scholarly journals Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation

2021 ◽  
Author(s):  
Thomas Prossliner ◽  
Michael Askvad Sørensen ◽  
Kristoffer Skovbo Winther
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
16S Rrna ◽  
Deletion Mutant ◽  
Growth Arrest ◽  
Physiological Role ◽  
Wild Type ◽  
Rnase R ◽  
Initial Stage ◽  
Target Sites

Abstract Ribosome hibernation is a universal translation stress response found in bacteria as well as plant plastids. The term was coined almost two decades ago and despite recent insights including detailed cryo-EM structures, the physiological role and underlying molecular mechanism of ribosome hibernation has remained unclear. Here, we demonstrate that Escherichia coli hibernation factors RMF, HPF and RaiA (HFs) concurrently confer ribosome hibernation. In response to carbon starvation and resulting growth arrest, we observe that HFs protect ribosomes at the initial stage of starvation. Consistently, a deletion mutant lacking all three factors (ΔHF) is severely inhibited in regrowth from starvation. ΔHF cells increasingly accumulate 70S ribosomes harbouring fragmented rRNA, while rRNA in wild-type 100S dimers is intact. RNA fragmentation is observed to specifically occur at HF-associated sites in 16S rRNA of assembled 70S ribosomes. Surprisingly, degradation of the 16S rRNA 3′-end is decreased in cells lacking conserved endoribonuclease YbeY and exoribonuclease RNase R suggesting that HFs directly block these ribonucleases from accessing target sites in the ribosome.

scholarly journals SdiA Aids Enterohemorrhagic Escherichia coli Carriage by Cattle Fed a Forage or Grain Diet

2013 ◽  
Vol 81 (9) ◽  
pp. 3472-3478 ◽  
Author(s):  
Haiqing Sheng ◽  
Y. N. Nguyen ◽  
Carolyn J. Hovde ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Rumen Fluid ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Dependent Manner ◽  
Bacterial Survival ◽  
Wild Type

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activatedgadgene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.

scholarly journals Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σB-Dependent General and Multiple Stress Response

2002 ◽  
Vol 184 (2) ◽  
pp. 459-467 ◽  
Author(s):  
Julia Elisabeth Bandow ◽  
Heike Brötz ◽  
Michael Hecker
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
Rna Polymerase ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Arrest ◽  
Wild Type ◽  
General Stress ◽  
Inhibition Of Growth ◽  
Increased Sensitivity

ABSTRACT Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment. l-[35S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The σB-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the β subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the σD regulon were also induced. The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the σB signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The sigB mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100. We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.

scholarly journals Formation and Metabolism of Methylmalonyl Coenzyme A in Corynebacterium glutamicum

2009 ◽  
Vol 191 (8) ◽  
pp. 2899-2901 ◽  
Author(s):  
Laure Botella ◽  
Nic D. Lindley ◽  
Lothar Eggeling
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Deletion Mutant ◽  
Coenzyme A ◽  
Growth Arrest ◽  
Dry Weight ◽  
Sequence Information ◽  
Carboxylase Activity ◽  
Genome Sequence Information ◽  
Number Of Bacteria

ABSTRACT Genome sequence information suggests that B12-dependent mutases are present in a number of bacteria, including members of the suborder Corynebacterineae like Mycobacterium tuberculosis and Corynebacterium glutamicum. We here functionally identify a methylmalonyl coenzyme A (CoA) mutase in C. glutamicum that is retained in all of the members of the suborder Corynebacterineae and is encoded by NCgl1471, NCgl1472, and NCgl1470. In addition, we observe the presence of methylmalonate in C. glutamicum, reaching concentrations of up to 757 nmol g (dry weight)−1 in propionate-grown cells, whereas in Escherichia coli no methylmalonate was detectable. As demonstrated with a mutase deletion mutant, the presence of methylmalonate in C. glutamicum is independent of mutase activity but possibly due to propionyl-CoA carboxylase activity. During growth on propionate, increased mutase activity has severe cellular consequences, resulting in growth arrest and excretion of succinate. The physiological context of the mutase present in members of the suborder Corynebacterineae is discussed.

scholarly journals Effect of MarA-Like Proteins on Antibiotic Resistance and Virulence in Yersinia pestis

2009 ◽  
Vol 78 (1) ◽  
pp. 364-371 ◽  
Author(s):  
Ida M. Lister ◽  
Joan Mecsas ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Mouse Model ◽  
Yersinia Pestis ◽  
Antibiotic Susceptibility ◽  
Deletion Mutant ◽  
Transcriptional Regulator ◽  
Drug Susceptibility ◽  
Wild Type ◽  
Lung Colonization

ABSTRACT MarA, an AraC/XylS transcriptional regulator in Escherichia coli, affects drug susceptibility and virulence. Two MarA-like proteins have been found in Yersinia pestis: MarA47 and MarA48. Deletion or overexpression of these proteins in the attenuated KIM 1001 Δpgm strain led to a change in multidrug susceptibility (including susceptibility to clinically relevant drugs). Additionally, lung colonization by the marA47 or marA48 deletion mutant was decreased about 10-fold in a pneumonic plague mouse model. Complementation of the deletions by replacing the deleted genes on the chromosome restored wild-type characteristics. These findings show that two MarA homologs in Y. pestis affect antibiotic susceptibility and virulence.

scholarly journals 16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

2005 ◽  
Vol 187 (11) ◽  
pp. 3708-3712 ◽  
Author(s):  
Lisa Nonaka ◽  
Sean R. Connell ◽  
Diane E. Taylor
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Drug Binding ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

scholarly journals Genetic Analysis of the Invariant Residue G791 in Escherichia coli 16S rRNA Implicates RelA in Ribosome Function

2009 ◽  
Vol 191 (7) ◽  
pp. 2042-2050 ◽  
Author(s):  
Hong-Man Kim ◽  
Sang-Mi Ryou ◽  
Woo-Seok Song ◽  
Se-Hoon Sim ◽  
Chang-Jun Cha ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
16S Rrna ◽  
Stringent Response ◽  
State Level ◽  
Synthetic Activity ◽  
Wild Type ◽  
Multicopy Suppressor ◽  
Invariant Residue ◽  
In Cells

ABSTRACT Previous studies identified G791 in Escherichia coli 16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified relA, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.

The direct synthesis of phospho enol pyruvate from pyruvate by Escherichia coli

1967 ◽  
Vol 168 (1012) ◽  
pp. 263-280 ◽  
Keyword(s):  
Escherichia Coli ◽  
Inorganic Phosphate ◽  
Direct Synthesis ◽  
Physiological Role ◽  
Wild Type ◽  
Pyruvate Kinase Activity ◽  
Ph Values ◽  
Direct Formation ◽  
Pyruvate Synthase

Extracts of Escherichia coli are shown to contain an enzyme system which in the presence of Mg 2+ catalyses the direct formation of phospho enol pyruvate from pyruvate and ATP with concomitant formation of AMP and inorganic phosphate. This enzyme, which has been designated 'phospho enol pyruvate synthase' ( PEP -synthase) has been purified 80-fold and is free of pyruvate kinase activity; PEP synthesis proceeded most rapidly at pH 8 to 8.5. At pH values between 6.2 and 7.5 the enzyme can catalyse the formation of ATP and pyruvate from PEP , AMP and inorganic phosphate; if arsenate is used instead of phosphate, pyruvate and ADP are produced instead. Studies of the enzymic formation of PEP with ATP specifically labelled with 32 P, and of the reverse reaction with [U -14 C] AMP , suggest that the PEP -synthase reaction involves the transfer of a pyrophosphoryl-group. The physiological role of PEP -synthase has been demonstrated with mutants of E. coli devoid of the enzyme: in contrast to wild-type organisms, such mutants neither grow on pyruvate, lactate or alanine, nor form glycogen from lactate. It is thus concluded that PEP -synthase plays an important role in the anaplerotic and the biosynthetic reactions which enable the organisms to grow on pyruvate as sole carbon source.

scholarly journals OmpA of Uropathogenic Escherichia coli Promotes Postinvasion Pathogenesis of Cystitis

2009 ◽  
Vol 77 (12) ◽  
pp. 5245-5251 ◽  
Author(s):  
Tracy F. Nicholson ◽  
Kristin M. Watts ◽  
David A. Hunstad
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protein A ◽  
Wild Type ◽  
Bladder Epithelium ◽  
Critical Determinant ◽  
Immune Effector ◽  
Effector Cells ◽  
Initial Stage ◽  
Epithelial Invasion

ABSTRACT Type 1 pilus directs bladder epithelial binding and invasion by uropathogenic Escherichia coli (UPEC) in the initial stage of cystitis, but the bacterial determinants of postinvasion events in the pathogenesis of cystitis are largely undetermined. We show here that the UPEC outer membrane protein A (OmpA), a monomeric, major, integral protein component of the bacterial outer membrane, functions as a critical determinant of intracellular virulence for UPEC, promoting persistent infection within bladder epithelium. Using a murine urinary tract infection (UTI) model, we demonstrate that whereas deletion of the UPEC ompA gene did not disrupt initial epithelial binding and invasion by UPEC, it did preclude completion of the intracellular bacterial community (IBC) pathway, accompanied by diminishing bacterial loads in the bladder. This defect in epithelial persistence of the ompA mutant was enhanced in competitive infections with wild-type UPEC. Microscopic examinations revealed that the ompA mutant formed significantly fewer IBCs, and those that were initiated were unable to progress past the early stages of maturation. These defects could be corrected by complementation of ompA. In addition, expression of ompA during wild-type UTI was sharply increased at time points correlated with IBC development and the arrival of host immune effector cells. Our findings establish OmpA as a key UPEC virulence factor that functions after epithelial invasion to facilitate IBC maturation and chronic bacterial persistence.

scholarly journals TheNeisseria gonorrhoeaeBiofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation

2011 ◽  
Vol 79 (4) ◽  
pp. 1504-1511 ◽  
Author(s):  
Christopher T. Steichen ◽  
Christine Cho ◽  
Jian Q. Shao ◽  
Michael A. Apicella
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Parent Strain ◽  
Deletion Mutant ◽  
Extracellular Dna ◽  
Wild Type ◽  
Multiple Forms ◽  
Phenotypic Analysis ◽  
Endogenous Nuclease ◽  
Biofilm Matrix

ABSTRACTNeisseria gonorrhoeaehas been shown to produce biofilms both in experimental flow chambers and in the human host. Our laboratory has shown that extracellular DNA is an essential component of the gonococcal matrix. We have also identified a gene inN. gonorrhoeae, which we designatednuc. This gene has homology with the staphylococcus-secreted thermonuclease. Our laboratory has characterizednucthrough phenotypic analysis of anucdeletion mutant. Biofilms grown with this strain are significantly thicker and of greater biomass than theN. gonorrhoeae1291 parent strain. Confocal microscopy indicates that the increased size of the mutant biofilms appears to be due to elevated amounts of extracellular DNA in the biofilm matrix. Chromosomal complementation of thenucmutation restored the wild-type biofilm phenotype. In addition, we have cloned and expressed the Nuc protein inEscherichia coli, and our data indicate that it has the ability to digest multiple forms of DNA and is a thermonuclease. The ability of Nuc to digest DNA also extends to its ability to disrupt established gonococcal biofilms through degradation of the DNA in the biofilm matrix. Our studies indicate that theN. gonorrhoeaebiofilm contains DNA and that the Nuc protein appears to play a role in biofilm formation and remodeling.

scholarly journals ISKpn40-Mediated Mobilization of the Colistin Resistance Gene mcr-3.11 in Escherichia coli

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Yu-Zhang He ◽  
Teng-Fei Long ◽  
Cai-Ping Chen ◽  
Bing He ◽  
Xing-Ping Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Wild Type ◽  
Colistin Resistance ◽  
Content Type ◽  
First Report ◽  
E Coli ◽  
Target Sites

ABSTRACT The mobile colistin resistance gene mcr-3 has globally disseminated since it was first reported in 2017 in Escherichia coli. In vitro mobilization assays in this study demonstrate the functionality of the composite transposon structure ISKpn40-mcr-3.11-dgkA-ISKpn40 in wild-type and recA− E. coli strains. These transpositions generated 4-bp duplications at the target sites. This is the first report demonstrating the mobility of the mcr-3.11 gene by transposition.

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