Browsing by Author "Liu, Shirley"

Now showing 1 - 4 of 4
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    Biochar and Microbial Signaling: Production Conditions Determine Effects on Microbial Communication
    (American Chemical Society, 2013) Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.
    Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell–cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.
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    Charcoal Disrupts Soil Microbial Communication through a Combination of Signal Sorption and Hydrolysis
    (American Chemical Society, 2016) Gao, Xiaodong; Cheng, Hsiao-Ying; Del Valle, Ilenne; Liu, Shirley; Masiello, Caroline A.; Silberg, Jonathan J.
    The presence of charcoal in soil triggers a range of biological effects that are not yet predictable, in part because it interferes with the functioning of chemical signals that microbes release into their environment to communicate. We do not fully understand the mechanisms by which charcoal alters the biologically available concentrations of these intercellular signals. Recently, charcoal has been shown to sorb the signaling molecules that microbes release, rendering them ineffective for intercellular communication. Here, we investigate a second, potentially more important mechanism of interference: signaling-molecule hydrolysis driven by charcoal-induced soil pH changes. We examined the effects of 10 charcoals on the bioavailable concentration of an acyl-homoserine lactone (AHL) used by many Gram-negative bacteria for cell–cell communication. We show that charcoals decrease the level of bioavailable AHL through sorption and pH-dependent hydrolysis of the lactone ring. We then built a quantitative model that predicts the half-lives of different microbial signaling compounds in the presence of charcoals varying in pH and surface area. Our model results suggest that the chemical effects of charcoal on pH-sensitive bacterial AHL signals will be fundamentally distinct from effects on pH-insensitive fungal signals, potentially leading to shifts in microbial community structures.
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    Lactose repressor hinge domain independently binds DNA
    (Wiley, 2018) Xu, Joseph S.; Hewitt, Madeleine N.; Gulati, Jaskeerat S.; Cruz, Matthew A.; Zhan, Hongli; Liu, Shirley; Matthews, Kathleen S.
    The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi-symmetric DNA sites for optimal contact by the N-terminal helix-turn-helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ∼4-fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.
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    The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation
    (American Chemical Society, 2016) Jones, Alicia M.; Mehta, Manan M.; Thomas, Emily E.; Atkinson, Joshua T.; Segall-Shapiro, Thomas H.; Liu, Shirley; Silberg, Jonathan J.; Systems, Synthetic, and Physical Biology Program
    Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement in which native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein’s functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AKs with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and it reveals a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection.