In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein.Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and 54 RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.Pseudomonas aeruginosa, like other fluorescent pseudomonads, is a metabolically versatile bacterium; it utilizes more than 100 different organic substrates for growth (37). This versatility requires a complex regulatory network that ensures the cellular carbon-nitrogen balance and determines the order in which growth substrates are degraded. In general, substrates that yield high energy and promote fast growth are degraded preferentially. For instance, intermediates of the tricarboxylic acid (TCA) cycle such as succinate prevent glucose degradation in P. aeruginosa (21). As a result, growth on a mixture of succinate and glucose is biphasic (diauxic) because the bacterium utilizes first succinate and then glucose (39). The underlying mechanism of carbon catabolite repression involves the CbrA/ CbrB two-component system (16,30), the small RNA (sRNA) CrcZ (36), and the RNA-binding protein Crc (6,22,25,26,33) as key regulatory elements. They are conserved in fluorescent pseudomonads (38; Pseudomonas Genome Database).
Quorum sensing has been implicated in the control of pathologically relevant bacterial behavior such as secretion of virulence factors, biofilm formation, sporulation, and swarming motility. The AI-2 quorum sensing pathway is found in both gram-positive and gram-negative bacteria. Therefore, antagonizing AI-2 quorum sensing is a possible approach to modifying bacterial behaviour. However, efforts in developing inhibitors of AI-2-mediated quorum sensing are especially lacking. High-throughput virtual screening using the V. harveyi LuxP crystal structure identified two compounds that were found to antagonize AI-2-mediated quorum sensing in V. harveyi without cytotoxicity. The sulfone functionality of these inhibitors was identified as critical to their ability to mimic the natural ligand in their interactions with Arg 215 and Arg 310 of the active site.
Recent
development in fluorescence-based molecular tools has contributed
significantly to developmental studies, including embryogenesis. Many
of these tools rely on multiple steps of sample manipulation, so obtaining large sample sizes presents a
major challenge as it can be labor-intensive and time-consuming. However,
large sample sizes are required to uncover critical aspects of embryogenesis,
for example, subtle phenotypic differences or gene expression dynamics.
This problem is particularly relevant for single-molecule fluorescence
in situ hybridization (smFISH) studies in Caenorhabditis
elegans embryogenesis. Microfluidics can help address
this issue by allowing a large number of samples and parallelization
of experiments. However, performing efficient reagent exchange on
chip for large numbers of embryos remains a bottleneck. Here, we present
a microfluidic pipeline for large-scale smFISH imaging of C. elegans embryos with minimized labor. We designed
embryo traps and engineered a protocol allowing for efficient chemical
exchange for hundreds of C. elegans embryos simultaneously. Furthermore, the device design and small
footprint optimize imaging throughput by facilitating spatial registration
and enabling minimal user input. We conducted the smFISH protocol
on chip and demonstrated that image quality is preserved. With one
device replacing the equivalent of 10 glass slides of embryos mounted
manually, our microfluidic approach greatly increases throughput.
Finally, to highlight the capability of our platform to perform longitudinal
studies with high temporal resolution, we conducted a temporal analysis
of par-1 gene expression in early C. elegans embryos. The method demonstrated here
paves the way for systematic high-temporal-resolution studies that
will benefit large-scale RNAi and drug screens and in systems beyond C. elegans embryos.
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