Biology subjects

Stubenrauch, C. J.

Publications and source records attributed to Stubenrauch, C. J..

3 recordsLinked to original sources

bioRxiv2021

Discovery of a conserved rule behind the assembly of β-barrel membrane proteins.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a "{beta}-signal" imprinted in the final {beta}-strand of the OMP engages the {beta}-barrel assembly machinery (BAM complex) to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule, that signals equivalent to the {beta}-signal are repeated in other, internal {beta}-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the {beta}-signal, arranging several {beta}-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults. TEASERBacterial outer membrane proteins are recognized and bound by BamD at specific signals located in multiple {beta}-strands at the C-terminus of these proteins.

biochemistry↗

bioRxiv2021

The component parts of bacteriophage virions accurately defined by a machine-learning approach built on evolutionary features.

Antimicrobial resistance (AMR) continues to evolve as a major threat to human health and new strategies are required for the treatment of AMR infections. Bacteriophages (phages) that kill bacterial pathogens are being identified for use in phage therapies, with the intention to apply these bactericidal viruses directly into the infection sites in bespoke phage cocktails. Despite the great unsampled phage diversity for this purpose, an issue hampering the roll out of phage therapy is the poor quality annotation of many of the phage genomes, particularly for those from infrequently sampled environmental sources. We developed a computational tool called STEP3 to use the "evolutionary features" that can be recognized in genome sequences of diverse phages. These features, when integrated into an ensemble framework, achieved a stable and robust prediction performance when benchmarked against other prediction tools using phages from diverse sources. Validation of the prediction accuracy of STEP3 was conducted with high-resolution mass spectrometry analysis of two novel phages, isolated from a watercourse in the Southern Hemisphere. STEP3 provides a robust computational approach to distinguish specific and universal features in phages to improve the quality of phage cocktails, and is available for use at http://step3.erc.monash.edu/. IMPORTANCEIn response to the global problem of antimicrobial resistance there are moves to use bacteriophages (phages) as therapeutic agents. Selecting which phages will be effective therapeutics relies on interpreting features contributing to shelf-life and applicability to diagnosed infections. However, the protein components of the phage virions that dictate these properties vary so much in sequence that best estimates suggest failure to recognize up to 90% of them. We have utilised this diversity in evolutionary features as an advantage, to apply machine learning for prediction accuracy for diverse components in phage virions. We benchmark this new tool showing the accurate recognition and evaluation of phage components parts using genome sequence data of phages from under-sampled environments, where the richest diversity of phage still lies.

microbiology↗

bioRxiv2021

Periplasm homeostatic regulation maintains spatial constraints essential for cell envelope processes and cell viability

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change the spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. Our findings demonstrate E. coli expressing elongated Lpp homeostatically counteracts periplasmic enlargement with a combination of tilting Lpp and reducing Lpp abundance. By genetic screening we identified all of the genes in E. coli that become essential in order to enact this homeostasis, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to enact this homeostasis. We observed increased levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation

cell biology↗