The cell walls of rod-shaped Gram-positive bacteria are thick, multilayered networks that chirally twist as cells elongate. The underlying basis of twisting is not known, but probing the processes underlying this phenomenon may give insights into how cell wall material is inserted, how it evolves during cleavage, and the mechanics within the sacculus. In Bacillus subtilis, we see cell chains lacking hydrolases twist far slower than chains of wild-type cells, indicating that cell wall cleavage modulates the twisting rate. We see that when cells within chains separate, the two nascent ends rotate as they separate. Together, this suggests there is torsional stress within the cell wall that, when unreleased, perturbs overall chain morphology. Unlike Escherichia coli, we see that twisting does not arise from MreB's angle of motion, as its angle is identical in both fast-twisting wild-type cells and slow-twisting hydrolase-deficient cells. Rather, the circumferential insertion of glycans appears to establish this torsional stress, as increasing Rod complex activity by deleting ponA causes cells to twist faster than wild-type cells. Together, these experiments suggest the twisting of B. subtilis cells arises from radial glycan insertion, which somehow causes torsional stress in the wall that is later released by hydrolase activity.

01 Apr 2025·Advanced Science

Cell Sizes Matter for Industrial Bioproduction, a Case of Polyhydroxybutyrate

Article

Author: Guo, Wei‐Ke ; Zhang, Li‐Zhan ; Wu, Qiong ; Wang, Jia‐Le ; Chen, Guo‐Qiang ; Chen, Yi‐Ling ; Liu, Xu ; Wu, Fu‐Qing ; Yang, Ji‐Shuai ; Yan, Xu ; Zheng, Shuang

AbstractMost bacterial cells are 1–2 microns in size, limiting intracellular products like polyhydroxyalkanoates (PHA) accumulation. Cell size is regulated by key genes such as mreB and minCD, which encode cellular skeleton protein and control cell fission ring location, respectively. Their expression changes significantly affect microbial growth. This study successfully redesigns the ClpXP protein degradation system by deleting the sspB gene and using mutated SsrA tags with different degradation rates to control MreB degradation. Dynamic degradation of MreB allows non‐model bacterium Halomonas bluephagenesis to grow normally and increase cell size simultaneously. Combined with overexpression of minCD, H. bluephagenesis with progressive MreB degradation increases the cell size further, albeit with a reduced growth rate. H. bluephagenesis CYL0307, with the PHB granule‐associated protein PhaP1 deleted and phaABRe overexpressed in the MreB‐degraded strain, increases cell volume more than nine times compared to the original strain. CYL0307 produces 149 g L−1 cell dry weight containing 82% PHB after 44 h in a 5000 L bioreactor, with cells containing single large PHB granules, simplifying recovery and purification. These results provide a post‐translational gene regulation method in H. bluephagenesis and a strategy for enhancing PHB production via morphological engineering.

01 Apr 2025·Accounts of Chemical Research

Biological Regulation Studied in Vitro and in Cellulo with Modified Proteins

Article

Author: Hecht, Sidney M. ; Chen, Shengxi ; Dedkova, Larisa M.

ConspectusProteins and peptides occur ubiquitously in organisms and play key functional roles, as structural elements and catalysts. Their major natural source is ribosomal synthesis, which produces polypeptides from 20 amino acid building blocks. Peptides containing noncanonical amino acids have long been prepared by chemical synthesis, which has provided a wealth of physiologically active compounds. Comparatively, preparing modified proteins has been more challenging. Site-directed mutagenesis provided an important advance but was initially limited to canonical amino acids. New techniques for tRNA activation with noncanonical amino acids subsequently increased the scope of site-directed mutagenesis.Our report in 2012 demonstrated that modification of bacterial ribosomes at key positions enabled the selection of ribosomes capable of introducing β-amino acids into proteins in vitro. The generality of the selection procedure was tested further. Ribosomes capable of incorporating dipeptides, conformationally constrained dipeptides, dipeptidometics with embedded fluorophores, contiguous nucleobase amino acids, and phosphorylated amino acids were successfully identified.In this Account, we focus on the application of the new technology to dramatically alter protein structure in ways that enable new strategies for understanding and altering protein function. To illustrate the robustness of the technology we have provided examples studied in vitro and in cellulo. The first category involves the introduction of nucleobase amino acids into proteins in support of specific interactions with RNA and DNA. The energetic differences between potential protein-nucleic acid complexes formed from two binding partners are often quite small. It seems logical to think that selective binding can be achieved by using a nucleobase moiety in each of the binding partners by utilizing known interactions between nucleic acid bases (located in the protein and nucleic acid) to achieve energetically favorable interactions. We do so both in vitro and in cellulo. A second focus has involved the design of small fluorescent probes not much larger than amino acids that are genetically encodable and which can be incorporated during protein biosynthesis, serving as detectable probes of protein trafficking and interaction with other macromolecules. We provide an in vitro example of strongly fluorescent tryptophan analogues positioned at single sites within dihydrofolate reductase, permitting selective communication with a FRET acceptor at a known position, even in the presence of several tryptophans. An oxazole amino acid, weakly fluorescent in aqueous solution, fluoresced more strongly following incorporation into MreB, a scaffold protein produced in cellulo. Finally, we describe the introduction of a single phosphorylated tyrosine into the p50 subunit of NF-κB. When present at either of two key positions, the resulting NF-κB significantly enhanced binding in vitro to the promoter DNA as well as subsequent mRNA transcription of its client protein CD40 in cellulo. In a separate expression in activated Jurkat cells, an increased production of CD40 protein was observed.

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Rod shape-determining protein mreB

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