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Journal articleMcQuail J, Carpousis AJ, Wigneshweraraj S,
The association between Hfq and RNase E in long-term nitrogen starved <i>Escherichia coli</i>
<jats:title>Abstract</jats:title><jats:p>Under conditions of nutrient adversity, bacteria adjust metabolism to minimise cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In <jats:italic>Escherichia coli</jats:italic>, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualised together in live bacteria. We now show that in long-term nitrogen starved <jats:italic>E. coli</jats:italic>, both RNase E and Hfq co-localise in a single, large focus. This subcellular assembly, which we refer to as the H-body, forms by a liquid-liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of an hitherto unreported subcellular compartmentalisation of a process(s) associated with RNA management in stressed bacteria.</jats:p>
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Journal articleThomson M, Nunta K, Cheyne A, et al.,
Modulation of the cAMP levels with a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria
<jats:title>Abstract</jats:title><jats:p>Antimicrobial tolerance (AMT) is the gateway to the development of antimicrobial resistance (AMR) and is therefore a major issue that needs to be addressed.</jats:p><jats:p>The second messenger cyclic-AMP (cAMP), which is conserved across all taxa, is involved in propagating signals from environmental stimuli and converting these into a response. In bacteria, such as<jats:italic>M. tuberculosis</jats:italic>,<jats:italic>P. aeruginosa</jats:italic>,<jats:italic>V. cholerae</jats:italic>and<jats:italic>B. pertussis</jats:italic>, cAMP has been implicated in virulence, metabolic regulation and gene expression. However, cAMP signalling in mycobacteria is particularly complex due to the redundancy of adenylate cyclases, which are enzymes that catalyse the formation of cAMP from ATP, and the poor activity of the only known phosphodiesterase (PDE) enzyme, which degrades cAMP into 5’- AMP.</jats:p><jats:p>Based on these two features, the modulation of this system with the aim of investigating cAMP signalling and its involvement in AMT in mycobacteria id difficult.</jats:p><jats:p>To address this pressing need, we identified a new cAMP-degrading phosphodiesterase enzyme (Rv1339) and used it to significantly decrease the intrabacterial levels of cAMP in mycobacteria. This analysis revealed that this enzyme increased the antimicrobial susceptibility of<jats:italic>M. smegmatis</jats:italic>mc<jats:sup>2</jats:sup>155. Using a combination of metabolomics, RNA-sequencing, antimicrobial susceptibility assays and bioenergetics analysis, we were able to characterize the molecular mechanism underlying this increased susceptibility.</jats:p><jats:p>This work represents an important milestone showing that the targeting of cAMP signalling is a promising new avenue for antimicrobial development and expan
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Journal articleKumar RK, Meiller-Legrand TA, Alcinesio A, et al.,
Droplet printing reveals the importance of micron-scale structure for bacterial ecology
<jats:title>Abstract</jats:title><jats:p>Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community<jats:sup>1,2</jats:sup>. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs<jats:sup>3–8</jats:sup>. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium <jats:italic>Escherichia coli</jats:italic> that naturally compete with one another using protein toxins<jats:sup>9,10</jats:sup>. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in t
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