Mary E. Moore, University of California, Davis
Introduction: Helicobacter pylori is a naturally competent gram negative pathogen that colonizes the harsh and dynamic environment of the gastric mucosa. Natural competence and homologous recombination are employed by H. pylori to adapt to this environment and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable. Some strains seem to have a very low baseline level of transformation efficiency. Here we sought to identify conditions that would increase natural competence in H. pylori making it easier to generate mutants in strains with low transformation efficiency.
Results: We first determined the transformation efficiency of H. pylori strain SS1 under low oxygen (5% oxygen, 7.6% carbon dioxide, 7.6% hydrogen) and high oxygen (15% oxygen, 2.9% carbon dioxide, 2.9% hydrogen) conditions using plasmid DNA containing an antibiotic resistance marker. H. pylori transformed under high oxygen conditions exhibited approximately ten-fold greater transformation efficiency than at low oxygen. Next, we targeted an antibiotic resistance marker to an intergenic region in SS1 and then repeated the experiments with H. pylori strain J166. This verified that our results were robust across different H. pylori strains and different gene loci. Growth phase was also affected by oxygen conditions. However, increased transformation efficiency in high oxygen conditions occurs regardless of growth phase. Additionally, total genomic DNA from H. pylori grown in either high or low oxygen was run on an agarose gel (0.7%) and stained with SYBR Green I to detect double strand breaks. This showed that growth conditions used in this study did not result in detectable double strand breaks in the H. pylori DNA. Changing the oxygen concentration for these experiments also changed the concentration of carbon dioxide and hydrogen. To test whether carbon dioxide or hydrogen were responsible for inducing increased transformation efficiency, H. pylori was transformed under conditions of high oxygen with high carbon dioxide and hydrogen (15% oxygen, 7.6% carbon dioxide, 7.6% hydrogen). Under these conditions, transformation efficiency and growth rate remained the same as in low oxygen conditions.
Conclusion: The increase in transformation efficiency observed in high oxygen, low carbon dioxide conditions is not due to increase in oxygen tension, incidence of double strand breaks or change in growth phase. H. pylori is a capnophile and is able to both produce and use carbon dioxide in processes involved in pH regulation. Additionally, H. pylori is able to effectively use molecular hydrogen, which is abundantly present in the gastric mucosa, as an energy source. We hypothesize changes in either carbon dioxide or hydrogen concentration result in stimulation of the stringent response which induces increased transformation efficiency in H. pylori.
Bioreactors – Everything is Bigger in Texas
Wednesday, 31 Aug 2016 17.08
Implementation of the Lifecycle Approach to Process Validation Statistics
Monday, 29 Aug 2016 17.08
Meet 2016 – 2017 ISPE International Board of Directors
Wednesday, 24 Aug 2016 19.08