Evidence for the Active Role of a Novel Nuclease from Helicobacter pylori in the Horizontal Transfer of Genetic Information

Abstract

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.Helicobacter pylori is a gram-negative bacterium that infects the gastric mucosa of more than one-half of the world's human population (10). It is the major cause of peptic ulcers and the main risk factor for the development of gastric cancer (10). Previous molecular genetic work and comparative analysis of the complete genomic sequences of two isolates of H. pylori revealed the highest degree of genetic diversity reported for any bacterial species (22, 36). The panmictic structure of H. pylori populations has been determined by mathematical and experimental approaches (11,35). A panmictic structure is not common in haploids, and it is expected that sequential selective sweeps and/or bottlenecks remove genetic variants resulting from mutational events from populations (35,39). However, extremely frequent horizontal genetic exchange could maintain most of the generated allelic variants in a population, in addition to creating new ones (11,35).H. pylori is naturally competent for transformation, and natural genetic transformation is believed to be essential for the genetic plasticity observed in this and other haploid species (11,22). Genetic variability is expected to be relevant in H. pylori adaptiveness given the diverse conditions to which this persistent pathogen is exposed (37). It has been widely reported that more than one strain can simultaneously colonize a human host, and it has also been proven that genetic recombination occurs during natural mixed infections in humans, yielding better-adapted pathogen variants (4,11,16,34). These experiments proved that horizontal genetic transfer occurs in vivo. Not only can the ability to tra...

Helicobacter pylori Salvages Purines from Extracellular Host Cell DNA Utilizing the Outer Membrane-Associated Nuclease NucT

“…It was also shown that NucT is not simply secreted but instead appears to be associated with the bacterial outer membrane (39). This seems to indicate that the bacterium requires the protein to be within close proximity of its membrane, possibly to avoid the nuclease from being removed (as a secreted protein is) by the turnover of gastric mucus into the gut.…”

“…H. pylori has long been known to possess robust external nuclease activity, similar to numerous campylobacters (38), but this ability to degrade DNA has been examined only with regard to natural transformation (39,40) and never in the context of purine salvage and metabolism. As the uptake of DNA by the ComB system appeared to play no role in purine utilization in metabolism, we next tested the possibility that external DNases were digesting external DNA.…”

“…This seems to indicate that the bacterium requires the protein to be within close proximity of its membrane, possibly to avoid the nuclease from being removed (as a secreted protein is) by the turnover of gastric mucus into the gut. This would allow H. pylori to surround itself with a perpetual nuclease cloud, which could aid not only in natural transformation, as has been previously shown (39,40), but also in the degradation of extracellular, host cell DNA. Once the DNA is broken down, purine bases can pass readily through the bacterium's porous outer membrane and subsequently be transported through the inner membrane by purine permeases, such as NupC, whose activity in H. pylori has been described previously in the literature (20,43).…”

“…In its initial characterization in H. pylori (39), NucT was shown to be capable of degrading nucleic acids endonucleolytically (as well as potentially exonucleolytically), degrading both singlestranded and double-stranded substrates (preferring single stranded), and of hydrolyzing RNA in addition to DNA (preferring DNA). It was also shown that NucT is not simply secreted but instead appears to be associated with the bacterial outer membrane (39).…”

“…4A). nucT encodes a previously identified thermostable, membrane-associated nuclease that has been shown to be involved in transformation competence in H. pylori (39,40). Both OD 600 measurements and CFU counts indicate a defect in growth for the ⌬nucT mutant over this time period under the conditions tested ( Fig.…”