Analysis of 5′-NAD capping of mRNAs in dormant spores of Bacillus subtilis

2020 ◽  
Vol 367 (17) ◽  
Author(s):  
D Levi Craft ◽  
George Korza ◽  
Yaqing Zhang ◽  
Jens Frindert ◽  
Andres Jäschke ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recent Work ◽  
Direct Synthesis ◽  
Subtilis Cell ◽  
Gram Positive Bacteria ◽  
Growing Cell ◽  
Spore Proteins ◽  
Spore Outgrowth ◽  
Almost All

ABSTRACT Spores of Gram-positive bacteria contain 10s–1000s of different mRNAs. However, Bacillus subtilis spores contain only ∼ 50 mRNAs at > 1 molecule/spore, almost all transcribed only in the developing spore and encoding spore proteins. However, some spore mRNAs could be stabilized to ensure they are intact in dormant spores, perhaps to direct synthesis of proteins essential for spores’ conversion to a growing cell in germinated spore outgrowth. Recent work shows that some growing B. subtilis cell mRNAs contain a 5′-NAD cap. Since this cap may stabilize mRNA in vivo, its presence on spore mRNAs would suggest that maintaining some intact spore mRNAs is important, perhaps because they have a translational role in outgrowth. However, significant levels of only a few abundant spore mRNAs had a 5′-NAD cap, and these were not the most stable spore mRNAs and had likely been fragmented. Even higher levels of 5′-NAD-capping were found on a few low abundance spore mRNAs, but these mRNAs were present in only small percentages of spores, and had again been fragmented. The new data are thus consistent with spore mRNAs serving only as a reservoir of ribonucleotides in outgrowth.

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

scholarly journals A Region of Bacillus subtilis CodY Protein Required for Interaction with DNA

2005 ◽  
Vol 187 (12) ◽  
pp. 4127-4139 ◽  
Author(s):  
Pascale Joseph ◽  
Manoja Ratnayake-Lecamwasam ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Dna Recognition ◽  
Transcriptional Regulators ◽  
Site Directed Mutagenesis ◽  
Regulation Of Transcription ◽  
Gram Positive Bacteria ◽  
Target Promoters

ABSTRACT Bacillus subtilis CodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G+C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.

scholarly journals AprlMutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2014 ◽  
Vol 197 (5) ◽  
pp. 932-942 ◽  
Author(s):  
Juliana Durack ◽  
Thomas P. Burke ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Atp Hydrolysis ◽  
Colony Morphology ◽  
Wild Type ◽  
Swarming Motility ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Culture Cells ◽  
Almost All

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogenListeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence inL. monocytogenes; however, the mechanism of export via this pathway is poorly understood.L. monocytogenessecA2mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility toL. monocytogenessecA2mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by thelmo2769operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation insecYwas identified that conferred smooth colony morphology tosecA2mutants, restored wild-type plaque formation, and increased virulence in mice. ThissecYmutation resembled aprlsuppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although theprlmutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1mutant was still less virulentin vivothan the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

scholarly journals In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon

2006 ◽  
Vol 72 (1) ◽  
pp. 327-333 ◽  
Author(s):  
Yoann Le Breton ◽  
Nrusingh Prasad Mohapatra ◽  
W. G. Haldenwang
Keyword(s):  
Bacillus Subtilis ◽  
Southern Hybridization ◽  
Random Mutagenesis ◽  
Temperature Sensitive ◽  
Transposase Gene ◽  
Gram Positive Bacteria ◽  
Insertion Sites ◽  
Random Transposition

ABSTRACT This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either σA- or σB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kanr cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Ermr gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10−2) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo− clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.

scholarly journals Effects of Major Spore-Specific DNA Binding Proteins on Bacillus subtilis Sporulation and Spore Properties

2000 ◽  
Vol 182 (24) ◽  
pp. 6906-6912 ◽  
Author(s):  
Barbara Setlow ◽  
Kelly A. McGinnis ◽  
Katerina Ragkousi ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Mother Cell ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Dna Binding Proteins ◽  
Specific Gene ◽  
Wild Type ◽  
Spore Proteins ◽  
Spore Outgrowth

ABSTRACT Sporulation of a Bacillus subtilis strain (termed α− β−) lacking the majority of the α/β-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in α− β− strains. The sporulation defects in α− β− strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, α/β-type SASP but not by a mutant SASP that binds DNA poorly. Spores from α− β− strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in α− β− spores were abolished by the synthesis of normal levels of α/β-type SASP. These results indicate that α/β-type SASP may well have global effects on gene expression during sporulation and spore outgrowth.

scholarly journals Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo

2021 ◽  
Author(s):  
John K. Crane ◽  
Cassandra L. Alvarado ◽  
Mark D. Sutton
Keyword(s):  
Antibiotic Resistance ◽  
Rabbit Model ◽  
Sos Response ◽  
Antimicrobial Drugs ◽  
Mutator Phenotype ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Almost All

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. In some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or “hypermutation.” As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of the SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic E. coli, strain E22. SOS -inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing emergence of resistance to new antimicrobial drugs.

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