sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of , encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent and in the range of 98 to 111 μM and 69 to 73 μmol · min · mg, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol. Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of spp. Predicted transmembrane domain and signal peptide sequences of were found to be functional in and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

High velocity jet impingement mixing is a unique operation of the reaction injection molding process. The quality of mixing depends on processing parameters for a given system, and can have significant effect on the properties of the product. We have compared three methods used for characterization of mixing quality reported in the literature for a cross-linking polyurethane system p r e cessed on an industrial scale high pressure RIM machine, with an Ltype mixhead. The methods are based on, (i) visual appearance of a molded sample, (ii) adiabatic temperature rise during reaction and, (iii) flexural modulus of elasticity of a molded sample. The visual appearance test was made quantitative by using a color matching optical system. The adiabatic temperature rise increases rapidly with nozzle Reynolds number (Re,,,,) for polyol. u p to a critical value (Re,,,, = 380) beyond which there is a slow increase. The latter is attributed to the geometry of the Ltype mixhead. A simple analysis is presented to explain the limiting extent of mixing at high Renzl. The flexural modulus and the visual appearance of molded samples are found to improve until Renzl= 500. implying a greater sensitivity to mixing as compared to the adiabatic temperature rise.

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis . The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at −87 to −84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended −10 promoter, it lacks a reasonable −35 element. Genetic analysis and structural simulations predicted that the absence of the −35 element might be compensated by interactions between σ A and αCTD, and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD likely participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new-type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors including the ubiquitous response regulators of two-component regulatory systems particularly in Gram-positive bacteria.

The transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidalSalmonella entericaserovar Typhimurium. During infection, phosphorylated SsrB activates genes onSalmonellaPathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain, confers pH sensitivity to SsrB allosterically. Acid-dependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB rendersSalmonellaavirulent and represents a potential means of controlling virulence.