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The Tol-Pal system spans the cell envelope of Gram-negative bacteria, transducing the potential energy of the proton motive force (PMF) into dissociation of the TolB-Pal complex at the outer membrane (OM), freeing the lipoprotein Pal to bind the cell wall. The primary physiological role of Tol-Pal is to maintain OM integrity during cell division through accumulation of Pal molecules at division septa. How the protein complex couples the PMF at the inner membrane into work at the OM is unknown. The effectiveness of this trans-envelope energy transduction system is underscored by the fact that bacteriocins and bacteriophages co-opt Tol-Pal as part of their import/infection mechanisms. Mechanistic understanding of this process has been hindered by a lack of structural data for the inner membrane TolQ-TolR stator, of its complexes with peptidoglycan (PG) and TolA, and of how these elements combined power events at the OM. Recent studies on the homologous stators of Ton and Mot provide a starting point for understanding how Tol-Pal works. Here, we combine ab initio protein modeling with previous structural data on sub-complexes of Tol-Pal as well as mutagenesis, crosslinking, co-conservation analysis and functional data. Through this composite pooling of in silico, in vitro, and in vivo data, we propose a mechanism for force generation in which PMF-driven rotary motion within the stator drives conformational transitions within a long TolA helical hairpin domain, enabling it to reach the TolB-Pal complex at the OM.

Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring the prevalence of SARS-CoV-2 on university campuses. However, concerns about effectiveness of raw sewage as a COVID-19 early warning system still exist, and it’s not clear how useful normalization by simultaneous comparison of Pepper Mild Mottle Virus (PMMoV) is in addressing variations resulting from fecal discharge dilution. This study aims to contribute insights into these aspects by conducting an academic-year field trial at the student residences on the University of Tennessee, Knoxville campus, raw sewage. This was done to investigate the correlations between SARS-CoV-2 RNA load, both with and without PMMoV normalization, and various parameters, including active COVID-19 cases, self-isolations, and their combination among all student residents. Significant positive correlations between SARS-CoV-2 RNA load a week prior, during the monitoring week, and the subsequent week with active cases. Despite these correlations, normalization by PMMoV does not enhance these associations. These findings suggest the potential utility of SARS-CoV-2 RNA load as an early warning indicator and provide valuable insights into the application and limitations of WBE for COVID-19 surveillance specifically within the context of raw sewage on university campuses.

Inadequate sampling approaches to wastewater analyses can introduce biases, leading to inaccurate results such as false negatives and significant over- or underestimation of average daily viral concentrations, due to the sporadic nature of viral input. To address this challenge, we conducted a field trial within the University of Tennessee residence halls, employing different composite sampling modes that encompassed different time intervals (1 h, 2 h, 4 h, 6 h, and 24 h) across various time windows (morning, afternoon, evening, and late-night). Our primary objective was to identify the optimal approach for generating representative composite samples of SARS-CoV-2 from raw wastewater. Utilizing reverse transcription-quantitative polymerase chain reaction, we quantified the levels of SARS-CoV-2 RNA and pepper mild mottle virus (PMMoV) RNA in raw sewage. Our findings consistently demonstrated that PMMoV RNA, an indicator virus of human fecal contamination in water environment, exhibited higher abundance and lower variability compared to pathogenic SARS-CoV-2 RNA. Significantly, both SARS-CoV-2 and PMMoV RNA exhibited greater variability in 1 h individual composite samples throughout the entire sampling period, contrasting with the stability observed in other time-based composite samples. Through a comprehensive analysis of various composite sampling modes using the Quade Nonparametric ANCOVA test with date, PMMoV concentration and site as covariates, we concluded that employing a composite sampler during a focused 6 h morning window for pathogenic SARS-CoV-2 RNA is a pragmatic and cost-effective strategy for achieving representative composite samples within a single day in wastewater-based epidemiology applications. This method has the potential to significantly enhance the accuracy and reliability of data collected at the community level, thereby contributing to more informed public health decision-making during a pandemic.

IntroductionAlthough severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA has been frequently detected in sewage from many university dormitories to inform public health decisions during the COVID-19 pandemic, a clear understanding of SARS-CoV-2 RNA persistence in site-specific raw sewage is still lacking. To investigate the SARS-CoV-2 RNA persistence, a field trial was conducted in the University of Tennessee dormitories raw sewage, similar to municipal wastewater.MethodsThe decay of enveloped SARS-CoV-2 RNA and non-enveloped Pepper mild mottle virus (PMMoV) RNA was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw sewage at 4°C and 20°C.ResultsTemperature, followed by the concentration level of SARS-CoV-2 RNA, was the most significant factors that influenced the first-order decay rate constants (k) of SARS-CoV-2 RNA. The mean k values of SARS-CoV-2 RNA were 0.094 day−1 at 4°C and 0.261 day−1 at 20°C. At high-, medium-, and low-concentration levels of SARS-CoV-2 RNA, the mean k values were 0.367, 0.169, and 0.091 day−1, respectively. Furthermore, there was a statistical difference between the decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA at different temperature conditions.DiscussionThe first decay rates for both temperatures were statistically comparable for SARS-CoV-2 RNA, which showed sensitivity to elevated temperatures but not for PMMoV RNA. This study provides evidence for the persistence of viral RNA in site-specific raw sewage at different temperature conditions and concentration levels.