Papers by Werner Pansegrau
Journal of Molecular Biology, 1998
The broad host range IncP plasmids are of particular interest because of their ability to promote... more The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial species. To facilitate study of these plasmids we have compiled the complete sequence of the IncPb plasmid R751. Comparison with the sequence of the IncPa plasmids con®rms the conservation of the IncP backbone of replication, conjugative transfer and stable inheritance functions between the two branches of this family. As in the IncPa genome the DNA of this backbone appears to have been enriched for the GCCG/CGGC motifs characteristic of the genome of organisms with a high G C content, such as P. aeruginosa, suggesting that IncPb plasmids have been subjected during their evolution to similar mutational and selective forces as IncPa plasmids and may have evolved in pseudomonad hosts. The IncP genome is consistently interrupted by insertion of phenotypic markers and/ or transposable elements between oriV and trfA and between the tra and trb operons. The R751 genome reveals a family of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA. Sequence analysis of the cryptic transposon Tn4321 revealed that it is not a member of the Tn21 family as we had proposed previously from an inspection of its ends. Rather it is a composite transposon de®ned by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distributed throughout the prokaryotes. The central unique region of Tn4321 encodes two predicted proteins, one of which is a regulatory protein while the other is presumably responsible for an as yet unidenti®ed phenotype. The most striking feature of the IncPa plasmids, the global regulation of replication and transfer by the KorA and KorB proteins encoded in the central control operon, is conserved between the two plasmids although there appear to be signi®cant differences in the speci®city of repressor-operator interactions. The importance of these global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts where the surrounding region, either a protein coding or intergenic sequence, has diverged considerably. There appears to be no equivalent of the parABCDE region which in the IncPa plasmids provides multimer resolution, lethality to plasmidfree segregants and active partitioning functions. However, we found that the continuous sector from co-ordinate 0 to 9100 bp, encoding the co-regulated klc and kle operons as well as the central control region, could confer a high degree of segregational stability on a low copy number test vector. Thus R751 appears to exhibit very clearly what was ®rst revealed by study of the IncPa plasmids, namely a fully functional
Infection and Immunity, 2010
Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus com... more Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the "head" of the protein, which contains the putative binding domains, whereas the elongated "stalk" was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the "head" domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.
Infection and Immunity, 2009
Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneu... more Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.
Infection and Immunity, 2007
Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that thi... more Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.
Journal of Bacteriology, 2008
Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification ... more Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.
Journal of Biological Chemistry, 2011
The Journal of biological chemistry, Jan 22, 2011
Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment... more Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution struc...
Progress in Nucleic Acid Research and Molecular Biology, 1996
Mitochondrial DNA, 1991
The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 r... more The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.
The Journal of biological chemistry, Jan 22, 2011
Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment... more Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution struc...
Proceedings of The National Academy of Sciences, 2000
RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range pl... more RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop). Helicobacter pylori HP0525 also belongs to the PulE superfamily and is encoded
Proceedings of the National Academy of Sciences, 1990
During tiatin of conjugative transfer of DNA containing the transfer origin (onTiT) of the promis... more During tiatin of conjugative transfer of DNA containing the transfer origin (onTiT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and Trai interact and assemble a specialized nucleoprotein complex (the relaxosome) at onT. The structure can be visualized on electron micrographs. Siteand strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraIl and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specifled TraJ protein cannot recognize the closely related onT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [
Proceedings of the National Academy of Sciences, 1993
Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by s... more Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (oriT). Cleavage results in covalent attachment of the plasmid-encoded relaxase (TraI) to the 5'-terminal 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the center of the catalytic site of TraI, mediating cleavage via formation of a phosphodiester between the DNA 5' phosphoryl and the aromatic hydroxyl group. The specificity of cleavage seen with form I oriT DNA was verified with short oligodeoxy-ribonucleotides embracing the nick region. The reaction requires TraI and Mg2+ but is independent of the relaxosome component TraJ. Cleavage produces one oligonucleotide fragment with a free 3' hydroxyl, the other part forms a covalent TraI-oligonucleotide adduct. Like nicking of form I oriT DNA, TraI-catalyzed oligonucleotide cleavage reaches an equilibrium when about 30% of the input TraI exists as a covalent protein-DNA complex. In the presence of two differently sized oligonucleotides, defined hybrid oligonucleotides are produced, demonstrating that TraI catalyzes recombination of two single strands at nic. This finding shows that TraI possesses cleaving-joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 nucleotides adjacent to nic. Only certain base changes in a few positions are tolerated, whereas the sequence of the 5' terminal nucleotides apparently is irrelevant for recognition by TraI. The reactions described here further support the hypothesis that DNA transfer via conjugation involves a rolling circle-like mechanism which generates the immigrant single strand while DNA-bound TraI protein scans for the occurrence of a second cleavage site at the donor-recipient interface.
Proceedings of the National Academy of Sciences, 1991
The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 o... more The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides
Proceedings of the National Academy of Sciences, 1993
As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked ... more As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked at the border sequences that delimit the T-DNA. Cleavage results in covalent attachment of VirD2 to the 5' termini of the nicked strand by a process resembling initiation of DNA transfer that occurs in the donor cell during bacterial conjugation. We
Proceedings of the National Academy of Sciences, 1989
To characterize protein-DNA interactions involved in the initiation of conjugative transfer repli... more To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (ori7) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the Abbreviation: ORF, open reading frame. tPresent address:
Plasmid, 1987
The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycosid... more The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type 1. The nucleotide sequence of the kanamycin resistance gene (Km? and the right end of the insertion element I!88 of plasmid RP4 has been determined. The gene (8 16 bp) is located between IS8 and the region (Tral ) encoding plasmid factors mediating bacterial conjugation. Km' and Tra I are transcribed toward each other. The nucleotide sequence has been compared to five related aphA genes originating from gram-negative and gram-positive organisms and from antibiotic producers. Among these that of Tn903 shares the highest degree of similarity (60%) with the RP4 gene. Significant similarities were also detected between the amino acid sequences of the six enzymes. The C-terminal domains of six different aminoglycoside 3'-phosphotransfer (APH(3')) are highly conserved. They are sub stantially similar to segments of a variety of enzymes using ATP as cofactor. The role of the C-terminal sequences of APH(3') as potential domains for ATP recognition and binding is diiUSS&.
Nucleic Acids Research, 1987
Uploads
Papers by Werner Pansegrau