Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Nutrient scarcity is a common condition in nature, but the resulting extremely low growth rates (below 0.025 h(-1) ) are an unexplored research area in Bacillus subtilis. To understand microbial life in natural environments, studying the adaptation of B. subtilis to near-zero growth conditions is relevant. To this end, a chemostat modified for culturing an asporogenous B. subtilis sigF mutant strain at extremely low growth rates (also named a retentostat) was set up, and biomass accumulation, culture viability, metabolite production and cell morphology were analysed. During retentostat culturing, the specific growth rate decreased to a minimum of 0.00006 h(-1) , corresponding to a doubling time of 470 days. The energy distribution between growth and maintenance-related processes showed that a state of near-zero growth was reached. Remarkably, a filamentous cell morphology emerged, suggesting that cell separation is impaired under near-zero growth conditions. To evaluate the corresponding molecular adaptations to extremely low specific growth, transcriptome changes were analysed. These revealed that cellular responses to near-zero growth conditions share several similarities with those of cells during the stationary phase of batch growth. However, fundamental differences between these two non-growing states are apparent by their high viability and absence of stationary phase mutagenesis under near-zero growth conditions.

Here, we developed a cell-based biosensor that can assess meat freshness using the Gram-positive model bacterium Bacillus subtilis as a chassis. Using transcriptome analysis, we identified promoters that are specifically activated by volatiles released from spoiled meat. The most strongly activated promoter was PsboA, which drives expression of the genes required for the bacteriocin subtilosin. Next, we created a novel BioBrick compatible integration plasmid for B. subtilis and cloned PsboA as a BioBrick in front of the gene encoding the chromoprotein amilGFP inside this vector. We show that the newly identified promoter could efficiently drive fluorescent protein production in B. subtilis in response to spoiled meat and thus can be used as a biosensor to detect meat spoilage.

Depleted gas fields are amongst the most probable candidates for subsurface storage of CO2. With proven reservoir and qualified seal these fields have retained gas over geological time scales. However, unlike methane, injection of CO2 changes the pH of the brine due to formation of carbonic acid. Subsequent dissolution/precipitation of minerals changes the porosity/permeability of reservoir and cap-rock. Thus, for adequate, safe and effective CO2 storage, the subsurface system needs to be fully understood. An important aspect for subsurface storage of CO2 is purity of this gas, which influences risk and cost of the process. In order to investigate the effects of CO2 plus impurities in a real case example, we have carried out medium-term (30 days) laboratory experiments on reservoir and cap-rock core samples from gas fields in northeast Netherlands. In addition, we attempted to determine the maximum allowable concentration of one of the possible impurities in the CO2 stream (H2S) in these fields. The injected gases were CO2, CO2+100 ppm H2S and CO2+5000 ppm H2S. Before and after the experiments the core samples were analyzed by scanning electron microscope (SEM) and X-ray diffraction (XRD) for mineralogical variations. Also the permeability of the samples was measured. Following the experiments, dissolution of feldspars, carbonates and kaolinite was observed as expected. In addition, we observed fresh precipitation of kaolinite. However, two significant results were obtained when adding H2S in the CO2 stream. Firstly we observed precipitation of sulfate minerals (anhydrite and pyrite). This differs from results after pure CO2 injection where dissolution of anhydrite was dominant in the samples. Secondly, severe salt precipitation took place in the presence of H2S. This is caused by the higher solubility of H2S in water and a higher water content of gas phase in the presence of H2S. This was confirmed by modeling using CMG-GEM (CMG, 2011) modeling software. The precipitation of salt, anhydrite and pyrite affects the permeability of the samples in different ways. After pure CO2 and CO2+100 ppm H2S injection, permeability of the reservoir samples increased by 10–30% and <3%, respectively. In caprock samples permeability increased by a factor of 3–10 and 1.3, respectively. However, after addition of 5000 ppm H2S the permeability of all samples decreased significantly. In the case of CO2+100 ppm H2S, salt precipitation did balance mineral dissolution, causing minimal variation in the permeability of samples.