21 research outputs found
Identification of neisserial DNA binding components
Neisseria meningitidis, a causative agent of meningitis and septicaemia, expresses type IV pili, a feature correlating with the uptake of exogenous DNA from the environment by natural transformation. The outer membrane complex PilQ, through which pili are extruded and retracted, has previously been shown to bind DNA in its pore region. In order to further elucidate how DNA is transported across the membranes, we searched for DNA binding proteins within the meningococcal inner membrane. Inner membrane fractions from a panel of neisserial strains were subjected to a solid-phase overlay assay with DNA substrates, and MS was subsequently employed to identify proteins that bind DNA. A number of DNA binding components were detected, including the pilus biogenesis component PilG, the competence protein ComL, and the cell division ATP-binding protein FtsE, as well as two hypothetical proteins. The DNA binding activity of these components was not dependent on the presence of the neisserial DNA uptake sequence. Null mutants, corresponding to each of the proteins identified, were constructed to assess their phenotypes. Only mutants defective in pilus biogenesis were non-competent and non-piliated. The DNA binding activity of the pilus biogenesis components PilQ and PilG and the phenotypes of their respective null mutants suggest that these proteins are directly involved as players in natural transformation, and not only indirectly, through pilus biogenesis
Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis
Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination
The outer membrane secretin PilQ from Neisseria meningitidis binds DNA
Neisseria meningitidis is naturally competent for transformation throughout its growth cycle. Transformation in neisserial species is coupled to the expression of type IV pili, which are present on the cell surface as bundled filamentous appendages, and are assembled, extruded and retracted by the pilus biogenesis components. During the initial phase of the transformation process, binding and uptake of DNA takes place with entry through a presumed outer-membrane channel into the periplasm. This study showed that DNA associates only weakly with purified pili, but binds significantly to the PilQ complex isolated directly from meningococcal membranes. By assessing the DNA-binding activity of the native complex PilQ, as well as recombinant truncated PilQ monomers, it was shown that the N-terminal region of PilQ is involved in the interaction with DNA. It was evident that the binding of ssDNA to PilQ had a higher affinity than the binding of dsDNA. The binding of DNA to PilQ did not, however, depend on the presence of the neisserial DNA-uptake sequence. It is suggested that transforming DNA is introduced into the cell through the outer-membrane channel formed by the PilQ complex, and that DNA uptake occurs by non-specific introduction of DNA coupled to pilus retraction, followed by presentation to DNA-binding component(s), including PilQ
Interactions between the Lipoprotein PilP and the Secretin PilQ in Neisseria meningitidis▿ †
Neisseria meningitidis can be the causative agent of meningitis or septicemia. This bacterium expresses type IV pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. Here we have studied the physical properties of the lipoprotein PilP and examined its interaction with PilQ. We found that PilP was an inner membrane protein required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. These mutant phenotypes were restored by the expression of PilP in trans. The pilP gene is located upstream of pilQ, and analysis of their transcripts indicated that pilP and pilQ were cotranscribed. Furthermore, analysis of the level of PilQ expression in pilP mutants revealed greatly reduced amounts of PilQ only in the deletion mutant, exhibiting a polar effect on pilQ transcription. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide. A three-dimensional reconstruction of the PilQ-PilP interacting complex was obtained at low resolution by transmission electron microscopy, and PilP was shown to localize around the cap region of the PilQ oligomer. These findings suggest a role for PilP in pilus biogenesis. Although PilQ does not need PilP for its stabilization or membrane localization, the specific interaction between these two proteins suggests that they might have another coordinated activity in pilus extrusion/retraction or related functions
DNA Uptake by the Nosocomial Pathogen Acinetobacter baumannii Occurs during Movement along Wet Surfaces
The emergence of Acinetobacter baumannii as an increasingly multidrug-resistant nosocomial pathogen largely relies on acquisition of resistance genes via horizontal gene transfer. Here, we demonstrate that many clinical isolates of A. baumannii take up DNA while they move along wet surfaces. We show that both motility and DNA uptake are abolished after inactivation of pilT, which putatively encodes the type 4 pilus (T4P) retraction ATPase, and comEC, which putatively encodes the DNA uptake channel, respectively. Inactivation of pilT correlates with an increase in the number and length of pili with an average diameter of 7.2 nm. In the Galleria mellonella infection model, the comEC mutant is significantly attenuated, whereas the pilT mutant is not, dissecting biologically distinct roles of T4P and the DNA uptake channel. Collectively, these findings promote our understanding of the mechanisms of DNA uptake and resistance development in A. baumannii, which may also apply to other important pathogens