YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress

yabA encodes a negative regulator of replication initiation in Bacillus subtilis and homologues are found in many other Gram-positive species. YabA interacts with the β-processivity clamp (DnaN) of DNA polymerase and with the replication initiator and transcription factor DnaA. Because of these interactions, YabA has been proposed to modulate the activity of DnaA. We investigated the role of YabA in regulating replication initiation and the activity of DnaA as a transcription factor. We found that YabA function is mainly limited to replication initiation at oriC. Loss of YabA did not significantly alter expression of genes controlled by DnaA during exponential growth or after replication stress, indicating that YabA is not required for modulating DnaA transcriptional activity. We also found that DnaN activates replication initiation apparently through effects on YabA. Furthermore, association of GFP-YabA with the replisome correlated with the presence of DnaN at replication forks, but was independent of DnaA. Our results are consistent with models in which YabA inhibits replication initiation at oriC, and perhaps DnaA function at oriC, but not with models in which YabA generally modulates the activity of DnaA in response to replication stress.United States. Public Health Service (Grant GM41934)National Institutes of Health (U.S) ( Kirschstein NRSA postdoctoral fellowship 5 F32 G-076950

TEnvR: MATLAB-Based Toolbox for Environmental Research

With the advancements in science and technology, datasets become larger and more multivariate, which warrants the need for programming tools for fast data processing and multivariate statistical analysis. Here, the MATLAB-based Toolbox for Environmental Research TEnvR (pronounced ten-ver ) is introduced. This novel toolbox includes 44 open-source codes for automated data analysis from a multitude of techniques, such as ultraviolet-visible, fluorescence, and nuclear magnetic resonance spectroscopies, as well as from ultrahigh resolution mass spectrometry. Provided are codes for processing data (e.g., spectral corrections, formula assignment), visualization of figures, calculation of metrics, multivariate statistics, and automated work-up of large datasets. TEnvR allows for efficient data analysis with minimal by-hand manual work by the user, which allows scientists to do research more efficiently. This manuscript is supplemented with a detailed tutorial, example data, and screenshots, which collectively provide instructions on how to use all codes. TEnvR is novice-friendly and experience in programming with MATLAB is not required. TEnvR fulfills the need for a concise MATLAB-based toolbox for working with environmental data and will be updated annually to keep pace with the latest advances and needs for computational work in the environmental sciences

Microbial Labilization and Diversification of Pyrogenic Dissolved Organic Matter

With the increased occurrence of wildfires around the world, interest in the chemistry of pyrogenic organic matter (pyOM) and its fate in the environment has increased. Upon leaching from soils by rain events, significant amounts of dissolved pyOM (pyDOM) enter the aquatic environment and interact with microbial communities that are essential for cycling organic matter within the different biogeochemical cycles. To evaluate the biodegradability of pyDOM, aqueous extracts of laboratory-produced biochars were incubated with soil microbes, and the molecular changes to the composition of pyDOM were probed using ultrahigh-resolution mass spectrometry (Fourier transform–ion cyclotron resonance–mass spectrometry). Given that solar irradiation significantly affects the composition of pyDOM during terrestrial-to-marine export, the effects of photochemistry were also evaluated in the context of pyDOM biodegradability. Ultrahigh-resolution mass spectrometry revealed that many different (both aromatic and aliphatic) compounds were biodegraded. New labile compounds were produced, 22 %–40 % of which were peptide-like. These results indicated that a portion of pyDOM has been labilized into microbial biomass during the incubations. Fluorescence excitation–emission matrix spectra revealed that some fraction of these new bio-produced molecules is associated with proteinaceous fluorophores. Two-dimensional 1H–1H total correlation nuclear magnetic resonance (NMR) spectroscopy identified a peptidoglycan-like backbone within the microbially produced compounds. These results are consistent with previous observations of peptidoglycans within the soil and ocean nitrogen cycles where remnants of biodegraded pyDOM are expected to be observed. Interestingly, the exact nature of the bio-produced organic matter was found to vary drastically among samples indicating that the microbial consortium used may produce different exudates based on the composition of the initial pyDOM. Another potential explanation for the vast diversity of molecules is that microbes only consume low molecular-weight compounds, but they also produce reactive oxygen species (ROS), which initiate oxidative and recombination reactions that degrade high molecular-weight compounds and produce new molecules. Some of the bio-produced molecules (212–308 molecular formulas) were identified in estuarine and marine (surface and abyssal oceanic), and 81–192 of these formulas were of molecular composition attributed to carboxyl-rich alicyclic molecules (CRAM). These results indicate that some of the pyDOM biodegradation products have an oceanic fate and can be sequestered into the deep ocean. The observed microbially mediated diversification of pyDOM suggests that pyDOM contributes to the observed large complexity of natural organic matter observed in riverine and oceanic systems. More broadly, our research shows that pyDOM can be substrate for microbial growth and be incorporated into environmental food webs within the global carbon and nitrogen cycles

Biolability of Fresh and Photodegraded Pyrogenic Dissolved Organic Matter From Laboratory-Prepared Chars

Pyrogenic dissolved organic matter (pyDOM) is known to be an important biogeochemical constituent of aquatic ecosystems and the carbon cycle. While recent studies have examined how pyDOM production, composition, and photolability varies with parent pyrogenic solid material type, we lack an understanding of potential microbial mineralization and transformation of pyDOM in the biogeosphere. Thus, leachates of oak, charred at 400 °C and 650 °C, as well as their photodegraded counterparts were incubated with a soil‐extracted microbial consortium over 96 days. During the incubation, significantly more carbon was biomineralized from the lower versus higher temperature char leachate (45% vs. 37% lost, respectively). Further, the photodegraded leachates were biomineralized to significantly greater extents than their fresh non‐photodegraded counterparts. Kinetic modeling identified the mineralizable pyDOC fractions to have half‐lives of 9–13 days. Proton nuclear magnetic resonance spectroscopy indicated that the majority of this loss could be attributed to low molecular weight constituents of pyDOM (i.e., simple alcohols and acids). Further, the quantification of benzenepolycarboxylic acid (BPCA) molecular markers indicated that condensed aromatic compounds in pyDOM were biomineralized to much lesser extents (4.4% and 10.1% decrease in yields of ΣBPCA‐C over 66 days from 400 °C and 650 °C oak pyDOM, respectively), but most of this loss could be attributed to the biomineralization of smaller condensed clusters (four aromatic rings or less). These results highlight the contrasting bioavailability of different portions of pyDOM, and the need to examine both to evaluate its role in soil or aquatic heterotrophy and its environmental fate in the hydrogeosphere

Biolability of Fresh and Photodegraded Pyrogenic Dissolved Organic Matter From Laboratory-Prepared Chars

Pyrogenic dissolved organic matter (pyDOM) is known to be an important biogeochemical constituent of aquatic ecosystems and the carbon cycle. While recent studies have examined how pyDOM production, composition, and photolability varies with parent pyrogenic solid material type, we lack an understanding of potential microbial mineralization and transformation of pyDOM in the biogeosphere. Thus, leachates of oak, charred at 400 °C and 650 °C, as well as their photodegraded counterparts were incubated with a soil‐extracted microbial consortium over 96 days. During the incubation, significantly more carbon was biomineralized from the lower versus higher temperature char leachate (45% vs. 37% lost, respectively). Further, the photodegraded leachates were biomineralized to significantly greater extents than their fresh non‐photodegraded counterparts. Kinetic modeling identified the mineralizable pyDOC fractions to have half‐lives of 9–13 days. Proton nuclear magnetic resonance spectroscopy indicated that the majority of this loss could be attributed to low molecular weight constituents of pyDOM (i.e., simple alcohols and acids). Further, the quantification of benzenepolycarboxylic acid (BPCA) molecular markers indicated that condensed aromatic compounds in pyDOM were biomineralized to much lesser extents (4.4% and 10.1% decrease in yields of ΣBPCA‐C over 66 days from 400 °C and 650 °C oak pyDOM, respectively), but most of this loss could be attributed to the biomineralization of smaller condensed clusters (four aromatic rings or less). These results highlight the contrasting bioavailability of different portions of pyDOM, and the need to examine both to evaluate its role in soil or aquatic heterotrophy and its environmental fate in the hydrogeosphere

Changes in Cell Morphology Are Coordinated with Cell Growth through the TORC1 Pathway

SummaryBackgroundGrowth rate is determined not only by extracellular cues such as nutrient availability but also by intracellular processes. Changes in cell morphology in budding yeast, mediated by polarization of the actin cytoskeleton, have been shown to reduce cell growth.ResultsHere we demonstrate that polarization of the actin cytoskeleton inhibits the highly conserved Target of Rapamycin Complex 1 (TORC1) pathway. This downregulation is suppressed by inactivation of the TORC1 pathway regulatory Iml1 complex, which also regulates TORC1 during nitrogen starvation. We further demonstrate that attenuation of growth is important for cell recovery after conditions of prolonged polarized growth.ConclusionsOur results indicate that extended periods of polarized growth inhibit protein synthesis, mass accumulation, and the increase in cell size at least in part through inhibiting the TORC1 pathway. We speculate that this mechanism serves to coordinate the ability of cells to increase in size with their biosynthetic capacity

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.United States. Public Health Service (Grant GM050895