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Simmons, L. A.

Publications and source records attributed to Simmons, L. A..

13 recordsLinked to original sources

Mapping the rRNA methylome reveals contributions of methyltransferases to ribosome function and antibiotic sensitivity

Ribosomal RNA (rRNA) folds into a complex structure used as the macromolecular core for protein synthesis. Chemical modification of rRNA contributes to ribosome structure, function, and susceptibility to antibiotics. Despite their importance, the enzymes responsible for specific rRNA modifications remain unknown in most species. In this work, we integrate genetics and biochemistry with sequencing and mass spectrometry to uncover enzymes responsible for rRNA methylation events in Bacillus subtilis. We characterize 17 enzymes responsible for 20 methylation modifications on the 16S and 23S rRNAs, 11 of which are encoded by previously uncharacterized genes. For each rRNA methyltransferase, we define the modification identity, location, and we determine the impact of loss of cognate rRNA methylation on ribosome biogenesis and antibiotic sensitivity. Our findings demonstrate that loss of nearly half of the 17 genes studied results in alterations to ribosome assembly or antibiotic sensitivity underscoring the importance of chemical modifications to ribosome function.

biochemistry↗

Bacillus subtilis RNase HII is inefficient at processing guanosine monophosphate and damaged ribonucleotides

During one round of DNA replication, nearly 2,000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphate (dNMP). Given their high rate of insertion, genomic DNA would contain rNMPs that are damaged or mismatched. Here, we tested the activity of Bacillus subtilis and Escherichia coli RNase HII on all four canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incised rAMP, rCMP, and rUMP, but was inefficient at processing rGMP in both a canonical and mismatched base pair. We tested damaged ribonucleotides and found that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.

biochemistry↗

TlyA is a 23S and 16S 2'-O-methylcytidine methyltransferase important for ribosome assembly in Bacillus subtilis

Ribosomal RNA (rRNA) is methylated in organisms ranging from bacteria to metazoans. Despite the pervasiveness of rRNA methylation in biology, the function of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2'-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside antibiotics that target the 16S rRNA. We show that{Delta} tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach and based on sequence alignments with other rRNA methyltransferases we tested the importance of potential catalytic residues and S-adenosyl-L-methionine (SAM) cofactor binding sites. We show that TlyA shares the common rRNA methyltransferase catalytic triad KDK and a SAM binding motif GxSxG which differs from Mtb TlyA. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since E. coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb and more divergent Gram-negative bacteria providing new insight into translation and antibiotic resistance mechanisms.

microbiology↗

DNA replication initiation timing is important for maintaining genome integrity

DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that {Delta}ccrZ and several ccrZ alleles under-initiate DNA replication while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA damaging agents. We show that cells under-initiating and over-initiating DNA replication were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA replication, we propose that an increase in the number of replication forks leads to replication fork stress which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled as changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material. IMPORTANCEThe regulation of DNA replication is fundamental to cell growth and cell cycle control. In eukaryotes under-initiation or over-initiation leads to genome instability. For bacteria, it is unclear how changes in replication initiation frequency impact DNA replication status and genome integrity. We show that tight regulation of DNA replication initiation is critical for maintaining genome integrity. Cells over-initiating or under-initiating DNA replication are sensitive to DNA damage. Further, cells over-initiating DNA replication experience replication fork stress at levels that phenocopy cells encountering DNA damage from the crosslinking agent mitomycin C. Our results establish the critical importance of properly regulating DNA replication initiation frequency because an imbalance in initiation results in replication fork perturbations, deficiencies in DNA repair, and genome instability.

microbiology↗

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 [A], in what appears to be an inactive state. In this conformation, a predicted -helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3-5 DNA exonuclease. We show that Leu113 aids in coordinating the 3 end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/580553v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@ac964borg.highwire.dtl.DTLVardef@bf1f81org.highwire.dtl.DTLVardef@1ad4e89org.highwire.dtl.DTLVardef@ff0641_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Two Distinct Regulatory Systems Control Pulcherrimin Biosynthesis in Bacillus subtilis

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MerR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important, and somewhat enigmatic, role for pulcherrimin in B. subtilis physiology. Author SummaryRegulation of gene expression is important for survival in ever changing environments. In the soil bacterium Bacillus subtilis, key developmental processes are controlled by overlapping networks of transcription factors, some of which are termed transition state regulators (TSRs). Despite decades of research, the scope of how TSRs influence B. subtilis physiology is still being uncovered. We found that three transcription factors, two of which are TSRs, converge to inhibit production of the iron-chelator pulcherrimin. Only when all three are missing is pulcherrimin production elevated. Finally, we demonstrate that expression of pulcherrimin biosynthesis genes occurs via direct and indirect regulation by the trio of transcription factors. Due to its iron chelating ability, pulcherrimin has been characterized as a modulator of niche development with antioxidant properties. Thus, our findings that TSRs control pulcherrimin, concurrently with other developmental phenotypes, provides new insight into how TSRs impact B. subtilis and its interaction with the environment.

microbiology↗

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome-wide

RNA:DNA hybrids such as R-loops affect genome integrity and DNA replication fork progression. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome-wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation, but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate most strongly in non-coding RNAs and 5'-UTRs of coding sequences. For {Delta}rnhB, hybrids accumulate preferentially in untranslated regions and early in coding sequences. Hybrid accumulation is particularly sensitive to gene expression in {Delta}rnhC; in cells lacking RnhC, DNA replication is disrupted leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts interact with replication to cause mutagenesis and shape genome organization.

genetics↗

Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids

Current models for Okazaki fragment maturation in eubacteria invoke RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on 5' flapped and nicked RNA-DNA hybrid substrates. We found that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that {Delta}polA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement {Delta}polA. Cells lacking FEN ({Delta}fenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model is similar to models for Okazaki fragment processing in eukaryotes, where Pol d catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Author Summary5' flap endo/exonuclease (FEN) activity provides an essential contribution to DNA replication and repair in all cellular life. In bacteria, DNA polymerase I is thought to be the central enzyme involved in Okazaki fragment processing, using its DNA polymerase and 5' nuclease activities to generate and then remove the 5 ssRNA segment of an Okazaki fragment. Many bacterial genomes encode a second, discrete FEN in addition to Pol I. We show that FEN is the primary 5' nuclease used by B. subtilis for primer removal. FEN activity exceeds that of Pol I on most substrates, including several that mimic Okazaki fragment intermediates. Additionally, we provide genetic evidence showing that FEN is involved in Okazaki fragment processing and that it is the DNA polymerase domain of Pol I rather than its 5' nuclease domain that is important in vivo. With our results, we propose a new model for Okazaki fragment processing in B. subtilis, which may be prevalent in a wider group of bacteria.

biochemistry↗

Maternal diet disrupts the placenta-brain axis in a sex-specific manner

High maternal weight is associated with a number of detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression, and communicative disorders (e.g. autism spectrum disorders)1-8. Despite widespread acknowledgement of sex-biases in the prevalence, incidence, and age of onset of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we use a mouse model to demonstrate how maternal high-fat diet, one contributor to overweight, causes endotoxin accumulation in fetal tissue, and subsequent perinatal inflammation influences sex-specific behavioral outcomes in offspring. In male high-fat diet offspring, increased macrophage toll like receptor 4 signaling results in excess phagocytosis of serotonin neurons in the developing dorsal raphe nucleus, decreasing serotonin bioavailability in the fetal and adult brain. Bulk sequencing from a large cohort of matched first trimester human fetal brain, placenta, and maternal decidua samples reveals sex-specific transcriptome-wide changes in placenta and brain tissue in response to maternal triglyceride accumulation (a proxy for dietary fat content). Further, we find that fetal brain serotonin levels decrease as maternal dietary fat intake increases in males only. These findings uncover a microglia-dependent mechanism through which maternal diet may impact offspring susceptibility for neuropsychiatric disorder development in a sex-specific manner.

neuroscience↗

A bacterial DNA repair pathway specific to a natural antibiotic

All organisms possess several DNA repair pathways to maintain the integrity of their genetic material. Although there are several DNA repair pathways that are well understood, we recently identified several genes in Bacillus subtilis that are important for surviving treatment with drugs that damage DNA. Here, we report a drug specific DNA repair pathway in B. subtilis. We identified genes coding for a previously uncharacterized helicase and exonuclease, mrfA and mrfB, respectively. Deletion of mrfA and mrfB resulted in sensitivity to the DNA damaging agent mitomycin C, but not other types of DNA damage. We found that MrfAB operate independently of canonical nucleotide excision repair, forming a novel excision repair pathway in bacteria. A phylogenetic analysis demonstrates that MrfAB homologs are present in diverse bacterial phyla, and a cross-complementation assay shows that MrfAB function is conserved in closely related species. Mitomycin C is a natural antibiotic that is produced by the soil dwelling bacterium Streptomyces lavendulae, and B. subtilis is also a soil dwelling organism. The specificity of the {Delta}mrfAB phenotype suggests that MrfAB have been adapted as a countermeasure to mitomycin producing bacteria.\n\nAbbreviated SummaryBacteria possess DNA repair pathways to maintain the integrity of their genetic material. The helicase MrfA and the exonuclease MrfB are part of a mitomycin C specific DNA repair pathway in Bacillus subtilis. Despite being present in many bacterial species, MrfAB activity in repairing MMC damaged DNA appears to be restricted to closely related species, suggesting that these proteins have likely been adapted to the specific needs of each bacterium.

microbiology↗

DdcA antagonizes a bacterial DNA damage checkpoint

Bacteria coordinate DNA replication and cell division, ensuring that a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage or impediments to DNA replication, a cell cycle checkpoint is activated to delay cell division by expressing a cell division inhibitor. The prevailing model for bacterial DNA damage checkpoints is that activation of the DNA damage response and protease mediated degradation of the cell division inhibitor is sufficient to regulate the checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to a broad spectrum of DNA damage. The ddcA deletion phenotypes are dependent on the checkpoint enforcement protein YneA. We found that expression of the checkpoint recovery proteases could not compensate for ddcA deletion. Similarly, expression if ddcA could not compensate for the absence of the checkpoint recovery proteases, indicating that DdcA function is distinct from the checkpoint recovery step. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels or stability, further supporting the conclusion that DdcA regulates YneA through a proteolysis independent mechanism. Using a functional GFP-YneA we found that DdcA inhibits YneA activity independent of YneA localization, suggesting that DdcA may regulate YneA access to its target. These results uncover a regulatory step that is important for controlling the DNA damage checkpoint in bacteria, and suggests that the typical mechanism of degrading the checkpoint enforcement protein is insufficient to control the rate of cell division in response to DNA damage.\n\nAuthor SummaryAll cells coordinate DNA replication and cell division. When cells encounter DNA damage, the process of DNA replication is slowed and the cell must also delay cell division. In bacteria, the process has long been thought to occur using two principle modes of regulation. The first, is RecA coated ssDNA transmits the signal of DNA damage through inactivation of the repressor of the DNA damage (SOS) response regulon, which results in expression of a cell division inhibitor establishing the checkpoint. The second principle step is protease mediated degradation of the cell division inhibitor relieving the checkpoint. Recent work by our lab and others has suggested that this process may be more complex than originally thought. Here, we investigated a gene of unknown function that we previously identified as important for survival when the bacterium Bacillus subtilis is exposed to DNA damage. We found that this gene negatively regulates the cell division inhibitor, but is functionally distinct from the checkpoint recovery process. We provide evidence that this gene functions as an antagonist to establishing the DNA damage checkpoint. Our study uncovers a novel layer of regulation in the bacterial DNA damage checkpoint process challenging the longstanding models established in the bacterial DNA damage response field.

microbiology↗

Cryptic adaptor protein interactions regulate DNA replication initiation

DNA replication is a fundamental biological process that is tightly regulated in all living cells. In bacteria, the master regulator DnaA controls when and where replication begins by building a step-wise complex that loads the replicative helicase onto chromosomal DNA. In many bacteria, DnaA requires the adaptor proteins DnaD and DnaB to aid DnaA during helicase loading. How DnaA, its adaptors, and the helicase form a complex at the origin is largely unknown. In this study, we addressed this long-standing question by disassembling the initiation proteins into their individual domains and testing all possible pair-wise combinations in a bacterial two-hybrid assay. Here we report a full description of the cryptic interaction sites used by the helicase loading machinery from Bacillus subtilis. In addition, we investigated how complex formation of the helicase loading machinery is regulated by the checkpoint protein SirA, which is a potent replication inhibitor in sporulating cells. We found that SirA and the DnaD adaptor bind overlapping sites on DnaA, and therefore SirA acts as a competitive inhibitor to block initiation. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this interface. Therefore, our approach has unveiled key protein interactions essential for initiation and is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces.\n\nAuthor SummaryIn order to proliferate, bacteria must first build a step-wise protein complex on their chromosomes that determines when and where DNA replication begins. This protein complex is assembled through dynamic interactions that have been difficult to study and remain largely uncharacterized. Here we show that by deconstructing the proteins into their constituent domains, the interactions used to build the initiation complex can be readily detected and mapped to single amino acid resolution. Using this approach, we demonstrate that DNA replication is controlled through conformational changes that dictate the availability of interaction surfaces. In addition, negative regulators can also block DNA replication by influencing complex formation so that cells survive inhospitable conditions. Initiation proteins from the model organism B. subtilis and the human pathogen S. aureus were both used to underscore the general applicability of the results to different bacterial systems. Furthermore, our general strategy for mapping dynamic protein interactions is suitable for many different signaling pathways that are controlled through cryptic interaction surfaces.

microbiology↗

Discovery of a two protease DNA damage checkpoint recovery mechanism

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells sense successful DNA repair and promote checkpoint recovery is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

molecular biology↗