Characterization and DNA Stable-Isotope Probing of Methanotrophic Bioaerosols.

The growth and activity of bacteria have been extensively studied in nearly every environment on Earth, but there have been limited studies focusing on the air. Suspended bacteria (outside of water droplets) may stay in the atmosphere for time frames that could allow for growth on volatile compounds, including the potent greenhouse gas methane. We investigated the ability of aerosolized methanotrophic bacteria to grow on methane in the airborne state in rotating gas-phase bioreactors. The physical half-life of the aerial bacterium-sized particles was 3 days. To assess the potential for airborne growth, gas-phase bioreactors containing the aerosolized cultures were amended with 1,500 ppmv 13CH4 or 12CH4. Three of seven experiments demonstrated 13C incorporation into DNA, indicating growth in air. Bacteria associated with the genera Methylocystis and Methylocaldum were detected in 13C-DNA fractions, thus indicating that they were synthesizing new DNA, suggesting growth in air. We conclude that methanotrophs outside of water droplets in the air can potentially grow under certain conditions. Based on our data, humidity seems to be a major limitation to bacterial growth in air. Furthermore, low biomass levels can pose problems for detecting 13C-DNA synthesis in our experimental system. IMPORTANCE Currently, the cellular activities of bacteria in the airborne state outside of water droplets have not been heavily studied. Evidence suggests that these airborne bacteria produce ribosomes and metabolize gaseous compounds. Despite having a potentially important impact on atmospheric chemistry, the ability of bacteria in the air to metabolize substrates such as methane is not well understood. Demonstrating that bacteria in the air can metabolize and grow on substrates will expand knowledge about the potential activities and functions of the atmospheric microbiome. This study provides evidence for DNA synthesis and, ultimately, growth of airborne methanotrophs.

Year-long wastewater monitoring for SARS-CoV-2 signals in combined and separate sanitary sewers.

COVID-19 wastewater-based epidemiology has been performed in catchments of various sizes and sewer types with many short-term studies available and multi-seasonal studies emerging. The objective of this study was to compare weekly observations of SARS-CoV-2 genes in municipal wastewater across multiple seasons for different systems as a factor of sewer type (combined, separate sanitary) and system size. Sampling occurred following the first wave of SARS-CoV-2 cases in the study region (June 2020) and continued through the third wave (May 2021), the period during which clinical testing was widely available and different variants dominated clinical cases. The strongest correlations were observed between wastewater N1 concentrations and the cumulative clinical cases reported in the 2 weeks prior to wastewater sampling, followed by the week prior, new cases, and the week after wastewater sampling. Sewer type and size did not necessarily explain the strength of the correlations, indicating that other non-sewer factors may be impacting the observations. In-system sampling results for the largest system sampled are presented for 1 month. Removing wet weather days from the data sets improved even the flow-normalized correlations for the systems, potentially indicating that interpreting results during wet weather events may be more complicated than simply accounting for dilution. PRACTITIONER POINTS: SARS-CoV-2 in wastewater correlated best with total clinical cases reported in 2 weeks before wastewater sampling at the utility level. Study performed when clinical testing was widespread during the year after the first COVID-19 wave in the region. Sewer type and size did not necessarily explain correlation strength between clinical cases and wastewater-based epidemiology results. Removing wet weather days improved correlations for 3/4 utilities studied, including both separate sanitary and combined sewers.

Drug Repositioning: A Unique Approach to Refurbish Drug Discovery.

For a decade, it has been observed that there is a remarkable decrease in the quantum of novel clinically approved drugs, in spite of modernization in the research and development process. We have highlighted repositioning of drugs as a methodology that has found new therapeutic implications for clinically approved drugs but with different indications. This can be considered as an upbringing strategy to deliver timely and cost-effective solutions, which still need exploration for getting over the shortage of novel drugs reaching the market. This review focuses on an activity-based drug repositioning approach, which is used to explore new uses of known drugs that are already approved for specific indications and are now being used for other indications on the basis that a single drug interacts with multiple targets. It also includes current research trends related to drug repositioning, which depends on strong knowledge of medicinal chemistry and involves elucidation of mechanisms of action and validation of novel targets. The review highlights the importance of computational tools and databases of various forms for drug repositioning purposes, which have enhanced the ability to pose reasonable and testable hypotheses. The critical nature of this aspect is obvious in cases where data gathered from in vitro, or animal models do not confirm in subsequent clinical trials. Hence, considering the positive outcomes of drug repositioning, it can be surmised that this approach can serve as a promising one that can develop into a robust drug discovery strategy.

The dynamic landscape of transcription initiation in yeast mitochondria.

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis.

Telomerase adds telomeric repeats to chromosome ends by processive copying of a template within the telomerase RNA bound to telomerase reverse transcriptase. Telomerase RNAs have single-stranded regions that separate the template from a 5' stem and 3' pseudoknot, and mammals gained additional stem P2a.1 separating the template from the pseudoknot. Using human telomerase, we show that the length of template 3'-flanking single-stranded RNA is a determinant of repeat addition processivity whereas template 5'-flanking single-stranded RNA and P2a.1 are critical for activity but not processivity. In comparison, requirements for the template sequence itself are confounding: different substitutions of the same position have strikingly different consequences, from improved processivity and activity to complete inactivation. We discovered that some altered-template sequences stabilize an alternative RNA conformation that precludes the pseudoknot by base-pairing of one pseudoknot strand to the template 3' end. Using mutations to reduce over-stability of the alternative conformation, we restore high activity and processivity to otherwise inactive altered-template telomerase ribonucleoproteins. In cells, over-stabilization or destabilization of the alternative state severely inhibited biogenesis of active telomerase. Our findings delineate roles for human telomerase RNA template-flanking regions, establish a biologically relevant pseudoknot-alternative RNA conformation, and expand the repertoire of human telomerase repeat synthesis.

The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

Interactions of the yeast mitochondrial RNA polymerase with the +1 and +2 promoter bases dictate transcription initiation efficiency.

Mitochondrial promoters of Saccharomyces cerevisiae share a conserved -8 to +1 sequence with +1+2 AA, AG or AT initiation sequence, which dictates the efficiency of transcription initiation by the mitochondrial RNA polymerase Rpo41 and its initiation factor Mtf1. We used 2-aminopurine fluorescence to monitor promoter melting and measured the kcat/Km of 2-mer synthesis to quantify initiation efficiency with systematic changes of the +1+2 base pairs to matched and mismatched pairs. We show that AA promoters are most efficient, followed by AG and then AT promoters, and the differences in their efficiencies stem specifically from differential melting of +1+2 region without affecting melting of the upstream -4 to -1 region. Inefficient +1+2 melting increases the initial NTPs Kms of the AG and AT promoters relative to AA or singly mispaired promoters. The 16-100-fold higher catalytic efficiency of AA initiation sequence relative to AG and AT, respectively, is partly due to Rpo41-Mtf1 interactions with the +1+2 non-template adenines that generate a stable pre-transcribing complex. We propose a model where the +2 base pair regulates the efficiency of initial transcription by controlling multiple steps including downstream promoter opening, +1+2 NTPs binding, and the rate of 2-mer synthesis.

Fluorescent methods to study transcription initiation and transition into elongation.

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.

Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase.

Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP "sensor" to regulate gene expression. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Transcription factor-dependent DNA bending governs promoter recognition by the mitochondrial RNA polymerase.

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

The N-terminal domain of the yeast mitochondrial RNA polymerase regulates multiple steps of transcription.

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1-270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1-380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.