Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy.

Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by cryo-EM. However, dynamic properties of the microtubule are key to its function, and this behavior has proved difficult to characterize at a structural level due to limitations in existing structure determination methods. We developed a high-resolution cryo-EM refinement method that divides an imaged microtubule into its constituent protofilaments, enabling deviations from helicity and other sources of heterogeneity to be quantified and corrected for at the single-subunit level. We demonstrate that this method improves the resolution of microtubule 3D reconstructions and substantially reduces anisotropic blurring artifacts, compared with methods that utilize helical symmetry averaging. Moreover, we identified an unexpected, discrete behavior of the m-loop, which mediates lateral interactions between neighboring protofilaments and acts as a flexible hinge between them. The hinge angle adopts preferred values corresponding to distinct conformations of the m-loop that are incompatible with helical symmetry. These hinge angles fluctuate in a stochastic manner, and perfectly cylindrical microtubule conformations are thus energetically and entropically penalized. The hinge angle can diverge further from helical symmetry at the microtubule seam, generating a subpopulation of highly distorted microtubules. However, the seam-distorted subpopulation disappears in the presence of Taxol, a microtubule stabilizing agent. These observations provide clues into the structural origins of microtubule flexibility and dynamics and highlight the role of structural polymorphism in defining microtubule behavior.

Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site.

RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bio-augmented with the commercially available KB-1TM microbial culture. The KB-1TM culture contains multiple strains of Dehalococcoides mccartyi (D. mccartyi) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1TM-bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter. Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi, chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase "DET1545"), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

Biomarkers' Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM.

Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10-12 to 5.9 × 10-10 microelectron equivalents per cell per hour (µeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R² = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.

SPINE: SParse eIgengene NEtwork linking gene expression clusters in Dehalococcoides mccartyi to perturbations in experimental conditions.

We present a statistical model designed to identify the effect of experimental perturbations on the aggregate behavior of the transcriptome expressed by the bacterium Dehalococcoides mccartyi strain 195. Strains of Dehalococcoides are used in sub-surface bioremediation applications because they organohalorespire tetrachloroethene and trichloroethene (common chlorinated solvents that contaminate the environment) to non-toxic ethene. However, the biochemical mechanism of this process remains incompletely described. Additionally, the response of Dehalococcoides to stress-inducing conditions that may be encountered at field-sites is not well understood. The constructed statistical model captured the aggregate behavior of gene expression phenotypes by modeling the distinct eigengenes of 100 transcript clusters, determining stable relationships among these clusters of gene transcripts with a sparse network-inference algorithm, and directly modeling the effect of changes in experimental conditions by constructing networks conditioned on the experimental state. Based on the model predictions, we discovered new response mechanisms for DMC, notably when the bacterium is exposed to solvent toxicity. The network identified a cluster containing thirteen gene transcripts directly connected to the solvent toxicity condition. Transcripts in this cluster include an iron-dependent regulator (DET0096-97) and a methylglyoxal synthase (DET0137). To validate these predictions, additional experiments were performed. Continuously fed cultures were exposed to saturating levels of tetrachloethene, thereby causing solvent toxicity, and transcripts that were predicted to be linked to solvent toxicity were monitored by quantitative reverse-transcription polymerase chain reaction. Twelve hours after being shocked with saturating levels of tetrachloroethene, the control transcripts (encoding for a key hydrogenase and the 16S rRNA) did not significantly change. By contrast, transcripts for DET0137 and DET0097 displayed a 46.8±11.5 and 14.6±9.3 fold up-regulation, respectively, supporting the model. This is the first study to identify transcripts in Dehalococcoides that potentially respond to tetrachloroethene solvent-toxicity conditions that may be encountered near contamination source zones in sub-surface environments.