Ole Herman Ambur

Genome dynamics in major bacterial pathogens.

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.

Dialects of the DNA uptake sequence in Neisseriaceae.

In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS–dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5′-CTG-3′ is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS–dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation in bacteria, and define the phylogenetic relationships within the Neisseriaceae family.

Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses

ABSTRACT Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture- and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop.

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.

Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ

DsbA ensures the correct folding of many exported bacterial proteins by forming intramolecular disulphide bonds in the bacterial periplasm. The pathogen Neisseria meningitidis is unusual in its possession of three different dsbA genes (dsbA1, dsbA2 and dsbA3), encoding two membrane-anchored (DsbA1 and DsbA2) and one periplasmic (DsbA3) thiol-disulphide oxidoreductase enzymes. In this study, the involvement of DsbA1 and DsbA2 in natural competence was confirmed and attributed to events in the early stages of the transformation process. Strains lacking both DsbA1 and DsbA2 were reduced in competence as a result of decreased DNA binding and uptake. Overexpression of DsbA3 could not overcome this defect, suggesting differences in substrate specificity and protein-folding abilities between the DsbA homologues. Competence in Neisseria is dependent on the expression of type IV pili, which are extruded and retracted through the outer-membrane secretin PilQ. Both DsbA1 and DsbA2 were able to specifically bind PilQ in solid-phase overlay assays. Consistent with this, deletion of both dsbA1 and dsbA2 resulted in reduced levels of PilQ, confirming inefficient folding of PilQ, while pilus expression was apparently unaffected. The secretin PilQ is involved in DNA binding and transport as well as pilus biogenesis, and the defect in PilQ folding resulting from the absence of DsbA1 and DsbA2 is revealed in the observed decreased DNA binding and uptake.

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.

Steady at the wheel: conservative sex and the benefits of bacterial transformation.

Many bacteria are highly sexual, but the reasons for their promiscuity remain obscure. Did bacterial sex evolve to maximize diversity and facilitate adaptation in a changing world, or does it instead help to retain the bacterial functions that work right now? In other words, is bacterial sex innovative or conservative? Our aim in this review is to integrate experimental, bioinformatic and theoretical studies to critically evaluate these alternatives, with a main focus on natural genetic transformation, the bacterial equivalent of eukaryotic sexual reproduction. First, we provide a general overview of several hypotheses that have been put forward to explain the evolution of transformation. Next, we synthesize a large body of evidence highlighting the numerous passive and active barriers to transformation that have evolved to protect bacteria from foreign DNA, thereby increasing the likelihood that transformation takes place among clonemates. Our critical review of the existing literature provides support for the view that bacterial transformation is maintained as a means of genomic conservation that provides direct benefits to both individual bacterial cells and to transformable bacterial populations. We examine the generality of this view across bacteria and contrast this explanation with the different evolutionary roles proposed to maintain sex in eukaryotes. This article is part of the themed issue ‘Weird sex: the underappreciated diversity of sexual reproduction’.

Structure–function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation

Neisseria meningitidis, the meningococcus, is naturally competent for transformation throughout its growth cycle. The uptake of exogenous DNA into the meningococcus cell during transformation is a multi-step process. Beyond the requirement for type IV pilus expression for efficient transformation, little is known about the neisserial proteins involved in DNA binding, uptake and genome integration. This study aimed to identify and characterize neisserial DNA binding proteins in order to further elucidate the multi-factorial transformation machinery. The meningococcus inner membrane and soluble cell fractions were searched for DNA binding components by employing 1D and 2D gel electrophoresis approaches in combination with a solid-phase overlay assay with DNA substrates. Proteins that bound DNA were identified by MS analysis. In the membrane fraction, multiple components bound DNA, including the neisserial competence lipoprotein ComL. In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant. The DNA binding activity of the recombinant ComL and SSB proteins purified to homogeneity was verified by electromobility shift assay, and the ComL–DNA interaction was shown to be Mg2+-dependent. In 3D models of the meningococcus ComL and SSB predicted structures, potential DNA binding sites were suggested. ComL was found to co-purify with the outer membrane, directly interacting with the secretin PilQ. The combined use of 1D/2D solid-phase overlay assays with MS analysis was a useful strategy for identifying DNA binding components. The ComL DNA binding properties and outer membrane localization suggest that this lipoprotein plays a direct role in neisserial transformation, while neisserial SSB is a DNA binding protein that contributes to the terminal part of the transformation process.

DprA from Neisseria meningitidis: Properties and role in natural competence for transformation

DNA processing chain A (DprA) is a DNA-binding protein that is ubiquitous in bacteria and expressed in some archaea. DprA is active in many bacterial species that are competent for transformation of DNA, but its role in Neisseriameningitidis (Nm) is not well characterized. An Nm mutant lacking DprA was constructed, and the phenotypes of the wild-type and ΔdprA mutant were compared. The salient feature of the phenotype of dprA null cells is the total lack of competence for genetic transformation shown by all of the donor DNA substrates tested in this study. Here, Nm wild-type and dprA null cells appeared to be equally resistant to genotoxic stress. The gene encoding DprANm was cloned and overexpressed, and the biological activities of DprANm were further investigated. DprANm binds ssDNA more strongly than dsDNA, but lacks DNA uptake sequence-specific DNA binding. DprANm dimerization and interaction with the C-terminal part of the single-stranded binding protein SSBNmwere demonstrated. dprA is co-expressed with smg, a downstream gene of unknown function, and the gene encoding topoisomerase 1, topA.

The Inner Membrane Protein PilG Interacts with DNA and the Secretin PilQ in Transformation

Expression of type IV pili (Tfp), filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.