Papers published from CBMNet projects

Below you can view papers that have been generated through CBMNet funded projects…

Part by part: synthetic biology parts used in solventogenic Clostridia – Gyulev IS, Willson BJ, Hennessy RC, Krabben P, Jenkinson ER, Thomas GH

From CBMNet Project DeToX.

The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the last decade, genetic tools to enable transformation and chromosomal modifications have been developed, and the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia and reveal particular categories where increased fundamental knowledge is needed to fully develop reliable parts, such as promoters, transcriptional terminators and ribosome binding sites. We also aim to define areas where improved toolboxes are needed in these industrially important bacteria.

General calibration of microbial growth in microplate readers – Keiran Stevenson, Alexander F. McVey, Ivan B. N. Clark, Peter S. Swain & Teuta Pilizota

Including reference to CBMNet funded Vacation Scholarship and Proof-of-Concept projects

Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from studies of antibiotic efficacy to investigations of growth under different nutritional or stress environments, to characterization of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions, and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.

Dynamics of Escherichia coli’s passive response to a sudden decrease in external osmolarity – Renata Budaa, Yunxiao Liub, Jin Yangb, Smitha Hegdea, Keiran Stevensona, Fan Baib and Teuta Pilizota

Including reference to CBMNet funded Vacation Scholarship and Proof-of-Concept projects

Mechanosensation is central to life. Bacteria, like the majority of walled cells, live and grow under significant osmotic pressure. By relying on mechanosensitive regulation, bacteria can adapt to dramatic changes in osmotic pressure. Studying such mechanical sensing and control is critical for understanding bacterial survival in a complex host and natural environment. Here, we investigate the fundamental design principles of Escherichia coli’s passive mechanosensitive response to osmotic downshocks by implementing single-cell high-resolution imaging. We explain the observed cell volume changes by modeling flux of water and solutes across the cell membrane. A better characterization of bacterial mechanosensitive response can help us map their reaction to environmental threats.

Biosurfactants – E-book (Frontiers in Microbiology) – Pattanathu K.S.M. Rahman

Including reference to CBMNet funded Proof-of-Concept project ‘Plants as Nanoparticle Producers’

Covers a compilation of original research articles, reviews and research commentary submitted by researchers enthusiastically working in the field of biosurfactants and highlights recent advances in our knowledge of the biosurfactants and understanding of the biochemical and molecular mechanisms involved in their production, scale-up and industrial applications. There are 11 manuscripts accepted for publication in this research topic contributed by 55 authors from UK, Denmark, Greece, Germany, South Africa, India, Brazil, Bahrain, Portugal, and China.

Layer-by-Layer Assembly of Supported Lipid Bilayer Poly-l-Lysine Multilayers – George R. HeathMengqiu LiIsabelle L. PolignanoJoanna L. Richens§Gianluca CatucciPaul O’Shea§Sheila J. SadeghiGianfranco GilardiJulea N. Butt, and Lars J. C. Jeuken*

Included a collaboration that was funded via a Early Career Researcher grant

Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.

Acoustic vibration can enhance bacterial biofilm formation – Mark F.Murphy12ThomasEdwards2GlynHobbs2JoannaShepherd3FredericBezombes1

A paper as a result of CBMNet members meeting

This paper explores the use of low-frequency-low-amplitude acoustic vibration on biofilm formation. Biofilm development is thought to be governed by a diverse range of environmental signals and much effort has gone into researching the effects of environmental factors including; nutrient availability, pH and temperature on the growth of biofilms. Many biofilm-forming organisms have evolved to thrive in mechanically challenging environments, for example soil yet, the effects of the physical environment on biofilm formation has been largely ignored. Exposure of Pseudomonas aeruginosa to vibration at 100, 800 and 1600 Hz for 48 h, resulted in a significant increase in biofilm formation compared with the control, with the greatest growth seen at 800 Hz vibration. The results also show that this increase in biofilm formation is accompanied with an increase in P. aeruginosa cell number. Acoustic vibration was also found to regulate the spatial distribution of biofilm formation in a frequency-dependent manner. Exposure of Staphylococcus aureus to acoustic vibration also resulted in enhanced biofilm formation with the greatest level of biofilm being formed following 48 h exposure at 1600 Hz. These results show that acoustic vibration can be used to control biofilm formation and therefore presents a novel and potentially cost effective means to manipulate the development and yield of biofilms in a range of important industrial and medical processes.

Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii – Okada U1, Yamashita E2, Neuberger A3, Morimoto M1, van Veen HW3, Murakami S4.

A paper as a result of CBMNet members meeting

The MacA-MacB-TolC tripartite complex is a transmembrane machine that spans both plasma membrane and outer membrane and actively extrudes substrates, including macrolide antibiotics, virulence factors, peptides and cell envelope precursors. These transport activities are driven by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. Here, we present the crystal structure of MacB at 3.4-Å resolution. MacB forms a dimer in which each protomer contains a nucleotide-binding domain and four transmembrane helices that protrude in the periplasm into a binding domain for interaction with the membrane fusion protein MacA. MacB represents an ABC transporter in pathogenic microorganisms with unique structural features.

Lemons, or Squeezed for Resources? Information Symmetry and Asymmetric Resources in Biotechnology – Arthur Neuberger1, Nektarios Oraiopoulos2 and Donald L. Drakeman2*

A paper as a result of CBMNet members meeting

Thousands of biotech companies are developing promising products, but have insufficient resources to complete the clinical testing process, while large, well-funded companies have increasingly focused on the need to access external innovation. As a result, licensing deals are an essential and growing part of this industry. Yet, casting a shadow over the licensing market is the classic Lemons Problem: Does asymmetrical information put licensees at a severe disadvantage, leading to a market dominated by inferior opportunities, with the best products retained for internal development? Our analysis of clinical stage products developed over three decades shows that there is no Lemons Problem. We discuss the results of this first apples-to-apples analysis of the biomedical licensing market, and suggest reasons why the Lemons Problem does not exist where it might be most expected—in a high technology, knowledge-based industry.

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump – Fitzpatrick AWP#1, Llabrés S2, Neuberger A3, Blaza JN4, Bai XC1, Okada U5, Murakami S5, van Veen HW3, Zachariae U2,6, Scheres SHW1, Luisi BF7, Du D#7.

A paper as a result of CBMNet members meeting

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.

Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions – Nair AV1, Singh H1, Raturi S1, Neuberger A1, Tong Z1, Ding N1, Agboh K1, van Veen HW1

A paper as a result of CBMNet members meeting

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.

Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP – Arthur Neuberger, Hendrik W. van Veen

A paper as a result of CBMNet members meeting

Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden “Janus” amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of “Hoechst 33342-like” drugs to cells and tissues in which these transporters are expressed.