We recently demonstrated that the futalosine pathway was operating in some bacteria for the biosynthesis of menaquinone and that futalosine was converted into dehypoxanthinyl futalosine (DHFL) by an MqnB of Thermus thermophilus. In this study, we found that aminodeoxyfutalosine, which has adenine instead of hypoxanthine in futalosine, was directly converted into DHFL by an MqnB of Helicobacter pylori. Therefore, this step is potentially an attractive target for the development of specific anti-H. pylori drugs.In prokaryotes, menaquinone (MK) is used for respiration. In Escherichia coli, MK is biosynthesized from chorismate by eight enzymes (4). However, we recently identified an alternative pathway (the futalosine pathway shown in Fig. 1) (2, 3, 5), which operates in bacteria, including some pathogens, such as Helicobacter pylori, Campylobacter jejuni, Chlamydia trachomatis, and Leptospira borgpetersenii. Disruption of this pathway in Streptomyces coelicolor has been shown to lead to the inhibition of bacteriostatic growth. We also showed that the Mqn genedisrupted S. coelicolor mutants required more than 100 g/ml menaquinone for their growth. Because we do not consume a diet containing this much menaquinone, the essential amount of MK is not supplied from our diet if the futalosine pathway is blocked. Moreover, humans and commensal intestinal bacteria, including lactobacilli, lack the futalosine pathway. Taking these results together, we think that the futalosine pathway is a potential attractive target for the development of specific anti-H. pylori drugs. However, the details of each of the biosynthetic steps in the futalosine pathway remain unclear. In this study, we investigated the second step of the futalosine pathway, which is the conversion of futalosine (FL) into dehypoxanthinyl futalosine (DHFL) by MqnB (futalosine hydrolase [EC 3.2.2.26]) (3), in Acidothermus cellulolyticus, H. pylori, and S. coelicolor. We found that aminodeoxyfutalosine (AFL), which has adenine instead of hypoxanthine in FL, was an intermediate in these microorganisms.To ascertain the details of each step in the futalosine pathway, we selected the second step catalyzed by MqnB, since FL, the substrate of MqnB, was the only compound that we could readily prepare from the culture broth of the MqnB-disrupted mutant (2). We first investigated the distribution of the futalosine pathway and the organization of the Mqn genes in microorganisms whose genomes had been completely sequenced.In almost all microorganisms, the Mqn genes were scattered throughout the genome. However, in A. cellulolyticus, a thermophilic actinobacterium, the Mqn genes, and other menaquinone-related genes, such as prenylation and methylation genes, were clustered in two loci (1). One cluster contained Acel_0105 and Acel_0106 genes, which encoded orthologs of MqnC and MqnB, respectively. The other was composed of 11 genes (Acel_0255 to Acel_0266) encoding MqnA (Acel_0261) and MqnC (Acel_0263) orthologs. To determine whether Acel_0106 did indeed encode MqnB, we prepare...

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery of MHV (0.070%–2.6%) and phi6 (0.078%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (7.9 × 10 8 to 5.5 × 10 9 ) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation ( r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

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