scholarly journals Pseudomonas stutzeri Has Two Closely RelatedpilA Genes (Type IV Pilus Structural Protein) with Opposite Influences on Natural Genetic Transformation

2001 ◽  
Vol 183 (7) ◽  
pp. 2359-2366 ◽  
Author(s):  
Stefan Graupner ◽  
Wilfried Wackernagel
Keyword(s):  
Genetic Transformation ◽  
Pseudomonas Stutzeri ◽  
Leader Peptide ◽  
Duplex Dna ◽  
Dna Uptake ◽  
Structural Protein ◽  
Dichelobacter Nodosus ◽  
Type Iv ◽  
Insertional Inactivation ◽  
Natural Genetic Transformation

ABSTRACT Pseudomonas stutzeri has type IV pili for which the pilA gene (here termed pilAI) provides the structural protein and which are required for DNA uptake and natural genetic transformation. Downstream of pilAIwe identified a gene, termed pilAII, coding for a deduced protein with a size similar to that of PilAI with 55% amino acid sequence identity and with a typical leader peptide including a leader peptidase cleavage site. Fusions to lacZ revealed that pilAII is expressed only about 10% compared topilAI, although the genes are cotranscribed as shown by reverse transcription-PCR. Surprisingly, insertional inactivation ofpilAII produced a hypertransformation phenotype giving about 16-fold-increased transformation frequencies. Hypertransformation also occurred in pilAI pilAII double mutants expressing heterologous pilA genes of nontransformable bacteria, like Pseudomonas aeruginosa or Dichelobacter nodosus. The overexpression of pilAII decreased transformation up to 5,000-fold compared to that of thepilAII mutant. However, neither inactivation ofpilAII nor its overexpression affected the amounts of [3H]thymidine-labeled DNA that were competence-specifically bound and taken up by the cells. In thepilAII mutant, the transformation by purified single-stranded DNA (which depends on comA andexbB, as does transformation by duplex DNA) was also increased 17-fold. It is concluded that PilAII suppresses a step in transformation after the uptake of duplex DNA into the cell and perhaps before its translocation into the cytoplasm. The idea that the degree of the transformability of cells could be permanently adjusted by the expression level of an antagonistic protein is discussed.

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.

Genetic Competence and Transformation in Oral Streptococci

2001 ◽  
Vol 12 (3) ◽  
pp. 217-243 ◽  
Author(s):  
D.G. Cvitkovitch
Keyword(s):  
Genetic Transformation ◽  
Genetic Information ◽  
Physiological State ◽  
Oral Streptococci ◽  
Dna Uptake ◽  
Genetic Competence ◽  
Gram Positive Bacteria ◽  
Oral Biofilms ◽  
Foreign Dna ◽  
Natural Genetic Transformation

The oral streptococci are normally non-pathogenic residents of the human microflora. There is substantial evidence that these bacteria can, however, act as "genetic reservoirs" and transfer genetic information to transient bacteria as they make their way through the mouth, the principal entry point for a wide variety of bacteria. Examples that are of particular concern include the transfer of antibiotic resistance from oral streptococci to Streptococcus pneumoniae. The mechanisms that are used by oral streptococci to exchange genetic information are not well-understood, although several species are known to enter a physiological state of genetic competence. This state permits them to become capable of natural genetic transformation, facilitating the acquisition of foreign DNA from the external environment. The oral streptococci share many similarities with two closely related Gram-positive bacteria. S. pneumoniae and Bacillus subtilis. In these bacteria, the mechanisms of quorum-sensing, the development of competence, and DNA uptake and integration are well-charaterized. Using this knowledge and the data available in genome databases allowed us to identify putative genes involved in these processes in the oral organism Streptococcus mutans. Models of competence development and genetic transformation in the oral streptococci and strategies to confirm these models are discussed. Future studies of competence in oral biofilms, the natural environment of oral streptococci, will be discussed.

scholarly journals An Unstable Competence-Induced Protein, CoiA, Promotes Processing of Donor DNA after Uptake during Genetic Transformation in Streptococcus pneumoniae

2006 ◽  
Vol 188 (14) ◽  
pp. 5177-5186 ◽  
Author(s):  
Bhushan V. Desai ◽  
Donald A. Morrison
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Transformation ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Specific Response ◽  
Dna Uptake ◽  
Gram Positive Bacteria ◽  
Specific Alternative ◽  
Natural Genetic Transformation

ABSTRACT Natural genetic transformation in Streptococcus pneumoniae entails transcriptional activation of at least two sets of genes. One set of genes, activated by the competence-specific response regulator ComE, is involved in initiating competence, whereas a second set is activated by the competence-specific alternative sigma factor ComX and functions in DNA uptake and recombination. Here we report an initial characterization of CoiA, a ComX-dependent gene product that is induced during competence and is required for transformation. CoiA is widely conserved among gram-positive bacteria, and in streptococci, the entire coiA locus composed of four genes is conserved. By use of immunoblot assay, we show that, similar to its message, CoiA protein is transient, appearing at 10 min and largely disappearing by 30 min post-competence induction. Using complementation analysis, we establish that coiA is the only gene of this induced locus needed for transformability. We find no indication of CoiA having a role in regulating competence. Finally, using 32P- and 3H-labeled donor DNA, we demonstrate that a coiA mutant can internalize normal amounts of donor DNA compared to the wild-type strain but is unable to process it into viable transformants, suggesting a role for CoiA after DNA uptake, either in DNA processing or recombination.

scholarly journals Properties and Biological Role of Streptococcal Fratricins

2012 ◽  
Vol 78 (10) ◽  
pp. 3515-3522 ◽  
Author(s):  
Kari Helene Berg ◽  
Truls Johan Biørnstad ◽  
Ola Johnsborg ◽  
Leiv Sigve Håvarstein
Keyword(s):  
Cell Wall ◽  
Genetic Transformation ◽  
Natural Transformation ◽  
Current View ◽  
Dna Uptake ◽  
Cell Wall Degrading Enzymes ◽  
Content Type ◽  
Streptococcal Species ◽  
Natural Genetic Transformation

ABSTRACTCompetence for natural genetic transformation is widespread in the genusStreptococcus. The current view is that all streptococcal species possess this property. In addition to the proteins required for DNA uptake and recombination, competent streptococci secrete muralytic enzymes termed fratricins. Since the synthesis and secretion of these cell wall-degrading enzymes are always coupled to competence development in streptococci, fratricins are believed to carry out an important function associated with natural transformation. This minireview summarizes what is known about the properties of fratricins and discusses their possible biological roles in streptococcal transformation.

scholarly journals Regulation of Type IV Fimbrial Biogenesis in Dichelobacter nodosus

2006 ◽  
Vol 188 (13) ◽  
pp. 4801-4811 ◽  
Author(s):  
Dane Parker ◽  
Ruth M. Kennan ◽  
Garry S. Myers ◽  
Ian T. Paulsen ◽  
J. Glenn Songer ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Extracellular Proteases ◽  
Western Blots ◽  
Dichelobacter Nodosus ◽  
Fimbrial Subunit ◽  
Type Iv ◽  
Genome Wide ◽  
Insertional Inactivation ◽  
Two Component Signal Transduction ◽  
Protease Secretion

ABSTRACT Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (σ54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and σ54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and σ54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and σ54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a σ54-dependent PilR/S system that also indirectly controls protease secretion.

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