Yi Chun Yeh

Assembly of the Caulobacter cell division machine

Cytokinesis in Gram‐negative bacteria is mediated by a multiprotein machine (the divisome) that invaginates and remodels the inner membrane, peptidoglycan and outer membrane. Understanding the order of divisome assembly would inform models of the interactions among its components and their respective functions. We leveraged the ability to isolate synchronous populations of Caulobacter crescentus cells to investigate assembly of the divisome and place the arrival of each component into functional context. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. Our results revealed a picture of divisome assembly with unprecedented temporal resolution. Specifically, we observed (i) initial establishment of the division site, (ii) recruitment of early FtsZ‐binding proteins, (iii) arrival of proteins involved in peptidoglycan remodelling, (iv) arrival of FtsA, (v) assembly of core divisome components, (vi) initiation of envelope invagination, (vii) recruitment of polar markers and cytoplasmic compartmentalization and (viii) cell separation. Our analysis revealed differences in divisome assembly among Caulobacter and other bacteria that establish a framework for identifying aspects of bacterial cytokinesis that are widely conserved from those that are more variable.

The Caulobacter Tol-Pal complex is essential for outer membrane integrity and the positioning of a polar localization factor

Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscopy images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor.

Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.

Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production.

BackgroundThe chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors.ResultsWe report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5’ mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts.ConclusionWe constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.

Quantitative analysis of multivalent interactions of carbohydrate-encapsulated gold nanoparticles with concanavalin A

Multivalent interactions between carbohydrate-encapsulated gold nanoparticles and Con A are found with high affinity and specificity.

One-step synthesis of water-soluble fluorescent copper nanoparticles for label-free detection of manganese ions

This paper reports a one-step method for the synthesis of water soluble fluorescent copper nanoparticles stabilized by 3-mercaptopropoic acid and histidine. The resulting Cu nanoparticles exhibit intense blue fluorescence (λem = 449 nm), and proved highly effective in the selective detection of manganese ions. The intensity of Cu nanoparticle fluorescence was shown to decrease with an increase in manganese ion concentration in the range of 0.25–250 μM.

A fluorescence-based microbial sensor for the selective detection of gold

This study presents a fluorescence-based microbial sensor for the detection of metal ions as a novel analytical tool for environmental applications. Our results demonstrate the effectiveness of whole-cell sensors in the selective detection of gold ions. Two heavy-metal-tolerant proteobacteria, Cupriavidus metallidurans and Ralstonia eutropha, were examined and showed great specificity. This work highlights the potential of employing engineered microbial strains as robust analytical tools.

Functionalizing bacterial cell surfaces with a phage protein.

Functionalization of bacterial cell surfaces has the potential to introduce new activities by chemical modification. Here we show that a bacteriophage-receptor complex can be used to functionalize the surface of two Gram-negative proteobacteria, Escherichia coli and Ralstonia eutropha with CdSe/ZnS nanoparticles. This work highlights the potential for using microbe-phage interactions to generate new functions on living cells.

Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli

This study used the biomolecule eumelanin as an agent for the reduction of metal ions. Our results demonstrate the effectiveness of synthesizing diverse metal nanoparticles through the use of recombinant E. coli expressing Rhizobium etli tyrosinase, MelA. Gold nanoparticles were recovered using cells with gold binding peptides on the surface. This study illustrates the possibility of using E. coli to produce and display diverse metal nanoparticles in a green chemistry synthetic route.

Development of a pigment-based whole-cell biosensor for the analysis of environmental copper

Using engineered microorganisms to detect heavy metals in the environment has proven to be highly effective and robust. This paper reports on the development of a novel microbial sensor for the detection of copper ions. To develop this microbial sensor, we screened and characterized various biological parts, including promoters, output signals, and hosts. In addition, we used the plant pigment betaxanthin to output fluorescent signals in order to reduce the detection time. The resulting whole-cell biosensor presented a good sensitivity when detecting copper ions in environmental samples including freshwater pond and tap water.