The effect of magnesium on transfection and transformation in Bacillus subtilis

1981 ◽  
Vol 27 (10) ◽  
pp. 991-997
Author(s):  
Paloma López ◽  
M. Teresa Pérez Ureña ◽  
Manolo Espinosa
Keyword(s):  
Bacillus Subtilis ◽  
Competence Development ◽  
Endonucleolytic Cleavage ◽  
Competent Cells ◽  
Phage Spp1 ◽  
Kinetics Of

The influence of Mg2+ on phage SPP1 DNA-mediated transfection as compared with chromosomal transformation was studied.A differential influence of this cation in both processes was detectable by analyzing the competence development and the kinetics of appearance of transformants and transfectants. Binding and uptake of DNA and release of acid-soluble products by competent cells in high- and low-Mg2+ media was measured for 3H-labelled SPP1 and 3H-labelled Bacillus subtilis DNAs. Phage SPP1 DNA was shown to be subjected to endonucleolytic cleavage after exposure to competent cells.

scholarly journals Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

2004 ◽  
Vol 186 (16) ◽  
pp. 5392-5399 ◽  
Author(s):  
Frank J. Grundy ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Assay System ◽  
Nascent Transcript ◽  
Vitro Assay ◽  
T Box ◽  
Transcription Antitermination ◽  
Transcriptional Pausing ◽  
Transcription Complexes ◽  
Kinetics Of

ABSTRACT Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNAGly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

scholarly journals Streptococcus pneumoniae Elaborates Persistent and Prolonged Competent State during Pneumonia-Derived Sepsis

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Jingjun Lin ◽  
Pyunghun Park ◽  
Hua Li ◽  
Myung Whan Oh ◽  
Iwona T. Dobrucki ◽  
...  
Keyword(s):  
Firefly Luciferase ◽  
Competence Development ◽  
Cell Contact ◽  
Bacterial Burden ◽  
Natural Competence ◽  
Host Infection ◽  
Kinetics Of ◽  
Competence Induction

ABSTRACT The competence regulon of pneumococcus regulates both genetic transformation and virulence. However, competence induction during host infection has not been examined. By using the serotype 2 strain D39, we transcriptionally fused the firefly luciferase (luc) to competence-specific genes and spatiotemporally monitored the competence development in a mouse model of pneumonia-derived sepsis. In contrast to the universally reported short transient burst of competent state in vitro, the naturally developed competent state was prolonged and persistent during pneumonia-derived sepsis. The competent state began at approximately 20 h postinfection (hpi) and facilitated systemic invasion and sepsis development and progressed in different manners. In some mice, acute pneumonia quickly led to sepsis and death, accompanied by increasing intensity of the competence signal. In the remaining mice, pneumonia lasted longer, with the competence signal decreasing at first but increasing as the infection became septic. The concentration of pneumococcal inoculum (1 × 106 to 1 × 108 CFU/mouse) and postinfection lung bacterial burden did not appreciably impact the kinetics of competence induction. Exogenously provided competence stimulating peptide 1 (CSP1) failed to modulate the onset kinetics of competence development in vivo. The competence shutoff regulator DprA was highly expressed during pneumonia-derived sepsis but failed to turn off the competent state in mice. Competent D39 bacteria propagated the competence signal through cell-to-cell contact rather than the classically described quorum-sensing mechanism. Finally, clinical pneumococcal strains of different serotypes were also able to develop natural competence during pneumonia-derived sepsis.

scholarly journals Dynamic Membrane Localization of RNase Y in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hamouche ◽  
Cyrille Billaudeau ◽  
Anna Rocca ◽  
Arnaud Chastanet ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Active Form ◽  
Model Organism ◽  
Living Cells ◽  
Cell Periphery ◽  
Rnase E ◽  
Content Type ◽  
Endonucleolytic Cleavage ◽  
Rnase Y

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

Some features of competent streptococci

1972 ◽  
Vol 18 (4) ◽  
pp. 487-491 ◽  
Author(s):  
R. Pakula ◽  
L. R. Spencer
Keyword(s):  
Cell Proliferation ◽  
Dna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Competence Development ◽  
Dna Uptake ◽  
Related Strain ◽  
Competent Cells ◽  
Insoluble Material ◽  
Competence Factor

The Wicky Streptococcus was rendered competent for deoxyribonucleic acid (DNA) uptake with the competence factor (CF) derived from a related strain. Initiation of competence resulted in an almost immediate decline of the rate of cell proliferation and of incorporation of 14C-uracil into acid-insoluble material. DNA synthesis was affected in the later stage of competence development. The competent cells were penicillin resistant.

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