scholarly journals Contribution of Phe-7 to Tat-Dependent Export of β-Lactamase in Xanthomonas campestris

2012 ◽  
Vol 56 (7) ◽  
pp. 3597-3602 ◽  
Author(s):  
Chen-Wei Lee ◽  
Yi-Hsuan Tseng ◽  
Fu-Seng Deng ◽  
Juey-Wen Lin ◽  
Yi-Hsiung Tseng ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Structure ◽  
Amino Acid ◽  
Xanthomonas Campestris ◽  
Constitutive Expression ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Content Type ◽  
Aromatic Interactions ◽  
Tat Pathway

ABSTRACTStrains ofXanthomonas campestrispv. campestris isolated in Taiwan are commonly resistant to ampicillin owing to the constitutive expression of a chromosomally encoded β-lactamase that is secreted into the periplasm. In this study, we found that levels of β-lactamase vary amongX. campestrispv. campestris strains, a difference that can be attributed to amino acid substitutions at least at positions 7 and 206, with the former having the major impact. Bioinformatic and PCR analyses indicated thatX. campestrispv. campestris possessestatABCgenes and that the signal peptide ofX. campestrispv. campestris pre-Bla contains the typical twin-arginine motif (N-R-R-Q-F-L at amino acid residues 3 to 8 in strainX. campestrispv. campestris strain 11), suggesting that Bla is secreted via the Tat pathway. To assess the importance of Phe7in the efficient export ofX. campestrispv. campestris Bla, we prepared mutant constructs containing amino acid substitutions and monitored their expression by measuring enzyme activity and detecting Bla protein by Western blotting. The results indicate that replacement of Phe7with Leu severely inhibited Bla export whereas replacement with Pro almost abolished it. Although a change to Arg caused moderate inhibition of export, replacement with Tyr had no effect. These results suggest that for efficient export of Bla byX. campestrispv. campestris, the aromatic-aromatic interactions and stability of protein structure around the twin-arginine motif are important, since only proteins that can attain a folded state in the cytoplasm are competent for export via the Tat pathway.

scholarly journals Mutations in the Regulatory Gene hrpG ofXanthomonas campestris pv. vesicatoria Result in Constitutive Expression of All hrp Genes

1999 ◽  
Vol 181 (21) ◽  
pp. 6828-6831 ◽  
Author(s):  
Kai Wengelnik ◽  
Ombeline Rossier ◽  
Ulla Bonas
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Xanthomonas Campestris ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Amino Acid Substitutions ◽  
Pathogenicity Genes ◽  
Hrp Genes ◽  
Related Proteins

ABSTRACT hrpG is a key regulatory gene for transcriptional activation of pathogenicity genes (hrp) ofXanthomonas campestris pv. vesicatoria. We identified three mutations in hrpG which render hrp gene expression constitutive in normally suppressing medium. The mutations in hrpG result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutatedhrpG enhances the timing and intensity of plant reactions in infection assays.

Effect of conserved intersubunit amino acid substitutions on Hfq protein structure and stability

2014 ◽  
Vol 79 (5) ◽  
pp. 469-477 ◽  
Author(s):  
V. N. Murina ◽  
B. S. Melnik ◽  
V. V. Filimonov ◽  
M. Ühlein ◽  
M. S. Weiss ◽  
...  
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Structure And Stability

Protein truncation is required for the activation of the c-myb proto-oncogene

1991 ◽  
Vol 11 (8) ◽  
pp. 3987-3996
Author(s):  
F A Grässer ◽  
T Graf ◽  
J S Lipsick
Keyword(s):  
Amino Acid ◽  
Bone Marrow Cells ◽  
Protein Product ◽  
Full Length ◽  
Carboxyl Terminus ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Avian Myeloblastosis Virus ◽  
Virally Encoded ◽  
Myb Proteins

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

scholarly journals OXA-48-Mediated Ceftazidime-Avibactam Resistance Is Associated with Evolutionary Trade-Offs

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Ane Molden Thomassen ◽  
Pål Jarle Johnsen ◽  
Hanna-Kirsti S. Leiros ◽  
...  
Keyword(s):  
Amino Acid ◽  
Inhibitory Activity ◽  
Treatment Options ◽  
Inhibitory Effect ◽  
Amino Acid Substitutions ◽  
Gram Negative ◽  
Content Type ◽  
Mortality And Morbidity ◽  
Trade Offs ◽  
Collateral Effects

ABSTRACTInfections due to carbapenemase-producing Gram-negative pathogens are associated with limited treatment options and consequently lead to increased mortality and morbidity. In response, combinations of existing β-lactams and novel β-lactamase inhibitors, such as ceftazidime-avibactam (CAZ-AVI), have been developed as alternative treatment options. To understand the development of resistance and evolutionary trajectories under CAZ-AVI exposure, we studied the effects of ceftazidime (CAZ) and CAZ-AVI on the carbapenemase OXA-48 and the epidemic OXA-48 plasmid inEscherichia coli. Exposure of CAZ and CAZ-AVI resulted in single (P68A) and double (P68A,Y211S) amino acid substitutions in OXA-48, respectively. The antimicrobial susceptibility data and enzyme kinetics showed that the P68A substitution was responsible for an increased activity toward CAZ, whereas P68A,Y211S led to a decrease in the inhibitory activity of AVI. X-ray crystallography and molecular modeling of the mutants demonstrated increased flexibility within the active site, which could explain the elevated CAZ hydrolysis and reduced inhibitory activity of AVI. Interestingly, these substitutions resulted in collateral effects compromising the activity of OXA-48 toward carbapenems and penicillins. Moreover, exposure to CAZ-AVI selected for mutations within the OXA-48-encoding plasmid that severely reduced fitness in the absence of antimicrobial selection. These evolutionary trade-offs may contribute to limit the evolution of OXA-48-mediated CAZ and CAZ-AVI resistance, as well as potentially resensitize isolates toward other therapeutic alternatives.IMPORTANCEThe recent introduction of novel β-lactam/β-lactamase inhibitor combinations like ceftazidime-avibactam has increased our ability to treat infections caused by multidrug-resistant Gram-negative bacteria, including carbapenemase-producingEnterobacterales. However, the increasing number of cases of reported resistance to ceftazidime-avibactam is a concern. OXA-48 is a carbapenemase that has no significant effect on ceftazidime, but is inhibited by avibactam. Since isolates with OXA-48 frequently harbor extended-spectrum β-lactamases that are inhibited by avibactam, it is likely that ceftazidime-avibactam will be used to treat infections caused by OXA-48-producingEnterobacterales.Our data show that exposure to ceftazidime-avibactam can lead to changes in OXA-48, resulting in increased ability to hydrolyze ceftazidime and withstand the inhibitory effect of avibactam. Thus, resistance toward ceftazidime-avibactam among OXA-48-producingEnterobacteralesshould be monitored. Interestingly, the compromising effect of the amino acid substitutions in OXA-48 on other β-lactams and the effect of ceftazidime-avibactam exposure on the epidemic OXA-48 plasmid indicate that the evolution of ceftazidime-avibactam resistance comes with collateral effects.

scholarly journals Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

2001 ◽  
Vol 355 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Chang Hoon LEE ◽  
Patrick Y. UM ◽  
Myung Hee PARK
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Binding Pocket ◽  
Complete Loss ◽  
Amino Acid Residues ◽  
Deoxyhypusine Synthase ◽  
Positive Identification ◽  
Insight Into

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

scholarly journals Gain-of-Function Mutations inUPC2Are a Frequent Cause ofERG11Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

2012 ◽  
Vol 11 (10) ◽  
pp. 1289-1299 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Katherine S. Barker ◽  
Elizabeth L. Berkow ◽  
Geoffrey Toner ◽  
Sean G. Chadwick ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Clinical Isolates ◽  
Transcriptional Analysis ◽  
Single Amino Acid ◽  
Ergosterol Biosynthesis ◽  
Amino Acid Substitutions ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTInCandida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documentedUPC2gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase inERG11expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressedERG11by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation inUPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations inUPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increasedERG11expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by theMDR1gene, and the uncharacterized ATP binding cassette transporterCDR11. These findings demonstrate that gain-of-function mutations inUPC2are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account forERG11overexpression in all such isolates ofC. albicans.

scholarly journals Elucidating the Role of Residue 67 in IMP-Type Metallo-β-Lactamase Evolution

2015 ◽  
Vol 59 (12) ◽  
pp. 7299-7307 ◽  
Author(s):  
Alecander E. LaCuran ◽  
Kevin M. Pegg ◽  
Eleanor M. Liu ◽  
Christopher R. Bethel ◽  
Ni Ai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hydrophobic Interactions ◽  
Amino Acid Substitutions ◽  
Binding Affinities ◽  
Content Type ◽  
Resistance Factors ◽  
Specific Effects ◽  
Docking Calculations ◽  
Rapid Dissemination ◽  
Imminent Threat

ABSTRACTAntibiotic resistance in bacteria is ever changing and adapting, as once-novel β-lactam antibiotics are losing their efficacy, primarily due to the production of β-lactamases. Metallo-β-lactamases (MBLs) efficiently inactivate a broad range of β-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring β-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of β-lactam antibiotics. Wild-type β-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs forEscherichia colicells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested β-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highestkcat/Kmvalues. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a β-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the β-lactam substrates.

scholarly journals Constitutive Expression of a Chromosomal Class A (BJM Group 2) β-Lactamase in Xanthomonas campestris

2004 ◽  
Vol 48 (1) ◽  
pp. 209-215 ◽  
Author(s):  
Shu-Fen Weng ◽  
Juey-Wen Lin ◽  
Chih-Hung Chen ◽  
Yih-Yuan Chen ◽  
Yi-Hsuan Tseng ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xanthomonas Campestris ◽  
Southern Hybridization ◽  
Constitutive Expression ◽  
Structural Features ◽  
Amino Acid Identity ◽  
Upstream Region ◽  
Class A ◽  
Group 2 ◽  
Lactamase Gene

ABSTRACT Sequencing of the upstream region of the β-lactamase gene from Xanthomonas campestris pv. campestris 11 (bla XCC-1) revealed the cognate ampR1 gene (289 amino acids, 31 kDa). It runs divergently from bla XCC-1 with a 100-bp intergenic region (IG) containing partially overlapped promoters with structural features typical of the bla-ampR IG. The deduced AmpR1 protein shows significant identity in amino acid sequence and conserved motifs with AmpR proteins of other species, e.g., of Pseudomonas aeruginosa (58.2% amino acid identity). Results of insertional mutation, complementation tests, and β-lactamase assays suggested that expression of bla XCC-1 was constitutive and dependent on AmpR1. Four bla genes and two ampR genes are present in the fully sequenced X. campestris pv. campestris ATCC 33913 genome, with XCC3039 and XCC3040 considered the analogues of bla XCC-1 and ampR1, respectively. An ampR1 homologue was detected by Southern hybridization in the ampicillin-resistant Xanthomonas strains, which appear to express β-lactamase constitutively. Although the significance remains to be studied, constitutive expression of β-lactamase by a widespread bacterial genus raises environmental concerns regarding the dissemination of resistance genes.

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