Margareta Krabbe

Self-perpetuating epigenetic pili switches in bacteria

Bacteria have developed an epigenetic phase variation mechanism to control cell surface pili–adhesin complexes between heritable expression (phase ON) and nonexpression (phase OFF) states. In the pyelonephritis-associated pili (pap) system, global regulators [catabolite gene activator protein (CAP), leucine-responsive regulatory protein (Lrp), DNA adenine methylase (Dam)] and local regulators (PapI and PapB) control phase switching. Lrp binds cooperatively to three pap DNA binding sites, sites 1–3, proximal to the papBA pilin promoter in phase OFF cells, whereas Lrp is bound to sites 4–6 distal to papBA in phase ON cells. Two Dam methylation targets, GATCprox and GATCdist, are located in Lrp binding sites 2 and 5, respectively. In phase OFF cells, binding of Lrp at sites 1–3 inhibits methylation of GATCprox, forming the phase OFF DNA methylation pattern (GATCdist methylated, GATCprox nonmethylated). Binding of Lrp at sites 1–3 blocks pap pili transcription and reduces the affinity of Lrp for sites 4–6. Together with methylation of GATCdist, which inhibits Lrp binding at sites 4–6, the phase OFF state is maintained. We hypothesize that transition to the phase ON state requires DNA replication to dissociate Lrp and generate a hemimethyated GATCdist site. PapI and methylation of GATCprox act together to increase the affinity of Lrp for sites 4–6. Binding of Lrp at the distal sites protects GATCdist from methylation, forming the phase ON methylation pattern (GATCdist nonmethyated, GATCprox methylated). Lrp binding at sites 4–6 together with cAMP-CAP binding 215.5 bp upstream of the papBA transcription start, is required for activation of pilin transcription. The first gene product of the papBA transcript, PapB, helps maintain the switch in the ON state by activating papI transcription, which in turn maintains Lrp binding at sites 4–6.

Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis by Pyrosequencing Technology

ABSTRACT We developed an assay for rapid detection of rifampin resistance in Mycobacterium tuberculosis based on Pyrosequencing technology, involving a technique for real-time sequencing. A 180-bp region of the rpoB gene was amplified in clinical isolates of both rifampin-resistant and -susceptible M. tuberculosis. The PCR products were subjected to Pyrosequencing analysis using four different sequencing primers in four overlapping reactions. These four sequencing reactions covered the 81-bp region where >96% of the mutations associated with rifampin resistance are located. The results were compared to those obtained with two other molecular methods, the line probe assay and cycle sequencing, and the phenotypic BACTEC method. The genotypic determination methods all detected the mutations that previously have been correlated with rifampin resistance. In addition, Pyrosequencing analysis and the two other molecular methods found additional mutations within the rpoB gene in phenotypically susceptible strains. We found that Pyrosequencing technology, in particular, offers high accuracy, short turnaround time, and a potentially high throughput in detection of rifampin resistance in M. tuberculosis.

Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene

ABSTRACT Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.

Inactivation of the replication-termination system affects the replication mode and causes unstable maintenance of plasmid R1

Two so‐called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini‐derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild‐type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling‐circle structures with double‐stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling‐circle replication.

Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

The NudA protein in the gastric pathogen Helicobacter pylori is an ubiquitous and constitutively expressed dinucleoside polyphosphate hydrolase.

The gastric pathogen Helicobacter pylori harbors one Nudix hydrolase, NudA, that belongs to the nucleoside polyphosphate hydrolase subgroup. In this work, the enzymatic activity of purified recombinant NudA protein was analyzed on a number of nucleoside polyphosphates. This predicted 18.6-kDa protein preferably hydrolyzes diadenosine tetraphosphate, Ap4A at a k cat of 0.15 s−1 and a K m of 80 μm, resulting in an asymmetrical cleavage of the molecule into ATP and AMP. To study the biological role of this enzyme inH. pylori, an insertion mutant was constructed. There was a 2–7-fold decrease in survival of the mutant as compared with the wild type after hydrogen peroxide exposure but no difference in survival after heat shock or in spontaneous mutation frequency. Western blot analyses revealed that NudA is constitutively expressed in H. pylori at different growth stages and during stress, which would indicate that this protein has a housekeeping function. Given thatH. pylori is a diverse species and that all the H. pylori strains tested in this study harbor the nudAgene and show protein expression, we consider NudA to be an important enzyme in this bacterium.

Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli

The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild‐type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni‐ to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC. Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi‐ or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.

Type I restriction-modification loci reveal high allelic diversity in clinical Helicobacter pylori isolates.

Background:  A remarkable variety of restriction‐modification (R‐M) systems is found in Helicobacter pylori. Since they encompass a large portion of the strain‐specific H. pylori genes and therefore contribute to genetic variability, they are suggested to have an impact on disease outcome. Type I R‐M systems comprise three different subunits and are the most complex of the three types of R‐M systems.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of HP1352, a putative DNA methyltransferase in Helicobacter pylori.

The putative methyltransferase gene HP1352 from Helicobacter pylori strain 26695 was heterologously expressed in Escherichia coli. The 359-amino-acid gene product was purified and crystallized. The crystals belong to space group I2(1)2(1)2(1) and show diffraction to at least 2.5 A resolution. The unit-cell parameters are a = 69.6, b = 86.6, c = 140.0 A. A greater than 90% complete native data set has been collected and structure determination using the molecular-replacement method is ongoing.