Prochiral hydrazones undergo efficient and highly selective hydrogenation in the presence of a chiral diphosphine ruthenium catalyst, yielding enantioenriched hydrazine products (up to 99% ee). The ...mild reaction conditions and broad functional group tolerance of this method allow access to versatile chiral hydrazine building blocks containing aryl bromide, heteroaryl, alkyl, cycloalkyl, and ester substituents. This method was also demonstrated on >150 g scale, providing a valuable hydrazine intermediate en route to an active pharmaceutical ingredient.
Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and ...small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S aureus and found that the LAC*ΔgdpP strain of S aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-β-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.
This manuscript describes the development of the first diastereoselective intermolecular synthesis of alkyl ethers via reductive etherification of diverse ketones or aldehydes with alcohols. Key to ...this development was the use of low-temperature high-throughput experimentation (HTE) technologies that enabled rapid reaction optimizations and parallel synthesis. A broad scope of pharmaceutically relevant substrates was surveyed, which formed alkyl ethers effectively. In addition, we demonstrated that the diastereoselectivity of this transformation can be readily modulated by prudent selection of the reductant.
Status quo of tet regulation in bacteria Bertram, Ralph; Neumann, Bernd; Schuster, Christopher F.
Microbial Biotechnology,
April 2022, Letnik:
15, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Summary
The tetracycline repressor (TetR) belongs to the most popular, versatile and efficient transcriptional regulators used in bacterial genetics. In the tetracycline (Tc) resistance determinant ...tet(B) of transposon Tn10, tetR regulates the expression of a divergently oriented tetA gene that encodes a Tc antiporter. These components of Tn10 and of other natural or synthetic origins have been used for tetracycline‐dependent gene regulation (tet regulation) in at least 40 bacterial genera. Tet regulation serves several purposes such as conditional complementation, depletion of essential genes, modulation of artificial genetic networks, protein overexpression or the control of gene expression within cell culture or animal infection models. Adaptations of the promoters employed have increased tet regulation efficiency and have made this system accessible to taxonomically distant bacteria. Variations of TetR, different effector molecules and mutated DNA binding sites have enabled new modes of gene expression control. This article provides a current overview of tet regulation in bacteria.
The tetracycline repressor (TetR) belongs to the most popular, versatile, and efficient transcriptional regulators used in bacterial genetics. Variations of TetR, different effector molecules and mutated DNA binding sites have enabled new modes of gene expression control. This article provides a current overview of tet regulation in bacteria.
Abstract
Background
Extended-spectrum β-lactamases (ESBLs) are enzymes that can render their hosts resistant to various β-lactam antibiotics. CTX-M-type enzymes are the most prevalent ESBLs and the ...main cause of resistance to third-generation cephalosporins in Enterobacteriaceae. The number of described CTX-M types is continuously rising, currently comprising over 240 variants. During routine screening we identified a novel blaCTX-M gene.
Objectives
To characterize a novel blaCTX-M variant harboured by a multidrug-resistant Escherichia coli isolate of sequence type ST354.
Methods
Antibiotic susceptibilities were determined using broth microdilution. Genome and plasmid sequences were reconstructed using short- and long-read sequencing. The novel blaCTX-M locus was analysed using long-read and Sanger sequencing. Plasmid polymorphisms were determined in silico on a single plasmid molecule level.
Results
The novel blaCTX-M-243 allele was discovered alongside a nearly identical blaCTX-M-104-containing gene array on a 219 kbp IncHI2A plasmid. CTX-M-243 differed from CTX-M-104 by only one amino acid substitution (N109S). Ultra-deep (2300-fold coverage) long-read sequencing revealed dynamic scaling of the blaCTX-M genetic contexts from one to five copies. Further antibiotic resistance genes such as blaTEM-1 also exhibited sequence heterogeneity but were stable in copy number.
Conclusions
We identified the novel ESBL gene blaCTX-M-243 and illustrate a dynamic system of varying blaCTX-M copy numbers. Our results highlight the constant emergence of new CTX-M family enzymes and demonstrate a potential evolutionary platform to generate novel ESBL variants and possibly other antibiotic resistance genes.
Here, we describe a protocol for a colony polymerase chain reaction (PCR) method for
The methodology involves the preparation of small
lysates by using the enzyme lysostaphin to degrade the ...peptidoglycan layer. These lysates are prepared using a small patch of bacteria grown on LB agar plates, and the lysates can subsequently be used for PCR analyses.
Staphylococcus aureus is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its ...high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long‐term exposure. In this study, we used transposon sequencing (TN‐seq) to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long‐term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene SAUSA300_0957 (gene 957) as essential under salt stress. Interestingly, a 957 mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin‐binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.
Staphylococcus aureus is able to grow in the presence of high concentrations of NaCl but the exact genetic factors contributing to this are unknown. Using a high‐throughput TN‐seq approach, we identified gene 957 as an important factor for the salt stress resistance in S. aureus. A 957‐mutant was not only salt sensitive but also showed increased peptidoglycan crosslinking, altogether highlighting the cell wall as an important barrier against osmotic stress.
This protocol continues a series of methods for the construction of an in-frame gene deletion in
strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for
We have ...previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-Δ
) can be constructed and isolated from
, then introduced into electrocompetent
cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (
* gene) and confers chloramphenicol resistance to
As a specific example, we present the construction of strain RN4220*Δ
from strain RN4220 carrying the pIMAY*-Δ
plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.
This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in
strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired ...gene deletion (in this case, pIMAY*-Δ
) can be constructed and isolated from
, we now present details of the next steps in this method-the preparation of electrocompetent
cells and introduction of the
mutant plasmid DNA into the
cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.
A Staphylococcus aureus strain deleted for the c‐di‐AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, ...encoding the main glycine‐betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild‐type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c‐di‐AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c‐di‐AMP signalling in S. aureus.
A large number of amino acid transporters and oligopeptide permeases are encoded in bacterial genomes. However, their actual substrate specificity and functions are hard to predict bioinformatically. In this study, we report that GltS and AlsT are main glutamate and glutamine transporters in Staphylococcus aureus, respectively, and show that glutamine and ammonium uptake inhibit the production of the nucleotide signalling molecule c‐di‐AMP.
PDF
