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"Smith, Janet"
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An antisense RNA regulates production of DnaA and affects sporulation in Bacillus subtilis
DnaA is the replication initiator and a transcription factor in virtually all bacteria. Although the synthesis and activity of DnaA are highly regulated, the mechanisms of regulation vary between organisms. We found that production of DnaA in Bacillus subtilis is regulated by an antisense RNA that overlaps with the 5’ untranslated region upstream of the dnaA open reading frame. We initially observed this RNA in in vitro transcription experiments and found that its production was inhibited by DnaA. This RNA, now called ArrA for a ntisense R NA r epressor of dna A , is made in vivo. We identified the arrA promoter and made a mutation that greatly reduced (or eliminated) production of ArrA RNA in vitro and in vivo. In vivo, this arrA promoter mutation caused an increase in the amount of mRNA and protein from dnaA and dnaN , indicating that arrA expression normally inhibits expression of the dnaA-dnaN operon. The arrA mutation also caused a delay in sporulation that was alleviated by loss of sda , a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in some members of the Bacillus genus, indicating that arrA has evolved in at least some endospore-forming bacteria to modulate production of DnaA and enable timely and robust sporulation.
Journal Article
Identification of insertion sites for the integrative and conjugative element Tn916 in the Bacillus subtilis chromosome
Integrative and conjugative elements (ICEs) are found in many bacterial species and are mediators of horizontal gene transfer. Tn 916 is an ICE found in several Gram-positive genera, including Enterococcus , Staphylococcus , Streptococcus , and Clostridioides (previously Clostridium ). In contrast to the many ICEs that preferentially integrate into a single site, Tn 916 can integrate into many sites in the host chromosome. The consensus integration motif for Tn 916 , based on analyses of approximately 200 independent insertions, is an approximately 16 bp AT-rich sequence. Here, we describe the identification and mapping of approximately 10 5 independent Tn 916 insertions in the Bacillus subtilis chromosome. The insertions were distributed between 1,554 chromosomal sites, and approximately 99% of the insertions were in 303 sites and 65% were in only ten sites. One region, between ykuC and ykyB ( kre ), was a ‘hotspot’ for integration with ~22% of the insertions in that single location. In almost all of the top 99% of sites, Tn 916 was found with similar frequencies in both orientations relative to the chromosome and relative to the direction of transcription, with a few notable exceptions. Using the sequences of all insertion regions, we determined a consensus motif which is similar to that previously identified for C. difficile . The insertion sites are largely AT-rich, and some sites overlap with regions bound by the nucleoid-associated protein Rok, a functional analog of H-NS of Gram-negative bacteria. Rok functions as a negative regulator of at least some horizontally acquired genes. We found that the presence or absence of Rok had little or no effect on insertion site specificity of Tn 916 .
Journal Article
An intramolecular macrocyclase in plant ribosomal peptide biosynthesis
The biosynthetic dogma of ribosomally synthesized and posttranslationally modified peptides (RiPP) involves enzymatic intermolecular modification of core peptide motifs in precursor peptides. The plant-specific BURP-domain protein family, named after their four founding members, includes autocatalytic peptide cyclases involved in the biosynthesis of side-chain-macrocyclic plant RiPPs. Here we show that AhyBURP, a representative of the founding Unknown Seed Protein-type BURP-domain subfamily, catalyzes intramolecular macrocyclizations of its core peptide during the sequential biosynthesis of monocyclic lyciumin I via glycine-tryptophan crosslinking and bicyclic legumenin via glutamine-tyrosine crosslinking. X-ray crystallography of AhyBURP reveals the BURP-domain fold with two type II copper centers derived from a conserved stapled-disulfide and His motif. We show the macrocyclization of lyciumin-C(
sp
3
)-N-bond formation followed by legumenin-C(
sp
3
)-O-bond formation requires dioxygen and radical involvement based on enzyme assays in anoxic conditions and isotopic labeling. Our study expands enzymatic intramolecular modifications beyond catalytic moiety and chromophore biogenesis to RiPP biosynthesis.
BURP-domain proteins are an unexplored family of plant-specific, copper-dependent peptide cyclases. Here the authors show that a BURP-domain protein has a particular protein fold, investigate its mechanism and provide evidence for intramolecular modification in RiPP biosynthesis.
Journal Article
Commentary on `Facing the phase problem' by Wayne Hendrickson
A commentary on Wayne Hendrickson's article 'Facing the phase problem'.
Journal Article
In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA
DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq). We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.
Journal Article
Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands
Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.
Journal Article