The N-terminal FleQ domain of the Vibrio cholerae flagellar master regulator FlrA plays pivotal structural roles in stabilizing its active state.

In Vibrio cholerae, the master regulator FlrA controls transcription of downstream flagellar genes in a σ54 -dependent manner. However, the molecular basis of regulation by VcFlrA, which contains a phosphorylation-deficient N-terminal FleQ domain, has remained elusive. Our studies on VcFlrA, four of its constructs, and a mutant showed that the AAA+ domain of VcFlrA, with or without the linker 'L', remains in ATPase-deficient monomeric states. By contrast, the FleQ domain plays a pivotal role in promoting higher-order functional oligomers, providing the required conformation to 'L' for ATP/cyclic di-GMP (c-di-GMP) binding. The crystal structure of VcFlrA-FleQ at 2.0 Å suggests that distinct structural features of VcFlrA-FleQ presumably assist in inter-domain packing. VcFlrA at a high concentration forms ATPase-efficient oligomers when the intracellular c-di-GMP level is low. Conversely, excess c-di-GMP locks VcFlrA in a non-functional lower oligomeric state, causing repression of flagellar biosynthesis.

Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria.

Reporter phage assays are a promising alternative to culture-based assays for rapidly detecting viable bacteria. The reporter systems used in phage-based detection are typically enzymes and their corresponding substrates that provide a signal following infection and expression. While several reporter systems have been developed, comparing reporter systems based on reported bacteria detection limits from literature can be challenging due to factors other than the reporter system that influence detection capabilities. To advance the development of phage-based assays, a systematic comparison and understanding of the components are necessary. The objective of this study was to directly compare two common enzyme-mediated luminescence reporter systems, NanoLuc/Nano-Glo and alkaline phosphatase (ALP*)/DynaLight, for phage-based detection of bacteria. The detection limits of the purified enzymes were determined, as well as the expression levels and bacteria detection capabilities following engineering of the coding genes into T7 phage and infection of E. coli BL21. When comparing the sensitivity of the purified enzymes, NLuc/Nano-Glo enzyme/substrate system demonstrated a lower detection limit than ALP*/DynaLight. In addition, the expression of the NLuc reporter following phage infection of E. coli was greater than ALP*. The lower detection limit combined with the higher expression resulted in a greater than 100-fold increase in sensitivity for the NLuc/Nano-Glo® reporter system compared to ALP*/DynaLight when used for the detection of E. coli in a model system. These findings provide a comparative analysis of two common reporter systems used for phage-based detection of bacteria and a foundational understanding of these systems for engineering future reporter phage assays.

Prospects of utilizing seawater as a reaction medium for pretreatment and saccharification of rice straw.

The transition towards a bio-based economy has led to an unprecedented surge in fresh water consumption that renders biofuel a high water footprint product. The depleting fresh water resources have exacerbated the situation which necessitates the exploration of non-potable water for biorefinery purposes. In the current study, seawater is used as a plausible alternative reaction medium for pretreatment and saccharification of rice straw. Response Surface Methodology (RSM) based on Box-Behnken Design (BBD) was employed to model, predict and validate cellulose release and reducing sugar yield from rice straw subjected to microwave-NaOH pretreatment. The optimized pretreatment conditions were determined to be 8.54% substrate loading, 1.94% NaOH and 4.09 min which resulted in the maximum cellulose release of 65.43% and reducing sugar yield of 0.554 g/g. Several physico-chemical studies of the raw and pretreated biomass were carried out using bomb calorimetry, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, Brunauer-Emmett-Teller (BET) analysis and thermal gravimetric analysis (TGA) to examine the efficacy of pretreatment. Evidences of an apparent delignification was substantiated by the increase in surface area from 7.719 to 44.188 m2 g-1and pore volume from 0.039 to 0.071 mlg-1 which was consistent with the decrease in energy density and distorted surface morphology of the pretreated biomass. Further, the FTIR revealed a reduced peak in the absorption spectral bands at 1636 cm-1 which confirmed the pretreatment mediated degradation of lignin and hemicellulose. This finding provides evidence on the prospects of utilizing abundantly available seawater resource as a reaction medium for sustainable biofuel production.

Functional insights into the role of C-terminal disordered domain of Sesbania mosaic virus RNA-dependent RNA polymerase and the coat protein in viral replication in vivo.

The C-terminal disordered domain of sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) interacts with the viral protein P10. The functional significance of this interaction in viral replication was examined by a comparative analysis of genomic and sub-genomic RNA levels (obtained by quantitative real time PCR) in the total RNA extracted from Cyamopsis plants agro-infiltrated with wild-type or mutant forms of SeMV infectious cDNA (icDNA). The sgRNA copy numbers were found to be significantly higher than those of gRNA in the wild-type icDNA transfected plants. Transfection of a mutant icDNA expressing an RdRp lacking the C-terminal disordered domain led to a drastic reduction in the copy numbers of both forms of viral RNA. This could be due to the loss of interaction between the disordered domain of RdRp and P10 and possibly other viral/host proteins that might be required for the assembly of viral replicase. The C-terminal disordered domain also harbours the motif E which is essential for the catalytic function of RdRp. Mutation of the conserved tyrosine within this motif in the full length icDNA resulted in complete inhibition of progeny RNA synthesis in the transfected plants confirming the importance of motif E in the polymerase function in vivo. The role of coat protein (CP) in viral infection was also investigated by agro-infiltration of a CP start codon mutant icDNA which suggested that CP is essential for the encapsidation of viral progeny RNAs at later stages of infection.

Interaction of the intrinsically disordered C-terminal domain of the sesbania mosaic virus RNA-dependent RNA polymerase with the viral protein P10 in vitro: modulation of the oligomeric state and polymerase activity.

The RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact with the viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociation constant for the interaction between the two proteins was determined to be 0.09 µM using surface plasmon resonance, and the disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 brought about a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly, unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantly as monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domain or its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residues of RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, located between 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance of motif E in RNA synthesis in vitro.

Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication.

Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp-p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, CΔ43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp.