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Variovorax paradoxus strain TBEA6 is one of the few organisms known to utilize 3,3’-thiodipropionate (TDP) as the only source of carbon and energy. It cleaves TDP to 3-mercaptopropionate (3MP), which is a direct precursor for polythioester synthesis. To establish this process in V. paradoxus TBEA6, it is crucial to unravel its TDP metabolism. Therefore, a proteomic approach with subsequent deletion of interesting genes in the bacterium was chosen. Cells were cultivated with D-gluconate, TDP or 3-sulfinopropionate as the only carbon sources. Proteins with high abundances in gels of cells cultivated with either of the organic sulfur compounds were analyzed further. Thereby, we did not only confirm parts of the already postulated TDP metabolism, but also eight new protein candidates for TDP degradation were detected. Deletions of the corresponding genes (two enoyl-CoA hydratases (Ech-20 and Ech-30), an FK506-binding protein, a putative acetolactate synthase, a carnitinyl-CoA dehydratase, and a putative crotonase family protein) were obtained. Only the deletions of both Ech-20 and Ech-30 led to a TDP negative phenotype. The deletion mutant of VPARA_05510, which encodes the putative crotonase family protein showed reduced growth with TDP. The three genes are located in one cluster with genes proven to be involved in TDP metabolism. Thermal shift assays showed an increased stability of Ech-20 with TDP-CoA but not with TDP. These results indicate that Ech-20 uses TDP-CoA as a substrate instead of TDP. Hence, we postulate a new putative pathway for TDP metabolism. Ech-30 interacts with neither TDP-CoA nor TDP but might interact with other CoA-activated intermediates of the proposed pathway. Further enzyme characterization is necessary to unravel the complete pathway from TDP to 3MP.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Recently, CxaP, a sugar acid substrate binding protein (SBP) from Advenella mimigardefordensis strain DPN7 T was identified as part of a novel sugar uptake strategy. In the current study, the protein was successfully crystallized. Although several SBP structures of TRAP transporters have already been solved, this is the first structure of an SBP accepting multiple sugar acid ligands. Protein crystals were obtained with bound D-xylonic acid, D-fuconic acid Dgalactonic and D-gluconic acid, respectively. The protein shows the typical structure of an SBP of a TRAP transporter consisting of two domains linked by a hinge and spanned by a long α-helix. By analysis of the structure, the substrate binding site of the protein was identified. The carboxylic group of the sugar acids interacts with Arg175, whereas the coordination of the hydroxylic groups at positions C2 and C3 are most probably realized by Arg154 and Asn151. Furthermore, it was observed that 2-keto-3-deoxy-D-gluconic acid is bound in protein crystals which were crystallized without the addition of any ligand, indicating that this molecule is pre-bound to the protein and is displaced by the other ligands if they are available.

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