Lai, T. F. | bioRxiv Science

bioRxiv2024

Quantitative proteome of bacterial periplasmic predation reveals a prey damaging protease

The rise of antimicrobial resistant pathogens calls for novel ways to kill and damage bacteria. A rich source for bacterial cell-damaging proteins is periplasmic predatory bacterium Bdellovibrio bacteriovorus, which invades, kills and subsequently exits the Gram-negative prey cell. An increased understanding of predatory protein function can be achieved by analyzing their relative abundance at key stages of predation. Here, we present the first quantitative proteome covering the complete predatory life cycle of the bacterial predator B. bacteriovorus killing Escherichia coli, quantifying 2195 predator proteins. From these proteins, nine protein clusters sharing similar expression patterns were identified. Towards the end of the life cycle when the predator exits prey remnants, we identified significant amounts of protease Bd2269. Gene knockout and heterologous expression experiments revealed that Bd2269 is involved in the prey exit process and damages E. coli from within. This quantitative predator proteome is a valuable resource to unravel bacterial predator-prey interactions and advances the search for novel antimicrobial enzymes.

microbiology↗

bioRxiv2017

Characterization of Azotobacter vinelandii and Kits for Its Synthetic Biology Applications

Azotobacter vinelandii, a Gram-negative aerobic bacterium with an intracellular anaerobic environment that maintains the oxygen-sensitive enzymatic cascades for nitrogen fixation, could be used to express oxygen-sensitive proteins. However, little is known about the properties of A. vinelandii for synthetic biology applications. We therefore first characterized and optimized the conditions for growing and screening BioBrick constructs in A. vinelandii in the presence of 2 antibiotics, ampicillin and chloramphenicol, and then developed two sets of BioBricks for regulated protein expression. The first kit used T7 RNA polymerase, whose expression is under the control of a nitrogen-repressible nifH promoter. The commonly used T7-dependent system in Escherichia coli can then be used in A. vinelandii. Because its intracellular anaerobic environment is favorable for processes such as magnetosome biogenesis, we attempted to migrate the biogenesis machineries from the magnetotactic bacterium Magnetospirillum gryphiswaldense to A. vinelandii. During this undertaking, another insertion kit construct was developed to allow protein conjugation onto magnetosomes. The kit consists of mamC, a gene encoding a transmembrane protein on magnetosomes, and multiple restriction sites downstream of mamC for fusing a gene of interest. This insertion kit allows the attachment of any desired protein onto the magnetosome membrane by fusing with the mamC protein. We demonstrated the function of this kit by fusing mamC to a GFP nanobody. This kit will facilitate the conjugation of any target protein onto magnetosomes for downstream applications in the future.\n\nFinancial DisclosureWe received sponsorship from the 2012-15 Teaching Development Grants Triennium, Faculty of Engineering and Biochemistry Program, School of Life Sciences, The Chinese University of Hong Kong. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting InterestsThe authors declare that no competing interests exist.\n\nEthics StatementN/A.\n\nData AvailabilityAll data are fully available without restriction.

synthetic biology↗