Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/PmmsA and PpHpdR/PhpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/PhpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops

Targeting RNS/caveolin-1/MMP signaling cascades to protect against cerebral ischemia-reperfusion injuries: potential application for drug discovery

Reactive nitrogen species (RNS) play important roles in mediating cerebral ischemia-reperfusion injury. RNS activate multiple signaling pathways and participate in different cellular events in cerebral ischemia-reperfusion injury. Recent studies have indicated that caveolin-1 and matrix metalloproteinase (MMP) are important signaling molecules in the pathological process of ischemic brain injury. During cerebral ischemia-reperfusion, the production of nitric oxide (NO) and peroxynitrite (ONOO-), two representative RNS, down-regulates the expression of caveolin-1 (Cav-1) and, in turn, further activates nitric oxide synthase (NOS) to promote RNS generation. The increased RNS further induce MMP activation and mediate disruption of the blood-brain barrier (BBB), aggravating the brain damage in cerebral ischemia-reperfusion injury. Therefore, the feedback interaction among RNS/Cav-1/MMPs provides an amplified mechanism for aggravating ischemic brain damage during cerebral ischemia-reperfusion injury. Targeting the RNS/Cav-1/MMP pathway could be a promising therapeutic strategy for protecting against cerebral ischemia-reperfusion injury. In this mini-review article, we highlight the important role of the RNS/Cav-1/MMP signaling cascades in ischemic stroke injury and review the current progress of studies seeking therapeutic compounds targeting the RNS/Cav-1/MMP signaling cascades to attenuate cerebral ischemia-reperfusion injury. Several representative natural compounds, including calycosin-7-O-β-D-glucoside, baicalin, Momordica charantia polysaccharide (MCP), chlorogenic acid, lutein and lycopene, have shown potential for targeting the RNS/Cav-1/MMP signaling pathway to protect the brain in ischemic stroke. Therefore, the RNS/Cav-1/MMP pathway is an important therapeutic target in ischemic stroke treatment.published_or_final_versio