine Tag on the Type IV Pilus Protein PilAI

2001 ◽  
Vol 183 (16) ◽  
pp. 4694-4701 ◽  
Author(s):  
Stefan Graupner ◽  
Nicole Weger ◽  
Monika Sohni ◽  
Wilfried Wackernagel
Keyword(s):  
Amino Acid ◽  
Natural Transformation ◽  
Pseudomonas Stutzeri ◽  
Amino Acid Identity ◽  
Binding Motif ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Insertional Inactivation

ABSTRACT The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termedpilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [3H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termedpilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When thepilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gmr cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT + in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

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