scholarly journals Serine-Threonine Kinases Encoded by SplithipAHomologs Inhibit Tryptophanyl-tRNA Synthetase

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Stine Vang Nielsen ◽  
Kathryn Jane Turnbull ◽  
Mohammad Roghanian ◽  
Rene Bærentsen ◽  
Maja Semanjski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Gene Family ◽  
Sequence Similarity ◽  
Trna Synthetase ◽  
Bacterial Toxin ◽  
Content Type ◽  
Multidrug Tolerance ◽  
K 12

ABSTRACTType II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact.hipBAofEscherichia colistrain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction ofhipAinhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed thattrpSin high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT ofE. coliO127 is a kinase that phosphorylates tryptophanyl-tRNA synthetasein vitroat a conserved serine residue. Consistently, induction ofhipTinhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream fromhipT, calledhipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipSin vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additionalhipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus,hipBSTconstitutes a novel family of tricomponent TA modules wherehipAhas been split into two genes,hipSandhipT, that function as a novel type of TA pair.IMPORTANCEBacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified washipAofEscherichia colistrain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. ThehipAgene encodes the toxin of thehipBATA module, whilehipBencodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family,hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

scholarly journals Escherichia coli Peptidase A, B, or N Can Process Translation Inhibitor Microcin C

2008 ◽  
Vol 190 (7) ◽  
pp. 2607-2610 ◽  
Author(s):  
Teymur Kazakov ◽  
Gaston H. Vondenhoff ◽  
Kirill A. Datsenko ◽  
Maria Novikova ◽  
Anastasia Metlitskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Trna Synthetase ◽  
Methionine Residue ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Translation Inhibitor ◽  
Rate Limiting ◽  
Broad Specificity

ABSTRACT The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.

scholarly journals Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Anne-Claire Mahérault ◽  
Harry Kemble ◽  
Mélanie Magnan ◽  
Benoit Gachet ◽  
David Roche ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Negative Impact ◽  
Fitness Cost ◽  
M Gene ◽  
Content Type ◽  
E Coli ◽  
Conjugative Plasmids ◽  
K 12

ABSTRACT Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length

2015 ◽  
Vol 197 (19) ◽  
pp. 3133-3141 ◽  
Author(s):  
Olga Bantysh ◽  
Marina Serebryakova ◽  
Inna Zukher ◽  
Alexey Kulikovsky ◽  
Darya Tsibulskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Functional Characterization ◽  
Trna Synthetase ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Target Cells ◽  
Content Type ◽  
Peptide Length

ABSTRACTEscherichia colimicrocin C (McC) consists of a ribosomally synthesized heptapeptide attached to a modified adenosine. McC is actively taken up by sensitiveEscherichia colistrains through the YejABEF transporter. Inside the cell, McC is processed by aminopeptidases, which release nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC is synthesized by the MccB enzyme, which terminally adenylates the MccA heptapeptide precursor MRTGNAN. Earlier, McC analogs with shortened peptide lengths were prepared by total chemical synthesis and were shown to have strongly reduced biological activity due to decreased uptake. Variants with longer peptides were difficult to synthesize, however. Here, we used recombinant MccB to prepare and characterize McC-like molecules with altered peptide moieties, including extended peptide lengths. We find that N-terminal extensions ofE. coliMccA heptapeptide do not affect MccB-catalyzed adenylation and that some extended-peptide-length McC analogs show improved biological activity. When the peptide length reaches 20 amino acids, both YejABEF and SbmA can perform facilitated transport of toxic peptide adenylates inside the cell. A C-terminal fusion of the carrier maltose-binding protein (MBP) with the MccA peptide is also recognized by MccBin vivoandin vitro, allowing highly specific adenylation and/or radioactive labeling of cellular proteins.IMPORTANCEEnzymatic adenylation of chemically synthesized peptides allowed us to generate biologically active derivatives of the peptide-nucleotide antibiotic microcin C with improved bioactivity and altered entry routes into target cells, opening the way for development of various McC-based antibacterial compounds not found in nature.

scholarly journals In Vitro Susceptibility Testing of GSK656 against Mycobacterium Species

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Wenzhu Dong ◽  
Shanshan Li ◽  
Shu’an Wen ◽  
Wei Jing ◽  
Jin Shi ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Mycobacterium Abscessus ◽  
Drug Binding ◽  
Mycobacterial Species ◽  
In Vitro Susceptibility ◽  
Content Type ◽  
Sequence Variations

ABSTRACT In this study, we aimed to assess the in vitro susceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitro susceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosis isolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitro susceptibility. For M. tuberculosis, 102 of 105 isolates had MICs of ≤0.5 mg/liter, demonstrating a MIC50 of 0.063 mg/liter and a MIC90 of 0.25 mg/liter. An epidemiological cutoff value of 0.5 mg/liter was proposed for identification of GSK656-resistant M. tuberculosis strains. For NTM, the MIC50 and MIC90 values were >8.0 mg/liter for both Mycobacterium intracellulare and Mycobacterium avium. In contrast, all Mycobacterium abscessus isolates had MICs of ≤0.25 mg/liter, yielding a MIC90 of 0.063 mg/liter. LeuRS from M. abscessus showed greater sequence similarity to M. tuberculosis LeuRS than to LeuRSs from M. avium and M. intracellulare. Sequence alignment revealed 28 residues differing between LeuRSs from M. avium and M. intracellulare and LeuRSs from M. tuberculosis and M. abscessus; among them, 15 residues were in the drug binding domain. Structure modeling revealed that several different residues were close to the tRNA-LeuRS interface or the entrance of the drug-tRNA binding pocket. In conclusion, our data demonstrate significant species diversity in in vitro susceptibility to GSK656 among various mycobacterial species. GSK656 has potent efficacy against M. tuberculosis and M. abscessus, whereas inherent resistance was noted for M. intracellulare and M. avium.

scholarly journals Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Anthony W. Kingston ◽  
Christine Ponkratz ◽  
Elisabeth A. Raleigh
Keyword(s):  
Escherichia Coli ◽  
Chromosomal Dna ◽  
Dna Endonuclease ◽  
Recombination Mechanism ◽  
Content Type ◽  
New Genes ◽  
The Family ◽  
K 12 ◽  
Acquisition Processes

ABSTRACT Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents recombination-promoting nuclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert. In vitro, RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes. IMPORTANCE Bacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo. For RpnA, we demonstrate in vitro action as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching.

scholarly journals Dual Function of Aar, a Member of the New AraC Negative Regulator Family, in Escherichia coli Gene Expression

2020 ◽  
Vol 88 (6) ◽  
Author(s):  
Abigail S. Mickey ◽  
James P. Nataro
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Negative Regulator ◽  
Dual Function ◽  
Content Type ◽  
Virulence Gene Expression ◽  
E Coli ◽  
K 12

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype associated with diarrhea and growth faltering. EAEC virulence gene expression is controlled by the autoactivated AraC family transcriptional regulator, AggR. AggR activates transcription of a large number of virulence genes, including Aar, which in turn acts as a negative regulator of AggR itself. Aar has also been shown to affect expression of E. coli housekeeping genes, including H-NS, a global regulator that acts at multiple promoters and silences AT-rich genes (such as those in the AggR regulon). Although Aar has been shown to bind both AggR and H-NS in vitro, functional significance of these interactions has not been shown in vivo. In order to dissect this regulatory network, we removed the complex interdependence of aggR and aar by placing the genes under the control of titratable promoters. We measured phenotypic and genotypic changes on downstream genes in EAEC strain 042 and E. coli K-12 strain DH5α, which lacks the AggR regulon. In EAEC, we found that low expression of aar increases aafA fimbrial gene expression via H-NS; however, when aar is more highly expressed, it acts as a negative regulator via AggR. In DH5α, aar affected expression of E. coli genes in some cases via H-NS and in some cases independent of H-NS. Our data support the model that Aar interacts in concert with AggR, H-NS, and possibly other regulators and that these interactions are likely to be functionally significant in vivo.

scholarly journals Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Steven J Sandler ◽  
Hardeep S Samra ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mutant Allele ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Dnab Helicase ◽  
K 12 ◽  
Extragenic Suppressors ◽  
Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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