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 Mycobacterium tuberculosis transcriptional landscape under genotoxic stress.

BACKGROUND: As an intracellular human pathogen, Mycobacterium tuberculosis (Mtb) is facing multiple stressful stimuli inside the macrophage and the granuloma. Understanding Mtb responses to stress is essential to identify new virulence factors and pathways that play a role in the survival of the tubercle bacillus. The main goal of this study was to map the regulatory networks of differentially expressed (DE) transcripts in Mtb upon various forms of genotoxic stress. We exposed Mtb cells to oxidative (H2O2 or paraquat), nitrosative (DETA/NO), or alkylation (MNNG) stress or mitomycin C, inducing double-strand breaks in the DNA. Total RNA was isolated from treated and untreated cells and subjected to high-throughput deep sequencing. The data generated was analysed to identify DE genes encoding mRNAs, non-coding RNAs (ncRNAs), and the genes potentially targeted by ncRNAs. RESULTS: The most significant transcriptomic alteration with more than 700 DE genes was seen under nitrosative stress. In addition to genes that belong to the replication, recombination and repair (3R) group, mainly found under mitomycin C stress, we identified DE genes important for bacterial virulence and survival, such as genes of the type VII secretion system (T7SS) and the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family. By predicting the structures of hypothetical proteins (HPs) encoded by DE genes, we found that some of these HPs might be involved in mycobacterial genome maintenance. We also applied a state-of-the-art method to predict potential target genes of the identified ncRNAs and found that some of these could regulate several genes that might be directly involved in the response to genotoxic stress. CONCLUSIONS: Our study reflects the complexity of the response of Mtb in handling genotoxic stress. In addition to genes involved in genome maintenance, other potential key players, such as the members of the T7SS and PE/PPE gene family, were identified. This plethora of responses is detected not only at the level of DE genes encoding mRNAs but also at the level of ncRNAs and their potential targets.

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.

Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing.

Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology's (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000-3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.

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.

AMR-Diag: Neural network based genotype-to-phenotype prediction of resistance towards ß-lactams in Escherichia coli and Klebsiella pneumoniae.

Antibiotic resistance poses a major threat to public health. More effective ways of the antibiotic prescription are needed to delay the spread of antibiotic resistance. Employment of sequencing technologies coupled with the use of trained neural network algorithms for genotype-to-phenotype prediction will reduce the time needed for antibiotic susceptibility profile identification from days to hours. In this work, we have sequenced and phenotypically characterized 171 clinical isolates of Escherichia coli and Klebsiella pneumoniae from Norway and India. Based on the data, we have created neural networks to predict susceptibility for ampicillin, 3rd generation cephalosporins and carbapenems. All networks were trained on unassembled data, enabling prediction within minutes after the sequencing information becomes available. Moreover, they can be used both on Illumina and MinION generated data and do not require high genome coverage for phenotype prediction. We cross-checked our networks with previously published algorithms for genotype-to-phenotype prediction and their corresponding datasets. Besides, we also created an ensemble of networks trained on different datasets, which improved the cross-dataset prediction compared to a single network. Additionally, we have used data from direct sequencing of spiked blood cultures and found that AMR-Diag networks, coupled with MinION sequencing, can predict bacterial species, resistome, and phenotype as fast as 1-8 h from the sequencing start. To our knowledge, this is the first study for genotype-to-phenotype prediction: (1) employing a neural network method; (2) using data from more than one sequencing platform; and (3) utilizing sequence data from spiked blood cultures.

Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing.

Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1-2 days for identification of bacterial agent and an additional 2-3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnostic approach with the ability to rapidly identify pathogens and AMR-encoding genes. Here we have used nanopore sequencing of bacterial DNA extracted from positive blood cultures for identification of pathogens, detection of plasmids and AMR-encoding genes. To our knowledge, this is the first study to gather the above-mentioned information from nanopore sequencing and conduct a comprehensive analysis for diagnostic purposes in real-time. Identification of pathogens was possible after 10 minutes of sequencing and all predefined AMR-encoding genes and plasmids from monoculture experiments were detected within one hour using raw nanopore sequencing data. Furthermore, we demonstrate the correct identification of plasmids and blaCTX-M subtypes using de novo assembled nanopore contigs. Results from this study hold great promise for future applications in clinical microbiology and for health care surveillance purposes.

Characterization of the meningococcal DNA glycosylase Fpg involved in base excision repair.

BACKGROUND: Neisseria meningitidis, the causative agent of meningococcal disease, is exposed to high levels of reactive oxygen species inside its exclusive human host. The DNA glycosylase Fpg of the base excision repair pathway (BER) is a central player in the correction of oxidative DNA damage. This study aimed at characterizing the meningococcal Fpg and its role in DNA repair. RESULTS: The deduced N. meningitidis Fpg amino acid sequence was highly homologous to other Fpg orthologues, with particularly high conservation of functional domains. As for most N. meningitidis DNA repair genes, the fpg gene contained a DNA uptake sequence mediating efficient transformation of DNA. The recombinant N. meningitidis Fpg protein was over-expressed, purified to homogeneity and assessed for enzymatic activity. N. meningitidis Fpg was found to remove 2,6-diamino-4-hydroxy-5-formamidopyrimidine (faPy) lesions and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8oxoG) opposite of C, T and G and to a lesser extent opposite of A. Moreover, the N. meningitidis fpg single mutant was only slightly affected in terms of an increase in the frequency of phase variation as compared to a mismatch repair mutant. CONCLUSION: Collectively, these findings show that meningococcal Fpg functions are similar to those of prototype Fpg orthologues in other bacterial species.

A genomic view of experimental intraspecies and interspecies transformation of a rifampicin-resistance allele into Neisseria meningitidis.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P<0.001). The recombination events were generally observed to be mosaic, with donor sequences interspersed with recipient sequence. Here, we present four models to explain these observations, by fragmentation of the transformed DNA, by interruptions of the recombination mechanism, by secondary recombination of endogenous self-DNA, or by repair/replication mechanisms.

The helicase DinG responds to stress due to DNA double strand breaks.

Neisseria meningitidis (Nm) is a Gram-negative nasopharyngeal commensal that can cause septicaemia and meningitis. The neisserial DNA damage-inducible protein DinG is a helicase related to the mammalian helicases XPD and FANCJ. These helicases belong to superfamily 2, are ATP dependent and exert 5' → 3' directionality. To better understand the role of DinG in neisserial genome maintenance, the Nm DinG (DinGNm) enzymatic activities were assessed in vitro and phenotypical characterization of a dinG null mutant (NmΔdinG) was performed. Like its homologues, DinGNm possesses 5' → 3' directionality and prefers DNA substrates containing a 5'-overhang. ATPase activity of DinGNm is strictly DNA-dependent and DNA unwinding activity requires nucleoside triphosphate and divalent metal cations. DinGNm directly binds SSBNm with a Kd of 313 nM. Genotoxic stress analysis demonstrated that NmΔdinG was more sensitive to double-strand DNA breaks (DSB) induced by mitomycin C (MMC) than the Nm wildtype, defining the role of neisserial DinG in DSB repair. Notably, when NmΔdinG cells grown under MMC stress assessed by quantitative mass spectrometry, 134 proteins were shown to be differentially abundant (DA) compared to unstressed NmΔdinG cells. Among the DNA replication, repair and recombination proteins affected, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly down regulated while TopA and SSB were upregulated under stress condition. Most of the other DA proteins detected are involved in metabolic functions. The present study shows that the helicase DinG is probably involved in regulating metabolic pathways as well as in genome maintenance.

Characterization of the Neisseria meningitidis Helicase RecG.

Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant.

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.

Reduced expression of aquaporins in human intestinal mucosa in early stage inflammatory bowel disease.

OBJECTIVES: The aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans. METHODS: Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled. RESULTS: AQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn's disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function. CONCLUSION: AQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is significantly reduced in patients with IBD, and they are differentially expressed in specific bowel segments in patients with Crohn's disease and ulcerative colitis. The data present a link between gut inflammation and water/solute homeostasis, suggesting that AQPs may play a significant role in IBD pathophysiology.

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.

Gut bacterial profile in patients newly diagnosed with treatment-naïve Crohn's disease.

OBJECTIVES: The aim of this study was to define the composition of the gut bacterial flora in Norwegian patients with early stage Crohn's disease (CD). METHODS: By using a nonselective metagenomics approach, the general bacterial composition in mucosal biopsies from the ileum and the colon of five subjects, four patients with different phenotypes of CD, and one noninflammatory bowel disease control, was characterized. After partial 16S ribosomal RNA (rRNA) gene sequencing, BLAST homology searches for species identification and phylogenetic analysis were performed. RESULTS: An overall biodiversity of 106 different bacterial operational taxonomic units (OTUs) was detected in the cloned libraries. Nearly all OTUs belonged to the phylae Bacteroidetes (42% in CD, 71% in the control) or Firmicutes (42% in CD, 28% in the control), except for some OTUs that belonged to the phylum Proteobacteria (15% in CD, 0% in the control) and a few OTUs that could not be assigned to a phylum (2% in CD, 1% in the control). CONCLUSION: Based on the high incidence of inflammatory bowel disease (IBD) in Norway, this pilot study represents a relevant determination of the gut microbiota in Norwegian patients compared to previous findings in other countries. The bacterial profile of Norwegian CD patients was found to be similar to that of CD patients in other countries. The findings do not support a particular bacterial composition as a predominant causative factor for the high incidence of IBD that exists in some countries.

DNA base excision repair gene polymorphisms modulate human cognitive performance and decline during normal life span.

To test the hypothesis that single nucleotide polymorphisms (SNPs) in DNA repair genes are associated with cognitive performance during normal aging, the relationship between SNPs in selected exons in DNA base excision repair (BER) genes and cognitive performance was examined in 712 healthy Norwegian individuals aged 20-75 years. SNPs examined included PolB(Pro242Arg), hOGG1(Ser326Cys), MutYH (Met22Val), MutYH(His324Gln), APE1(Gln51His), APE1(Glu148Asp), XRCC1(Lys298Asn), XRCC1(Arg7Leu), NEIL1(Asp252Asn), and NEIL2(Arg257Leu). XRCC1(Arg7Leu) and PolB(Pro242Arg) were characterized by single nucleotide variations (≤0.1% homozygote SNPs). hOGG1(Ser326Cys) (Ser/Cys 40.8%/Cys/Cys 5.7%), MutYH(His324Gln) (His/Gln37%/Gln/Gln 6.0%) and APE1(Glu148Asp) (Glu/Asp 51.3%/Asp/Asp 23.0%) were characterized by higher SNP frequencies. MutYH(Met22Val), APE1(Gln51His) and NEIL2(Arg257Leu) occurred at intermediate SNP frequencies of 11.5, 7.6 and 5.3%, respectively. Interestingly, hOGG1(Ser326Cys) and APE1(Gln51His) had genotype by age interactions with general cognitive function, reasoning, control and speed of processing in cross-sectional analysis and a significant effect on longitudinal decline. Dispersed association effects involving MutYH(His324Gln), MutYH(Met22Val), PolB(Pro242Arg) and NEIL2(Arg257Leu) were also detected when APOE or CHRNA4, were included in the statistical model, a result consistent with proposed involvement of the latter markers in human cognitive decline and/or function. In summary, the results support the notion that polymorphisms in BER genes modulate cognitive performance in healthy elderly individuals.

Molecular diagnostics in tuberculosis: basis and implications for therapy.

The processing of clinical specimens in the mycobacterial diagnostic laboratory has undergone remarkable improvements during the last decade. While microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis on a worldwide basis, new methods including molecular diagnostic tests have evolved over the last two decades. The majority of molecular tests have been focused on (i) detection of nucleic acids, both DNA and RNA, that are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR); and (ii) detection of mutations in the genes that are associated with resistance to antituberculosis drugs by sequencing or nucleic acid hybridization. Recent developments in direct and rapid detection of mycobacteria, with emphasis on M. tuberculosis species identification by 16S rRNA gene sequence analysis or oligohybridization and strain typing, as well as detection of drug susceptibility patterns, all contribute to these advances. Generally, the balance between genome instability and genome maintenance as the basis for evolutionary development, strain diversification and resistance development is important, because it cradles the resulting M. tuberculosis phenotype. At the same time, semi-automated culture systems have contributed greatly to the increased sensitivity and reduced turnaround time in the mycobacterial analysis of clinical specimens. Collectively, these advances are particularly important for establishing the diagnosis of tuberculosis in children. More basic and operational research to appraise the impact and cost effectiveness of new diagnostic technologies must, however, be carried out. Furthermore, the design and quality of clinical trials evaluating new diagnostics must be improved to allow clinical and laboratory services that would provide rapid response to test results. Thus, important work remains before the new diagnostic tools can be meaningfully integrated into national tuberculosis control programs of high-burden countries.

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.

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.

New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators.

The frequently occurring DNA uptake sequence (DUS), recognized as a 10-bp repeat, is required for efficient genetic transformation in the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae. Genome scanning for DUS occurrences in three different species of Neisseria demonstrated that 76% of the nearly 2,000 neisserial DUS were found to have two semiconserved base pairs extending from the 5' end of DUS to constitute a 12-mer repeat. Plasmids containing sequential variants of the neisserial DUS were tested for their ability to transform N. meningitidis and N. gonorrhoeae, and the 12-mer was found to outperform the 10-mer DUS in transformation efficiency. Assessment of meningococcal uptake of DNA confirmed the enhanced performance of the 12-mer compared to the 10-mer DUS. An inverted repeat DUS was not more efficient in transformation than DNA species containing a single or direct repeat DUS. Genome-wide analysis revealed that half of the nearly 1,500 12-mer DUS are arranged as inverted repeats predicted to be involved in rho-independent transcriptional termination or attenuation. The distribution of the uptake signal sequence required for transformation in the Pasteurellaceae was also biased towards transcriptional terminators, although to a lesser extent. In addition to assessing the intergenic location of DUS, we propose that the 10-mer identity of DUS should be extended and recognized as a 12-mer DUS. The dual role of DUS in transformation and as a structural component on RNA affecting transcription makes this a relevant model system for assessing significant roles of repeat sequences in biology.