scholarly journals Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium Thermus thermophilus Strain HB27

2002 ◽  
Vol 68 (2) ◽  
pp. 745-755 ◽  
Author(s):  
Alexandra Friedrich ◽  
Christina Prust ◽  
Thomas Hartsch ◽  
Anke Henne ◽  
Beate Averhoff
Keyword(s):  
Deinococcus Radiodurans ◽  
Natural Transformation ◽  
Genomic Sequence ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Open Reading Frames ◽  
Gram Positive Bacteria ◽  
Type Iv ◽  
Competence Proteins ◽  
High Competence

ABSTRACT Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.

scholarly journals Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis

2003 ◽  
Vol 69 (7) ◽  
pp. 3695-3700 ◽  
Author(s):  
Alexandra Friedrich ◽  
Judit Rumszauer ◽  
Anke Henne ◽  
Beate Averhoff
Keyword(s):  
Natural Transformation ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Type Iv Pili ◽  
Dna Uptake ◽  
Significant Similarity ◽  
High Frequencies ◽  
Type Iv ◽  
Genes Encoding ◽  
Pilus Biogenesis

ABSTRACT The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Δkat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.

scholarly journals Natural Transformation in Mesophilic and Thermophilic Bacteria: Identification and Characterization of Novel, Closely Related Competence Genes in Acinetobacter sp. Strain BD413 andThermus thermophilus HB27

2001 ◽  
Vol 67 (7) ◽  
pp. 3140-3148 ◽  
Author(s):  
Alexandra Friedrich ◽  
Thomas Hartsch ◽  
Beate Averhoff
Keyword(s):  
Natural Transformation ◽  
Thermophilic Bacteria ◽  
Thermus Thermophilus ◽  
Kanamycin Resistance ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Competence Proteins ◽  
And Function ◽  
Product Displays

ABSTRACT The mesophile Acinetobacter sp. strain BD413 and the extreme thermophile Thermus thermophilus HB27 display high frequencies of natural transformation. In this study we identified and characterized a novel competence gene in Acinetobacter sp. strain BD413, comA, whose product displays significant similarities to the competence proteins ComA and ComEC inNeisseria and Bacillus species. Transcription of comA correlated with growth phase-dependent transcriptional regulation of the recently identified pilin-like factors of the transformation machinery. This finding strongly suggests that comA is part of a competence regulon. Examination of the genome sequence of T. thermophilus HB27 led to detection of a comA/comEC-like open reading frame (ORF) which is flanked by an ORF whose product shows significant similarities to the Bacillus subtilis competence protein ComEA. To examine whether these two ORFs, designated comEC andcomEA, are implicated in natural transformation of T. thermophilus HB27, both were disrupted by using a thermostable kanamycin resistance marker. Natural transformation in comEC mutants was reduced 1,000-fold, whereas in comEA mutants the natural transformation phenotype was completely eliminated. These results strongly suggest that both genes, comEC and comEA, are required for natural transformation in T. thermophilus HB27. Several transmembrane α-helices are predicted based on the amino acid sequences of ComA in Acinetobacter sp. strain BD413 and ComEC in T. thermophilus HB27, which suggests that ComA and ComEC are located in the inner membrane and function in DNA transport through the cytoplasmic membrane.

Identification and Characterization of ComE and ComF, Two Novel Pilin-Like Competence Factors Involved in Natural Transformation of Acinetobacter sp. Strain BD413

1999 ◽  
Vol 65 (10) ◽  
pp. 4568-4574 ◽  
Author(s):  
Silke Busch ◽  
Christine Rosenplänter ◽  
Beate Averhoff
Keyword(s):  
Natural Transformation ◽  
Open Reading Frames ◽  
Dna Translocation ◽  
Secretion Pathway ◽  
Coding Regions ◽  
Type Iv ◽  
Chromosomal Walking ◽  
General Protein ◽  
High Level ◽  
Overlapping Open Reading Frames

ABSTRACT Although the high level of competence for natural transformation ofAcinetobacter sp. strain BD413 has been the subject of numerous studies, only two competence genes, comC andcomP, have been identified to date. By chromosomal walking analysis we found two overlapping open reading frames, designatedcomE and comF, starting 61 bp downstream ofcomC. comE and comF are expressed as stable proteins in Escherichia coli, thus proving that they are indeed coding regions, but expression was successful only with 5′-deleted genes. ComE and ComF are similar to pilins and pilin-like components. Both genes were mutated, and the phenotypes of the mutants were analyzed. Natural transformation in comF mutants is 1,000-fold reduced, whereas comE mutants exhibit 10-fold-reduced transformation frequencies. This is clear evidence thatcomE and comF are involved in natural transformation. However, ComE and ComF are specific for DNA translocation, since comE and comF defects affected neither piliation nor lipase secretion. These results suggest that the type IV pili, the general protein secretion pathway, and the DNA translocation machinery in Acinetobacter sp. strain BD413 are evolutionary related but functionally distinct systems.

scholarly journals Genomic Sequence and Receptor for the Vibrio cholerae Phage KSF-1Φ: Evolutionary Divergence among Filamentous Vibriophages Mediating Lateral Gene Transfer

2005 ◽  
Vol 187 (12) ◽  
pp. 4095-4103 ◽  
Author(s):  
Shah M. Faruque ◽  
Iftekhar Bin Naser ◽  
Kazutaka Fujihara ◽  
Pornphan Diraphat ◽  
Nityananda Chowdhury ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Vibrio Cholerae ◽  
Genomic Sequence ◽  
Genetic Material ◽  
Open Reading Frames ◽  
Evolutionary Divergence ◽  
Type Iv ◽  
High Level ◽  
Filamentous Phages

ABSTRACT KSF-1Φ, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1Φ infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1Φ infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1Φ. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1Φ, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXΦ, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.

scholarly journals Type IV Pilus Biogenesis, Twitching Motility, and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF, PilT1, and PilT2

2013 ◽  
Vol 80 (2) ◽  
pp. 644-652 ◽  
Author(s):  
Ralf Salzer ◽  
Friederike Joos ◽  
Beate Averhoff
Keyword(s):  
Natural Transformation ◽  
Thermophilic Bacteria ◽  
Bacterial Species ◽  
Thermus Thermophilus ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Content Type ◽  
Ecological Diversification ◽  
Type Iv

ABSTRACTNatural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species.Thermus thermophilusHB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whetherT. thermophilushas any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that inT. thermophilus, T4P and natural transformation are linked but distinct systems.

scholarly journals Natural competence in Chlorogloeopsis fritschii PCC 6912 and other ramified cyanobacteria

2020 ◽  
Author(s):  
Benjamin L. Springstein ◽  
Fabian Nies ◽  
Tal Dagan
Keyword(s):  
Natural Transformation ◽  
Photosynthetic Apparatus ◽  
Arid Environments ◽  
Natural Competence ◽  
Type Iv ◽  
Chlorogloeopsis Fritschii ◽  
Competence Proteins ◽  
Global Biogeochemical Cycles ◽  
Dna Acquisition

AbstractLateral DNA transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA acquisition via transformation involves the uptake of DNA from the environment. The ability of recipient cells to actively transport DNA into the cytoplasm – termed natural competence – depends on the presence of type IV pili and competence proteins. Natural competence has been described in cyanobacteria for several organisms including unicellular and filamentous species. However, the presence of natural competence in ramified cyanobacteria, which are considered the peak of cyanobacterial morphological complexity, remains unknown. Here we show that ramified cyanobacteria harbour the genes essential for natural competence and experimentally demonstrate natural competence in the ramified cyanobacterium Chlorogloeopsis fritschii PCC 6912 (hereafter Chlorogloeopsis). Searching for homologs to known natural competence genes in ramified cyanobacteria showed that these genes are conserved in the majority of tested isolates. Experimental validation of natural competence using several alternative protocols demonstrates that Chlorogloeopsis could be naturally transformed with a replicative plasmid. Our results show that natural competence is a common trait in ramified cyanobacteria and that natural transformation is likely to play an important role in cyanobacteria evolution.ImportanceCyanobacteria are crucial players in the global biogeochemical cycles where they contribute to CO2- and N2-fixation. Their main ecological significance is the oxygen-producing photosynthetic apparatus that contributes to contemporary food chains. Ramified cyanobacteria form true-branching and multiseriate cell filament structures that represent a peak of prokaryotic multicellularity. Species in that group inhabit fresh and marine water habitats, thermal springs, arid environments, as well as endolithic and epiphytic habitats. Here we show that ramified cyanobacteria harbor the mechanisms required for DNA acquisition via natural transformation. The prevalence of mechanisms for natural uptake of DNA has implications for the role of DNA acquisition in the evolution of cyanobacteria. Furthermore, presence of mechanisms for natural transformation in ramified cyanobacteria opens up new possibilities for genetic modification of ramified cyanobacteria.

scholarly journals Investigating bacterial ribosomal sequence variation in regards to future structural and antibiotic research.

2021 ◽  
Author(s):  
Helena B Cooper ◽  
Kurt L Krause ◽  
Paul P. Gardner
Keyword(s):  
Pathogenic Bacteria ◽  
Deinococcus Radiodurans ◽  
Markov Models ◽  
Genomic Sequence ◽  
Thermus Thermophilus ◽  
Campylobacter Pylori ◽  
Haloarcula Marismortui ◽  
Bacterial Ribosome ◽  
Pathogenic Escherichia Coli ◽  
Species Specific

Ribosome-targeting antibiotics comprise over half of antibiotics used in medicine, but our fundamental knowledge of their binding sites is derived primarily from ribosome structures from non-pathogenic species. These include Thermus thermophilus, Deinococcus radiodurans and Haloarcula marismortui, as well as the commensal or pathogenic Escherichia coli. Advancements in electron cryomicroscopy have allowed for the determination of more ribosome structures from pathogenic bacteria, with each study highlighting species-specific differences that had not been observed in the non-pathogenic structures. These observed differences suggest that more novel ribosome structures, particularly from pathogens, are required to get a more accurate understanding of the level of diversity in the bacterial ribosome, leading to potential advancements in antibiotic research. In this study, covariance and hidden Markov models were used to annotate ribosomal RNA and protein sequences respectively from genomic sequence, allowing us to determine the underlying ribosomal sequence diversity using phylogenetic methods. This analysis provided evidence that the current non-pathogenic ribosome structures are not sufficient representatives of some pathogenic bacteria, such as Campylobacter pylori, or of whole phyla such as Bacteroidetes.

scholarly journals A new twist on bacterial motility – two distinct type IV pili revealed by cryoEM

2019 ◽  
Author(s):  
Alexander Neuhaus ◽  
Muniyandi Selvaraj ◽  
Ralf Salzer ◽  
Julian D. Langer ◽  
Kerstin Kruse ◽  
...  
Keyword(s):  
Natural Transformation ◽  
Thermus Thermophilus ◽  
Distinct Type ◽  
Type Iv Pili ◽  
Dna Uptake ◽  
Molecular Architecture ◽  
Surface Adhesion ◽  
Cellular Functions ◽  
Type Iv ◽  
New Type

SummaryMany bacteria express flexible protein filaments on their surface that enable a variety of important cellular functions. Type IV pili are examples of such filaments and are comprised of a helical assembly of repeating pilin subunits. Type IV pili are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development.By a complement of experimental approaches, we show that the bacterium Thermus thermophilus produces two different forms of type IV pilus. We have determined the structures of both and built atomic models. The structures answer key unresolved questions regarding the molecular architecture of type IV pili and identify a new type of pilin. We also delineate the roles of the two filaments in promoting twitching and natural transformation.

scholarly journals Cryo-EM structure of the bifunctional secretin complex of Thermus thermophilus

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Edoardo D'Imprima ◽  
Ralf Salzer ◽  
Ramachandra M Bhaskara ◽  
Ricardo Sánchez ◽  
Ilona Rose ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Natural Transformation ◽  
Thermus Thermophilus ◽  
Homology Model ◽  
Type Iv Pili ◽  
Gram Negative Bacteria ◽  
Dna Uptake ◽  
Type Iv ◽  
Periplasmic Domain

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA–DNA junction to the RNA/DNA heteroduplex

2008 ◽  
Vol 412 (3) ◽  
pp. 517-526 ◽  
Author(s):  
Naoto Ohtani ◽  
Masaru Tomita ◽  
Mitsuhiro Itaya
Keyword(s):  
Metal Ion ◽  
Deinococcus Radiodurans ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Rnase H ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Thermus Thermophilus Hb8

The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA–DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5′ side of the last ribonucleotide at the RNA–DNA junction. In fact, the TTHA0198 protein prefers the RNA–DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA–DNA junction of the RNA–DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5′ terminus of the RNA–DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA–DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity.

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