scholarly journals Tackling Intrinsic Antibiotic Resistance in Serratia marcescens with a Combination of Ampicillin/Sulbactam and Phage SALSA

Antibiotics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 371
Author(s):  
Lorenz Weber ◽  
Mathias Jansen ◽  
Alex Krüttgen ◽  
Eva Miriam Buhl ◽  
Hans-Peter Horz
Keyword(s):  
Drug Resistance ◽  
Treatment Options ◽  
Open Reading Frames ◽  
Clinical Isolates ◽  
Gene Encoding ◽  
Acquired Drug Resistance ◽  
Genome Wide ◽  
Colony Forming Units ◽  
Intrinsic Drug Resistance ◽  
Reading Frames

During the antibiotic crisis, bacteriophages (briefly phages) are increasingly considered as potential antimicrobial pillars for the treatment of infectious diseases. Apart from acquired drug resistance, treatment options are additionally hampered by intrinsic, chromosomal-encoded resistance. For instance, the chromosomal ampC gene encoding for the AmpC-type β-lactamases is typically present in a number of nosocomial pathogens, including S. marcescens. In this study, phage SALSA (vB_SmaP-SALSA), with lytic activity against clinical isolates of S. marcescens, was isolated from effluent. Besides phage characterization, the aim of this study was to evaluate whether a synergistic effect between the antibiotic ampicillin/sulbactam (SAM) and phage can be achieved despite intrinsic drug resistance. Phage SALSA belongs to the Podoviridae family and genome-wide treeing analysis groups this phage within the phylogenetic radiation of T7-like viruses. The genome of Phage SALSA consists of 39,933 bp, which encode for 49 open reading frames. Phage SALSA was able to productively lyse 5 out of 20 clinical isolates (25%). A bacterial challenge with phage alone in liquid medium revealed that an initial strong bacterial decline was followed by bacterial re-growth, indicating the emergence of phage resistance. In contrast, the combination of SAM and phage, together at various concentrations, caused a complete bacterial eradication, confirmed by absorbance measurements and the absence of colony forming units after plating. The data show that it is principally possible to tackle the axiomatic condition of intrinsic drug resistance with a dual antimicrobial approach, which could be extended to other clinically relevant bacteria.

scholarly journals Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1

2009 ◽  
Vol 75 (10) ◽  
pp. 3106-3114 ◽  
Author(s):  
Jessica Rehdorf ◽  
Christian L. Zimmer ◽  
Uwe T. Bornscheuer
Keyword(s):  
Pseudomonas Putida ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Open Chain ◽  
Aromatic Ketones ◽  
Heteroaromatic Compounds ◽  
Aliphatic Ketones ◽  
Expression Characterization ◽  
Reading Frames ◽  
Villiger Oxidation

ABSTRACT While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).

scholarly journals Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

2015 ◽  
Author(s):  
David E Weinberg ◽  
Premal Shah ◽  
Stephen W Eichhorn ◽  
Jeffrey A Hussmann ◽  
Joshua B Plotkin ◽  
...  
Keyword(s):  
Translational Control ◽  
Nucleotide Composition ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Coding Regions ◽  
Genome Wide ◽  
Upstream Open Reading Frames ◽  
Improved Methods ◽  
Reading Frames

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′- untranslated regions. Collectively, our results reveal key features of translational control in yeast and provide a framework for executing and interpreting ribosome- profiling studies.

scholarly journals Ferrous Iron- and Sulfur-Induced Genes in Sulfolobus metallicus

2007 ◽  
Vol 73 (8) ◽  
pp. 2491-2497 ◽  
Author(s):  
Stephan Bathe ◽  
Paul R. Norris
Keyword(s):  
Reverse Transcription ◽  
Ferrous Iron ◽  
Subtractive Hybridization ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Induced Genes ◽  
Reading Frames ◽  
Sulfur Oxygenase Reductase

ABSTRACT Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

scholarly journals Sequence of the Genome of the Temperate, Serotype-Converting,Pseudomonas aeruginosa Bacteriophage D3

2000 ◽  
Vol 182 (21) ◽  
pp. 6066-6074 ◽  
Author(s):  
Andrew M. Kropinski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sequence Similarity ◽  
Complete Sequence ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Virus Family ◽  
Double Stranded Dna ◽  
High Degree ◽  
Phage Proteins ◽  
Reading Frames

ABSTRACT Temperate bacteriophage D3, a member of the virus familySiphoviridae, is responsible for serotype conversion in its host, Pseudomonas aeruginosa. The complete sequence of the double-stranded DNA genome has been determined. The 56,426 bp contains 90 putative open reading frames (ORFs) and four genes specifying tRNAs. The latter are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The tRNAs may function in the translation of certain highly expressed proteins from this relatively AT-rich genome. D3 proteins which exhibited a high degree of sequence similarity to previously characterized phage proteins included the portal, major head, tail, and tail tape measure proteins, endolysin, integrase, helicase, and NinG. The layout of genes was reminiscent of lambdoid phages, with the exception of the placement of the endolysin gene, which parenthetically also lacked a cognate holin. The greatest sequence similarity was found in the morphogenesis genes to coliphages HK022 and HK97. Among the ORFs was discovered the gene encoding the fucosamine O-acetylase, which is in part responsible for the serotype conversion events.

Genome-Wide Search for Translated Upstream Open Reading Frames in Arabidopsis Thaliana

2016 ◽  
Vol 15 (2) ◽  
pp. 148-157 ◽  
Author(s):  
Qiwen Hu ◽  
Catharina Merchante ◽  
Anna N. Stepanova ◽  
Jose M. Alonso ◽  
Steffen Heber
Keyword(s):  
Arabidopsis Thaliana ◽  
Open Reading Frames ◽  
Genome Wide ◽  
Genome Wide Search ◽  
Upstream Open Reading Frames ◽  
Reading Frames

Protection against bacteriocin 28b in Serratia matcescens is apparently not related to the expression of an immunity gene

1995 ◽  
Vol 41 (3) ◽  
pp. 217-226 ◽  
Author(s):  
Margarita Beatriz Viejo ◽  
Josefina Enfedaque ◽  
Joan Francesc Guasch ◽  
Santiago Ferrer ◽  
Miguel Regué
Keyword(s):  
Nucleotide Sequence ◽  
Serratia Marcescens ◽  
Structural Gene ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Immunity Gene ◽  
Release Analysis ◽  
Reading Frames

The gene encoding bacteriocin 28b from Serratia marcescens N28b (bss gene) has been cloned in Escherichia coli and its nucleotide sequence has been determined. The genetic determinants coding for other well-characterized bacteriocins from enterobacteria (colicins) are located in plasmids and they have always been shown to contain a gene responsible for immunity located downstream from the bacteriocin structural gene. In some cases there is another gene located downstream from the immunity gene, which is responsible for bacteriocin release. Analysis of bacteriocin 28b release and the sensitivity to this bacteriocin of E. coli strains harbouring recombinant plasmids containing the bss gene showed that bacteriocin 28b is not released from the cell in these strains and that their phenotypic insensitivity is not associated with any region close to the structural gene. The nucleotide sequence of the region downstream from the bss gene contains two putative open reading frames transcribed in the opposite direction to the bss gene. These open reading frames apparently encode proteins that seem not to be involved in bacteriocin immunity or release. Moreover, a S. marcescens N28b genomic library was screened and no immunity gene was found. Therefore, bacteriocin 28b differs greatly from the bacteriocins from other enterobacteria, and in the following senses it is unique: firstly, the gene encoding bacteriocin 28b seems to be located on the chromosome, and secondly, insensitivity to this bacteriocin in S. marcescens N28b is not associated with the expression of an immunity gene.Key words: bacteriocin, pore-forming colicins, immunity, Serratia marcescens.

scholarly journals A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2, the Gene Encoding a Fungal Aminodeoxychorismate Lyase

2007 ◽  
Vol 6 (11) ◽  
pp. 2102-2111 ◽  
Author(s):  
Javier Botet ◽  
Laura Mateos ◽  
José L. Revuelta ◽  
María A. Santos
Keyword(s):  
Large Scale ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Cellular Processes ◽  
Founder Member ◽  
Yeast Genes ◽  
Vacuole Biogenesis ◽  
Reading Frames

ABSTRACT Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.

scholarly journals Perturbations in 3D genome organization can promote acquired drug resistance

2021 ◽  
Author(s):  
Anna G Manjón ◽  
Daan Peric Hupkes ◽  
Ning Qing Liu ◽  
Anoek Friskes ◽  
Stacey Joosten ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Drug Resistance ◽  
Genome Organization ◽  
Gene Activation ◽  
Nuclear Lamina ◽  
Tumor Evolution ◽  
Gene Encoding ◽  
Acquired Drug Resistance ◽  
H3k27 Methylation ◽  
3D Genome

AbstractAcquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

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