Metabolism of pyrithiamine by the pyrithiamine-requiring mutant of Staphylococcus aureus.
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ABSTRACT:
SUBMITTER: Sinha AK
PROVIDER: S-EPMC1198641 | biostudies-other | 1968 Mar
REPOSITORIES: biostudies-other
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Project description:1. Amino acid metabolism and protein synthesis in a Staphylococcus aureus mutant strain that requires pyrithiamine for optimum growth were studied and compared with those in the thiamine-requiring parent S. aureus. 2. The mutant strain utilized amino acids at a higher rate than did the parent strain. The utilization of glutamic acid, serine and glycine was much stimulated in the mutant strain. 3. The rate of oxidation of glutamic acid, aspartic acid, isoleucine and glycine was higher in the mutant strain. 4. The mutant strain contained serine, glycine, tyrosine, glutamic acid, aspartic acid, arginine and histidine as free amino acids, whereas the parent strain possessed lysine, arginine, histidine, aspartic acid and glutamic acid. 5. The mutant strain possessed slightly higher glutamate-oxalo-acetate transaminase activity, whereas the activities of glutamate-pyruvate transaminase were similar in both strains. 6. The incorporation of (14)C from [2-(14)C]-acetate into individual amino acids of the cell protein was greater in the mutant strain. 7. The incorporation of (14)C-labelled amino acids into the cell proteins of the mutant strain was not much different from that in the parent strain. 8. Induction of beta-d-galactosidase in the mutant strain did not occur, whereas induction of this enzyme is possible in the parent strain. Thiamine or pyrithiamine has no direct effect on the induction of beta-d-galactosidase.
| S-EPMC1271229 | biostudies-other
Project description:The transcription level of a rex-deficient S. aureus mutant in comparison to its parental strain S. aureus SH1000 was analyzed using DNA microarrays.
2009-12-31 | GSE17128 | GEO
Project description:As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K(+)) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K(+) uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K(+) deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K(+) uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K(+) uptake in S. aureus revealed that the Ktr-mediated K(+) transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K(+) uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K(+) uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K(+) uptake in establishing efficient carbon utilization.
| S-EPMC4911797 | biostudies-literature
Project description:1. As a result of the mutation of Staphylococcus aureus by pyrithiamine, deletion of the enzyme thiaminokinase in the system occurs. Some properties of thiaminokinase including the effects of pH, pyrophosphate donor nucleotides and metal ions on the enzyme in the parent S. aureus have been studied. Cell-free extract from mutant strain has been studied under similar conditions and thiaminokinase activity was found to be absent. Addition of thiamine (10mug./ml.) to the medium containing pyrithiamine (required for the growth of the mutant strain) did not give rise to thiaminokinase activity in the mutant bacteria. 2. The parent and the mutant strains of S. aureus have been studied for the fermentative production of acetylmethylcarbinol (3-hydroxybutan-2-one) and the mutant strain did not produce acetylmethylcarbinol under the conditions used.
| S-EPMC1271212 | biostudies-other
Project description:The transcription level of a rex-deficient S. aureus mutant in comparison to its parental strain S. aureus SH1000 was analyzed using DNA microarrays. S. aureus N315 microarrays were purchased from Scienion (Scienion AG, Berlin, Germany) and were produced by spotting 2,338 PCR products of the 2,593 ORFs comprising annotated genome of S. aureus N315 [reference identification: NC_002745] on a glass slide. Each ORF is present in duplicate on the microarray (further details can be found at http://www.scienion.com), cDNA was synthesized from total RNA with the LabelStar Array Kit from QIAGEN using the QIAGEN protocol with slight modifications: Random hexamer primer were used (Invitrogen, Karlsruhe, Germany) and Cy3- and Cy5-dCTP were purchased from Perkin-Elmer (Rodgau - Juegesheim, Germany). As recommended by Scienion, 10 µg RNA from either SH1000 or AK1 were used for cDNA synthesis. After hybridisation for 72 h, the microarrays were washed as recommended by the manufacturer. Data analysis. The hybridized microarrays were scanned with a GenePix 4000B microarray scanner (MDS Analytical Technologies GmbH, Ismaning, Germany). A geometric raster was laid over the resulting microarray picture to distinguish the signals from the background. After localization of single spots, intensities and global background were calculated automatically. The hybridization patterns and intensities were quantitatively analyzed using the Imagene 6 software (BioDiscovery, El Segundo, CA). The replicates were averaged, and the spots identified by Imagene 6 (BioDiscovery) as flawed were omitted. The data set was normalized by application of the LOWESS algorithm. In a next step, the intensity values of all arrays for each time point as well as for all time points combined were used for t tests. Genes with a change of <0.5- or â?¥2.0-fold were characterized as having significantly differing amounts of transcripts based on t tests with a P value cut-off of at least 0.05. Gene functions were assigned to the respective accession numbers and annotations as compiled on DOGAN, a web page for S. aureus N315 (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=n315G1). The parental strain SH1000 and the Rex deficient mutant AK1 were applied on full-genome microarrays to get a detailed view on the differences in the transcriptional profiles which are caused â?? directly or indirectly â?? by the introduced mutation. More specifically, expression levels were compared at five time points, covering different growth phases. To highlight the general changes in the expression profile between SH1000 and the rex mutant, the microarray data of all five time points were also analyzed in a combined way using standard statistical methods. In further experiments, we focused on those genes, which seemed to flag the general difference between the investigated strains.
2010-05-16 | E-GEOD-17128 | biostudies-arrayexpress
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) strains are distinct from general Staphylococcus strains with respect to the composition of the membrane, ability to form a thicker biofilm, and, importantly, ability to modify the target of antibiotics to evade their activity. The agr gene is an accessory global regulator of gram-positive bacteria that governs virulence or resistant mechanisms and therefore an important target for the control of resistant strains. However, the mechanism by which agr impacts resistance to ?-lactam antibiotics remains unclear. In the present study, we found the ?agr mutant strain having higher resistance to high concentrations of ?-lactam antibiotics such as oxacillin and ampicillin. To determine the influence of variation in the microenvironment of cells between the parental and mutant strains, fatty acid analysis of the supernatant, total lipids, and phospholipid fatty acids were compared. The ?agr mutant strain tended to produce fewer fatty acids and retained lower amounts of C16, C18 fatty acids in the supernatant. Phospholipid analysis showed a dramatic increase in the hydrophobic longer-chain fatty acids in the membrane. To target membrane, we applied several surfactants and found that sorbitan monolaurate (Span20) had a synergistic effect with oxacillin by decreasing biofilm formation and growth. These findings indicate that agr deletion allows for MRSA to resist antibiotics via several changes including constant expression of mecA, fatty acid metabolism, and biofilm thickening.
| S-EPMC7138893 | biostudies-literature
Project description:RNAIII from Staphylococcus lugdunensis (RNAIII-sl) in a Staphylococcus aureus agr mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5' end of RNAIII-sl and the 3' end of RNAIII from S. aureus restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.
| S-EPMC107643 | biostudies-literature
Project description:Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment) or genetic (Deltafur) alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB), a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.
| S-EPMC1557832 | biostudies-literature
Project description:Ozenoxacin (OZN) belongs to a new generation of non-fluorinated quinolones for the topical treatment of skin infections which has shown to be effective in the treatment of susceptible and resistant Gram-positive cocci. The mutant prevention concentration (MPC) of ozenoxacin, levofloxacin and ciprofloxacin was determined in quinolone-susceptible and -resistant strains including methicillin-susceptible S. aureus, methicillin-resistant S. aureus, methicillin-susceptible S. epidermidis and methicillin-resistant S. epidermidis with different profile of mutation in the quinolone resistance determining regions (QRDR). The MPC value of OZN for the methicillin-susceptible S. aureus strain susceptible to quinolones, without mutations in QRDR, was 0.05 mg/L, being 280-fold lower than that observed with ciprofloxacin and levofloxacin. In methicillin-susceptible and-resistant S. aureus strains with mutations in the gyrA or/and grlA genes the MPC of OZN went from 0.1 to 6 mg/L, whereas the MPC of levofloxacin and ciprofloxacin was > 50 mg/L for the same strains. For methicillin-susceptible and-resistant S. epidermidis the results were similar to those abovementioned for S. aureus. According to our results, the MPC of OZN was far below the quantity of ozenoxacin achieved in the epidermal layer, suggesting that the in vivo selection of mutants, if it occurs, will take place at low frequency. Ozenoxacin is an excellent candidate for the treatment of bacterial infections caused by susceptible and quinolone-resistant staphylococci isolated usually from skin infections.
| S-EPMC6785070 | biostudies-literature
Project description:In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureus?mvaS mutant can revert to prototrophy. This mutant couldn't grow in the absence of MVA. However, after a long lag-phase of 4-6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ?mvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ?mvaS mutant.
| S-EPMC6052127 | biostudies-literature