We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that ...only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Genomic Content of Neisseria Species TOBIASON, Deborah M; SEIFERT, H. Steven
Journal of Bacteriology,
04/2010, Letnik:
192, Številka:
8
Journal Article
The PilB protein of Neisseria gonorrhoeae has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine ...sulfoxide reductase family of enzymes, specifically MsrA and MsrB from Escherichia coli. MsrA and MsrB in E. coli are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of Neisseria gonorrhoeae is not involved in pilus expression. Additionally, we show that wild-type N. gonorrhoeae produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage.
Summary
The penC resistance gene was previously characterized in an FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. ...We show here that antibiotic resistance mediated by penC is the result of a Glu‐666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS‐resistant high‐molecular‐mass PilQ secretin complex, disrupts piliation and decreases transformation frequency by 50‐fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Site‐saturation mutagenesis of Glu‐666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues for stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence and absence of SDS‐resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm.
Piv, a site‐specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid ...motifs that define this novel family of both transposases and site‐specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.
Pilus-mediated adherence to mucosal epithelial cells is a critical step for Neisseria gonorrhoeae to establish an infection in the human host. CD46, the defined receptor for the gonococcal pilus, is ...a complement-regulatory protein that is expressed on all human nucleated cells. It was observed that a piliated, Opa(-) variant of gonococcal strain FA1090 adhered with different efficiencies to the human epithelial cell lines tested (Chang, ME180, HEC-1B and PC-3). Surprisingly, these differences in adherence levels did not correlate with levels of CD46 expressed by these cell lines. In fact, there was an inverse relationship between total surface-exposed CD46 and gonococcal adherence. Four major isoforms of CD46 are produced due to alternative RNA splicing of a surface-exposed region and the cytoplasmic tail. The relative isoform surface expression of each cell line was determined, and each was found to express different ratios of the four CD46 isoforms. No correlation could be derived between CD46 isoform surface expression and pilus-mediated gonococcal adherence, indicating that CD46 does not act as a classic receptor for gonococcal pili.
The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis . Piv shows significant homology with the transposases of ...the IS 110 /IS 492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the λ Int or Hin/Res
families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin
to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized
the interaction of Piv with the DNA inversion region of M. lacunata . Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies
indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside
of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the
recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with
apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.
Four bacteriophages infecting
mc
155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their ...genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages.
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