scholarly journals Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

2016 ◽  
Vol 198 (24) ◽  
pp. 3287-3295 ◽  
Author(s):  
Lauren R. Walling ◽  
J. Scott Butler
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Antibiotic Treatment ◽  
Bacterial Growth ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Bacterial Persistence ◽  
Content Type ◽  
Single Amino Acid Change ◽  
Specificity Determinants

ABSTRACT Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCE Toxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae , as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.

scholarly journals De NovoAmino Acid Biosynthesis Contributes to Salmonella enterica Growth in Alfalfa Seedling Exudates

2014 ◽  
Vol 81 (3) ◽  
pp. 861-873 ◽  
Author(s):  
Grace Kwan ◽  
Tippapha Pisithkul ◽  
Daniel Amador-Noguez ◽  
Jeri Barak
Keyword(s):  
Amino Acid ◽  
Bacterial Growth ◽  
Salmonella Enterica ◽  
Metabolic Networks ◽  
Single Amino Acid ◽  
Content Type ◽  
Growth Defects ◽  
Proteomic Data ◽  
Metabolic Functions ◽  
Metabolic Reactions

ABSTRACTSalmonella entericais a member of the plant microbiome. Growth ofS. entericain sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements ofS. entericaduring growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305S. entericaproteins extracted 24 h postinoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over one-quarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important forS. entericagrowth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor duringS. entericagrowth in association with roots. Collectively, these data suggest thatS. entericametabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth.

scholarly journals In Vitro Activities of Garenoxacin (BMS-284756) against Haemophilus influenzae Isolates with Different Fluoroquinolone Susceptibilities

2003 ◽  
Vol 47 (11) ◽  
pp. 3539-3541 ◽  
Author(s):  
María Pérez-Vázquez ◽  
Federico Román ◽  
Belen Aracil ◽  
Rafael Cantón ◽  
José Campos
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Amino Acid Change ◽  
Antimicrobial Agents ◽  
Acquired Resistance ◽  
Single Amino Acid ◽  
Intrinsic Activity ◽  
Single Amino Acid Change ◽  
Amoxicillin Clavulanate

ABSTRACT The in vitro activity of garenoxacin (BMS-284756) against 62 clinical Haemophilus influenzae isolates with different fluoroquinolone susceptibilities was determined by the microdilution susceptibility testing method and compared with the activities of other oral quinolones and nonquinolone oral antimicrobial agents. Cefixime presented the highest intrinsic activity (MIC at which 50% of the isolates tested were inhibited [MIC50], 0.01 μg/ml), followed by garenoxacin, moxifloxacin, and ciprofloxacin (MIC50, 0.06 μg/ml), levofloxacin (MIC50, 0.12 μg/ml), cefuroxime (MIC50, 1.0 μg/ml), and amoxicillin-clavulanate (MIC50, 1.0/0.5 μg/ml), amoxicillin (MIC50, 2 μg/ml), azithromycin (MIC50, 4 μg/ml), and erythromycin (MIC50, 8 μg/ml). In strains with ciprofloxacin MICs of ≤0.06 μg/ml, ciprofloxacin and garenoxacin displayed similar MIC50s and MIC90s, one dilution lower than those of moxifloxacin and levofloxacin. For strains for which ciprofloxacin MICs were ≥0.12 μg/ml, MIC50s were similar for the four quinolones tested, although garenoxacin presented the widest activity range (0.03 to 32 μg/ml) and the highest MIC at which 90% of the isolates tested were inhibited (16.0 μg/ml). For strains without amino acid changes in the quinolone resistance determining region (QRDR) of GyrA and ParC, garenoxacin MICs were ≤0.03 μg/ml; with a single amino acid change in GyrA, garenoxacin MICs were 0.06 to 0.12 μg/ml; with one amino acid change each in GyrA and ParC, garenoxacin MICs were 0.5 to 2.0 μg/ml; one amino acid change in ParC combined with two amino acid changes in GyrA increased the MICs to ≥4 μg/ml for all assayed quinolones. We conclude that garenoxacin has excellent activity against H. influenzae, although progressive acquired resistance was observed by step-by-step mutation in the QRDR of gyrA and parC.

scholarly journals Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

2014 ◽  
Vol 58 (8) ◽  
pp. 4762-4766 ◽  
Author(s):  
Aurélie Jayol ◽  
Laurent Poirel ◽  
Adrian Brink ◽  
Maria-Virginia Villegas ◽  
Mesut Yilmaz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Amino Acid Change ◽  
Sequence Type ◽  
Single Amino Acid ◽  
Wild Type ◽  
Two Component System ◽  
Content Type ◽  
Single Amino Acid Change ◽  
Two Component

ABSTRACTA series of colistin-resistantKlebsiella pneumoniaeisolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by thepmrBgene, part of thepmrCABoperon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of thepmrA(70-fold),pmrB(70-fold),pmrC(170-fold), andpmrK(40-fold) genes in thepmrB-mutated isolate compared to expression of thepmrBwild-type isogenicK. pneumoniaeisolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression ofpmrCABandpmrHFIJKLMoperons, leading to resistance to colistin.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides

FEBS Letters ◽  
2000 ◽  
Vol 470 (2) ◽  
pp. 135-138 ◽  
Author(s):  
H. Vais ◽  
S. Atkinson ◽  
N. Eldursi ◽  
A.L. Devonshire ◽  
M.S. Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sodium Channel ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Pyrethroid Insecticides ◽  
Highly Sensitive ◽  
Single Amino Acid Change

Further characterization and determination of the single amino acid change in thetsI138reovirus thermosensitive mutant

2012 ◽  
Vol 58 (5) ◽  
pp. 589-595
Author(s):  
Guy Lemay ◽  
Martin Bisaillon
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Genetic Alterations ◽  
Biological Properties ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Gene Encoding ◽  
Viral Particles ◽  
Single Amino Acid Change

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

1993 ◽  
Vol 13 (7) ◽  
pp. 3850-3859
Author(s):  
T A Coleman ◽  
C Kunsch ◽  
M Maher ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Dna Binding Specificity ◽  
Dna Motif ◽  
Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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