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The capsular polysaccharide synthesis (cps) locus of Neisseria meningitidis is implicated in invasive meningococcal disease. The synthesis (synABCD) and transport (ctrABCD) operons are transcribed in opposite directions from a common intergenic region, and the expression is negatively regulated by the bacterial two-component system (TCS) misR/misS and thermosensitive RNA folding. However, these mechanisms do not fully explain the stationary phase responses, and the cis-acting elements remain to be fully characterized. Using the GFP reporter gene and site-directed mutagenesis, cis-regulatory elements in the 134 bp intergenic region, NmIR, were investigated. While confirming a known RpoD promoter, an additional potential promoter element and putative binding sites for the transcription factors fis and lexA were identified through sequence analysis. Deletion of the putative lexA binding site led to an increase in GFP fluorescence. The N. meningitidis genome carries only one lexA homolog, the helix-turn-helix regulator XRE family member (GenBank-NMB0910, HTH_XRE). Trans-complementation of the NmIR-GFP reporter with the N. meningitidis HTH_XRE expression plasmid led to increased fluorescence. Trans-complementation with either misR/misS or nusG decreased reporter gene expression. Consistent with previous reports, deletion of the RpoD promoter reduced expression by 50%, suggesting the redundancy of promoter elements in the intergenic region. Thus, the results confirm the functioning of an exogenous N. meningitidis capsule synthesis promoter in Escherichia coli and demonstrate its regulation through trans-complementation by misR/misS, HTH_XRE, and nusG. 

A novel rhodamine-6G derivative, RdN, was synthesized by condensing rhodamine glyoxal and 3-hydroxy-2-naphthoic hydrazide using a microwave irradiation-assisted reaction. Colorimetric and photophysical studies have demonstrated that the molecule produced can selectively sense Pb2+ and Cu2+ ions in a solution of CH3CN/H2O (9:1, v/v). The spirolactam ring of RdN opens upon complexation with the cations, forming a highly fluorescent complex and a visible color change in the solution. The compound RdN was further studied with the help of computational methods such as the Density Functional Theory (DFT) method and time-dependent density functional theory (TD-DFT) calculations to study the binding interactions and properties of the molecule. DFT calculations and job plot data supported the 2:1 complex formation between RdN and Pb2+/Cu2+. The limit of detection for Pb2+ was determined to be 0.112 µM and 0.130 µM for Cu2+. The probe RdN was applied to the image of Pb2+ and Cu2+ ions in living cells and is safe for biomedical applications. It is used to monitor Pb2+ in environmental water samples. 

Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of target metal ions and overall binding capacity. Microorganisms are a key component in wastewater treatment plants, and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose–sialic acid (Gal–NeuNAc) heteropolysaccharide binds lead ions effectively, thereby demonstrating the potential for its use in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis of N-acetylgalactosamine–sialic acid (GalNAc–NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics (MD) simulations with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated.