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Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His 6 )-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu 2+ ions gave the best results regarding His 6 -tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N-His 6 -ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL -1 . High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His 6 -tagged ATA constructs. The highest turnover number within the indicated time was 7.23·10 6 , which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation., Competing Interests: Declaration of Competing Interest On behalf of all the authors of a submission I hereby disclose no competing interest. Prof. Dr. Polona Žnidaršič-Plazl., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC-MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but is absent in mammals. Yet, it is not the target of any existing antibiotics. Hence, this enzyme is an attractive target for developing novel antimicrobial agents. A wealth of structural and mechanistic information has provided solid basis for structure-based design of HPPK inhibitors. Our bisubstrate inhibitors were initially created by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups (HP n A, n = 2, 3, or 4), among which HP 4 A exhibited the highest binding affinity (K d  = 0.47 ± 0.04 μM). Further development was carried out based on high-resolution structures of HPPK in complex with HP 4 A. Replacing the phosphate bridge with a piperidine linked thioether eliminated multiple negative charges of the bridge. Substituting the pterin moiety with 7,7-dimethyl-7,8-dihydropterin improved the binding affinity. Arming the piperidine ring with a carboxyl group and oxidizing the thioether further enhanced the potency, resulting in a druglike inhibitor of HPPK (K d  = 0.047 ± 0.007 μM). None of these inhibitors, however, exhibits bacterial cell permeability. It is most likely due to the lack of active folate transporters in bacteria. Replacing the pterin moiety with a 7-deazagaunine moiety, we have obtained a novel bisubstrate inhibitor (HP-101) showing observable cell permeability toward a Gram-positive bacterium. Here, we report the in vitro activity of HP-101 and its structure in complex with HPPK, providing a framework for structure-based further development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

N 6-Methyladenine (m6A) RNA methylation plays a key role in RNA processing and translational regulation, influencing both normal physiological and pathological processes. Yet, current techniques for studying RNA methylation struggle to isolate the effects of individual m6A modifications. Engineering of RNA methyltransferases (RNA MTases) could enable development of improved synthetic biology tools to manipulate RNA methylation, but it is challenging due to limited understanding of structure-function relationships in RNA MTases. Herein, using ancestral sequence reconstruction, we explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA. We thereby created an efficient non-specific RNA MTase termed SUPer RNA EcoGII Methyltransferase (SUPREM), which exhibits 8-fold higher expression levels, 7°C higher thermostability and 12-fold greater m6A RNA methylation activity compared with M.EcoGII. Immunofluorescent staining and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed SUPREM's higher RNA methylation activity compared with M.EcoGII in mammalian cells. Additionally, Nanopore direct RNA sequencing highlighted that SUPREM is capable of methylating a larger number of RNA methylation sites than M.EcoGII. Through phylogenetic and mutational analysis, we identified a critical residue for the enhanced RNA methylation activity of SUPREM. Collectively, our findings indicate that SUPREM holds promise as a versatile tool for in vivo RNA methylation and labeling., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA n ) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA 4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA 4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity., Competing Interests: Declaration of interests G. Tamulaitis is an inventor on patents and patent applications related to Csm and CARF protein applications., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We consider a Markovian model for the kinetics of RNA Polymerase (RNAP) which provides a physical explanation for the phenomenon of cooperative pushing during transcription elongation observed in biochemical experiments on Escherichia coli and yeast RNAP. To study how backtracking of RNAP affects cooperative pushing we incorporate into this model backward (upstream) RNAP moves. With a rigorous mathematical treatment of the model we derive conditions on the mutual static and kinetic interactions between RNAP under which backtracking preserves cooperative pushing. This is achieved by exact computation of several key properties in the steady state of this model, including the distribution of headway between two RNAP along the DNA template and the average RNAP velocity and flux., (Creative Commons Attribution license.)

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N 3 CDP) to trap a wild-type α2β2 complex of Escherichia coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a midturnover state. Instead of N 3 CDP in the active site, forward RT has resulted in N 2 loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-β interface. This N 3 CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process., Competing Interests: Competing interests statement:The authors declare no competing interest.

The extended-spectrum β-lactamase (ESBL) gene, bla VEB-1 , was identified for the first time in an Escherichia coli clinical isolate, JARB-RN-0061, from blood cultures in a Japanese general hospital in 2021. The isolate exhibited high resistance to broad-spectrum cephalosporins, including ceftazidime (MIC >128 mg/L) and cefepime (MIC = 16 mg/L). bla VEB-1 was identified during whole-genome sequencing and characterization of the isolate. JARB-RN-0061 belonged to the B2-O2:K1:H7-ST95- fimH 41 lineage and was classified as presumptive extraintestinal pathogenic E. coli (ExPEC) and uropathogenic E. coli (UPEC). Moreover, the strain harbored multiple virulence genes on the chromosome. The Col156/IncFIB(AP001918)/IncFII(29)-type plasmid (114,216 bp), with clbB and tcpC genes involved in bacteremia, was unique to the fimH 41 subclone. The bla VEB-1 gene was located on a non-typeable and non-conjugative plasmid, pJARB-RN-0061_VEB-1 (17,093 bp). It was embedded in the class 1 integron In1883-like, with multidrug resistance gene cassettes for aacA4 , aadB , cmlA5 , qnrVC4 , and dfrA14 . Notably, comparative analysis of the complete sequence of plasmid pJARB-RN-0061_VEB-1 revealed that it was highly homologous to the bla VEB-1 -harboring plasmid, pMS2H7VEB-1 (100% coverage and 99.99% identity), except for the Tn3 family transposon (4,931 bp) and the plasmid pRHBSTW-00138_5 (97% coverage and 100% identity) harbored by Klebsiella quasipneumoniae subsp. similipneumoniae strains from hospital sewage in Japan and wastewater influent in the United Kingdom, respectively. The emergence of a human pathogenic E. coli clinical isolate with the bla VEB-1 -carrying plasmid in the B2-ST95 worldwide pandemic lineage, characterized by the virulence potential of ExPEC or UPEC but a low prevalence of antimicrobial resistance, would raise public health concerns., Importance: ESBLs are plasmid-mediated enzymes that confer resistance to clinically significant antimicrobial agents, such as broad-spectrum cephalosporins. Recently, the rapid spread of CTX-M-type ESBL-producing E. coli has become a global issue, including in Japan, where ESBL production in human pathogenic E. coli , such as the ExPEC and UPEC lineages, which typically harbor several virulence genes, is a severe public health concern. To date, VEB (Vietnamese extended-spectrum β-lactamase) producers have been found only in hospital wastewater and rivers in Japan. Thus, we describe the first detection of a very rare human-derived blaVEB-1 gene in the E. coli B2-ST95 pandemic clonal lineage that is highly associated with ExPEC and UPEC in a Japanese clinical setting. Furthermore, we characterized the genomic features of plasmids harboring the class 1 integron-borne blaVEB-1. Our findings highlight the significance of closely monitoring ESBL-producing E. coli isolates to prevent the potential dissemination of this resistance determinant in Japan., Competing Interests: The authors declare no conflict of interest.

Antibiotics, particularly the β-lactams, are a cornerstone of modern medicine. However, the rise of bacterial resistance to these agents, particularly through the actions of β-lactamases, poses a significant threat to our continued ability to effectively treat infections. Metallo-β-lactamases (MBLs) are of particular concern due to their ability to hydrolyze a wide range of β-lactam antibiotics including carbapenems. For this reason there is growing interest in the development of MBL inhibitors as well as novel antibiotics that can overcome MBL-mediated resistance. Here, we report the synthesis and evaluation of novel conjugates that combine a carbapenem (meropenem or ertapenem) with a recently reported MBL inhibiting indole carboxylate scaffold. These hybrids were found to display potent inhibition against MBLs including NDM-1 and IMP-1, with IC 50 values in the low nanomolar range. However, their antibacterial potency was limited. Mechanistic studies suggest that despite maintaining effective MBL inhibiting activity in live bacteria, the new carbapenem/MBL inhibitor conjugates have a reduced ability to engage with the bacterial target of the β-lactams., (© 2024 The Author(s). ChemMedChem published by Wiley-VCH GmbH.)

The intensification of livestock farming has led to the widespread use of massive amounts of antibiotics worldwide. Poultry production, including white meat, eggs and the use of their manure as fertiliser, has been identified as one of the most crucial reservoirs for the emergence and spread of resistant bacteria, including E. coli in poultry as an important opportunistic pathogen representing the greatest biological hazard to human and wildlife health. Thus, this study aimed to analyse E. coli in the faecal carriage of healthy poultry flocks and to investigate the phenotypic and genotypic characteristics of antimicrobial resistance, including integrons genes and phylogenetic groups. A total of 431 cloacal swabs from apparently healthy poultry from four regions in Eastern Algeria from December 2021 to October 2022. 360 E. coli were isolated; from broilers (n = 151), broiler breeders (n = 91), laying hens (n = 72), and breeding hens (n = 46). Among this, 281 isolates exhibited multidrug resistance (MDR) phenotype, 17 of the 360 E. coli isolates exhibited ESBL, and one isolate exhibited both ESBL/pAmpC. A representative collection of 183 among 281 MDR E. coli was selected for further analysis by PCR to detect genes encoding resistance to different antibiotics, and sequencing was performed on all positive PCR products of bla CTX-M and bla CMY-2 genes. Phylogenetic groups were determined in 80 E. coli isolates (20 from each of the four kinds of poultry). The bla CTX-M gene was found in 16 (94.11 %) ESBL-producing E. coli isolates within 11 strains co-expressing the bla SHV gene and 8 strains co-expressing the bla TEM gene. Sequence analysis showed frequent diversity in CTX-M-group-1, with bla CTX-M-15 being the most predominant (n = 11), followed by bla CTX-M-1 (n = 5). The bla CMY-2 gene was detected only in one ESBL/pAmpC isolate. Among the 183 tested isolates, various antimicrobial resistance genes were found (number of strains) bla TEM (n = 121), bla SHV (n = 12), tetA (n = 100), tetB (n = 29), sul1(n = 67), sul2 (n = 32), qnrS (n = 45), qnrB (n = 10), qnrA (n = 1), catA1(n = 13), aac-(6')-Ib (n = 3). Furthermore, class 1 and class 2 integrons were found in 113 and 2 E. coli, respectively. The isolates were classified into multiple phylogroups, including A (35 %), B1 (27.5 %), B2 and D each (18.75 %). The detection of integrons and different classes of resistance genes in the faecal carriage of healthy poultry production indicates that commensal E. coli could potentially act as a reservoir for antimicrobial resistance, posing a significant One Health challenge encompassing the interconnected domains of human, animal health and the environment. Here, we present the first investigation to describe the diversity of bla CTX-M producing E. coli isolates with widespread detection of CTX-M-15 and CTX-M-1 in healthy breeders (Broiler and breeding hens) in Eastern Algeria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present study aimed to determine the presence of Klebsiella pneumoniae and Escherichia coli with extended-spectrum β-lactamase (ESBL)s property from treated wastewater effluents. Treated effluent samples were collected from two major water treatment plants which located at Avaniyapuram and Sakkimangalam, Madurai, Tamil Nadu, India. Among the 51 isolates, 56.86 % represented E. coli (18 from Avaniyapuram and 11 from Sakkimangalam) and 43.14 % were K. pneumoniae (7 from Avaniyapuram and 15 from Sakkimangalam). Based on the ESBL propensity, E. coli was overrepresented in the present study. All the isolates turned positive for ESBL, while 5.88 % of K. pneumoniae and 7.84 % of E. coli were positive for carbapenemases. Further, K. pneumoniae isolates from both sites showed 100 % resistance to beta-lactams, with resistance to other antibiotics such as tetracycline and meropenem. E. coli isolates were 100 % resistant to ceftazidime and cefuroxime, and 88.9 % were resistant to amoxicillin/clavulanate and ceftriaxone. The MAR indices observed in the present study for E. coli and K. pneumoniae were above the threshold value of 0.2 suggested a high risk of environmental contamination. These findings highlighted the need for routine surveillance at appropriate intervals for the presence of ESBL producing pathogens and other MDR pathogens in the environment to provide proper clinical management, develop various counter measures and policies to address and halt the spread of such potential threats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b 558 , heme b 595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O 2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b 558 is pH dependent, with a maximum rate constant (~10 4  s -1 ) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pK a  ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58 B ➔Asn and Asp105 B ➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105 B ➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105 B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pK a proton donor, and the conservation pattern of the Asp-105 B in the cyt. bd superfamily., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nature's two redox cofactors, nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN + ) as a noncanonical cofactor orthogonal to NAD(P) + . Here we present the development of Nox Ortho, a reduced NMN + (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN + ratio together with an NMN + -specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~10 3 -10 6 -fold cofactor specificity switch from NAD(P) + to NMN + . We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)'s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H)., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Escherichia coli and Klebsiella pneumoniae rank among the primary bacterial culprits in neonatal infections and fatalities in sub-Saharan Africa. This study characterized the phenotypic and genotypic features of E coli and K pneumoniae in a labor ward in Yaoundé, Cameroon., Methods: A prospective and cross-sectional study spanning 5months, from February 21, 2022 to June 30, 2022. Rectovaginal swabs were obtained from expectant mothers, and nasopharyngeal swabs were collected from their babies. Hand swabs of health care workers and environmental samples were also collected. The samples were cultured on eosin methylene blue agar. Extended-spectrum ß-lactamase (ESBL) production was assessed using CHROMAgar ESBL and the double-disk synergy test. A polymerase chain reaction was employed to detect ß-lactamase genes., Results: A total of 93 mothers and 90 neonates were collected. Almost all pregnant women (90%) were colonized by one or more multidrug-resistant (MDR) isolates with 58% being concomitantly ESBL producers. Altogether, 14 of 22 (64%) neonates were colonized by MDR isolates, while out of the 5 workers positive to Enterobacterales, all were colonized by MDR isolates. E coli predominated in pregnant women (55%) and neonates (73%), while K pneumoniae (83%) predominated in health care workers. The blaCTX-M (75%) was the leading ß-lactamase gene detected., Conclusions: Our study suggests that drug-resistant E coli and K pneumoniae are circulating at high prevalence in the labor ward in Yaoundé and emphasizes the necessity for effective infection prevention and control along with antimicrobial stewardship measures., (Copyright © 2024 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been studied, is a model system in the study of protein dynamics. Myriad previous works have identified networks of residues in positions near to and remote from the active site that are involved in the dynamics. For example, specific mutations on the Met20 loop in Escherichia coli DHFR (N23PP/S148A) are known to disrupt millisecond-time scale motions as well as reduce catalytic activity. However, how and if networks of dynamically coupled residues influence the evolution of DHFR is still an unanswered question. In this study, we first identify, by statistical coupling analysis and molecular dynamic simulations, a network of coevolving residues that possesses increased correlated motions. We then go on to show that allosteric communication in this network is knocked down in N23PP/S148A mutant E. coli DHFR. We also identify two sites in the human DHFR sector which may accommodate the Met20 loop double proline motif. Finally, we demonstrate a concerted evolutionary change in the human DHFR allosteric networks, which maintains dynamic communication. These findings strongly implicate protein dynamics as a driving force for evolution.

Inosine 5'-monophosphate dehydrogenase (IMPDH), known as GuaB in bacteria, catalyzes the rate-limiting step in de novo guanine biosynthesis and is conserved from humans to bacteria. We developed a series of potent inhibitors that selectively target GuaB over its human homolog. Here, we show that these GuaB inhibitors are bactericidal, generate phenotypic signatures that are distinct from other antibiotics, and elicit different time-kill kinetics and regulatory responses in two important Gram-negative pathogens: Acinetobacter baumannii and Escherichia coli . Specifically, the GuaB inhibitor G6 rapidly kills A. baumannii but only kills E. coli after 24 h. After exposure to G6, the expression of genes involved in purine biosynthesis and stress responses change in opposite directions while siderophore biosynthesis is downregulated in both species. Our results suggest that different species respond to GuaB inhibition using distinct regulatory programs and possibly explain the different bactericidal kinetics upon GuaB inhibition. The comparison highlights opportunities for developing GuaB inhibitors as novel antibiotics.IMPORTANCE A. baumannii is a priority bacterial pathogen for which development of new antibiotics is urgently needed due to the emergence of multidrug resistance. We recently developed a series of specific inhibitors against GuaB, a bacterial inosine 5'-monophosphate dehydrogenase, and achieved sub-micromolar minimum inhibitory concentrations against A. baumannii . GuaB catalyzes the rate-limiting step of de novo guanine biosynthesis and is highly conserved across bacterial pathogens. This study shows that inhibition of GuaB induced a bacterial morphological profile distinct from that of other classes of antibiotics, highlighting a novel mechanism of action. Moreover, our transcriptomic analysis showed that regulation of de novo purine biosynthesis and stress responses of A. baumannii upon GuaB inhibition differed significantly from that of E. coli ., Competing Interests: The authors declare no conflict of interest.

Polycyclic aromatic hydrocarbons (PAHs) are chemically stable pollutants that are poorly degraded by microorganisms in anoxic sediments. The anaerobic degradation pathway of PAHs such as phenanthrene starts with a carboxylation reaction forming phenanthroic acid. In this study, we identified and characterized the next enzyme in the pathway, the 2-phenanthroate:CoA ligase involved in the ATP-dependent formation of 2-phenanthroyl-CoA from cell-free extracts of the sulfate-reducing enrichment culture TRIP grown anaerobically with phenanthrene. The identified gene sequence indicated that 2-phenanthroate:CoA ligase belongs to the phenylacetate:CoA ligase-like enzyme family. Based on the sequence, we predict a two-domain structure of the 2-phenanthroate:CoA ligase with a typical large N-terminal and a smaller C-terminal domain. Partial purification of 2-phenanthroate:CoA ligase allowed us to identify the coding gene in the genome. 2-Phenanthroate:CoA ligase gene was heterologously expressed in Escherichia coli . Characterization of the 2-phenanthroate:CoA ligase was performed using the partially purified enzyme from cell-free extract and the purified recombinant enzyme. Testing all possible phenanthroic acid isomers as substrate for the ligase reaction showed that 2-phenanthroic acid is the preferred substrate and only 3-phenanthroic acid can be utilized to a minor extent. This also suggests that the product of the prior carboxylase reaction is 2-phenanthroic acid. 2-Phenanthroate:CoA ligase has an optimal activity at pH 7.5 and is oxygen-insensitive, analogous to other aryl-CoA ligases. In contrast to aryl-Coenzyme A ligases reported in the literature, which need Mg 2+ as cofactor, 2-phenanthroate:CoA ligase showed greatest activity with a combination of 5 mM MgCl 2 and 5 mM KCl. Furthermore, a substrate inhibition was observed at ATP concentrations above 1 mM and the enzyme was also active with ADP., Importance: Polycyclic aromatic hydrocarbons (PAHs) constitute a class of very toxic and persistent pollutants in the environment. However, the anaerobic degradation of three-ring PAHs such as phenanthrene is barely investigated. The initial degradation step starts with a carboxylation followed by a CoA‑thioesterification reaction performed by an aryl-CoA ligase. The formation of a CoA-thioester is an important step in the degradation pathway of aromatic compounds because the CoA-ester is needed for all downstream biochemical reactions in the pathway. Furthermore, we provide biochemical proof for the identification of the first genes for anaerobic phenanthrene degradation. Results presented here provide information about the biochemical and structural properties of the purified 2‑phenanthroate:CoA ligase and expand our knowledge of aryl-CoA ligases., Competing Interests: The authors declare no conflict of interest.

Background: Production of extended-spectrum β-lactamases (ESBLs) is a common resistance mechanism in Enterobacteriaceae, leading to serious hospital-acquired infections. This study aimed to assess phenotypic, phylogenetic, and antibiotic resistance patterns among ESBL-producing Escherichia coli isolates recovered from two rural tertiary hospitals in Thailand., Results: Among 467 Enterobacteriaceae isolates, E. coli was the most prevalent 356 (76.2%) followed by K. pneumoniae 88 (18.8%), K. aerogenes 8 (1.7%), K. variicola 3 (0.6%), K. quasipneumoniae 1 (0.2%%), K. oxytoca 1 (0.2%), and unidentified 9 (1.9%). Of the 202 cephalosporin-resistant E. coli isolates, 195 (96.5%) were ESBL-producing and 7 (3.5%) were non-ESBL-producing. Clermont typing revealed that phylogroup B2 was predominant (43.3%), followed by phylogroups F (11.3%), D (10.3%), C (9.7%), and A (8.7%). Among the beta-lactamase-encoding genes, bla CTX-M (83.6%) and bla TEM (81.0%) were widely found among the isolates, and bla CTX-M-1 (60.7%) was the most common among the five bla CTX-M subgroups detected. The predominant ESBL was bla CTX-M-15 (58.3%). All isolates were resistant to cefotaxime (100%) and ampicillin (100%), followed by ciprofloxacin (91.3 %), ceftazidime (72.8 %), and tetracycline (64.1%)., Conclusion: Our findings show that phylogroup B2 was the most prevalent phylogroup among ESBL-producing E. coli isolates in northeastern Thailand. Notably, the isolates mostly carried the bla CTX-M gene(s)., (© 2024. The Author(s).)

L-rhamnose isomerase (L-RhI) plays a key role in the microbial L-rhamnose metabolism by catalyzing the reversible isomerization of L-rhamnose to L-rhamnulose. Additionally, the enzyme exhibits activity on various other aldoses and ketoses, and its broad substrate specificity has attracted attention for its potential application in the production of rare sugars; however, improvement of the enzyme properties is desirable, such as thermal stability, enzymatic activity, and a pH optimum suitable for industrial usage. This review summarizes our current insights into L-RhIs with respect to their substrate recognition mechanism and their relationship with D-xylose isomerase (D-XI) based on structural and phylogenetic analyses. These two enzymes are inherently different, but recognize distinctly different substrates, and share common features that may be phylogenetically related. For example, they both have a flexible loop region that is involved in shaping active sites, and this region may also be responsible for various enzymatic properties of L-RhIs, such as substrate specificity and thermal stability. KEY POINTS: •L-RhIs share structural features with D-XI. •There are two types of L-RhIs: E. coli L-RhI-type and D-XI-type. •Flexible loop regions are involved in the specific enzyme properties., (© 2024. The Author(s).)

Bio-processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten metagenomes and retrieved 48 genes coding for deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence-based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA-61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA-61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non-natural substrates, compared to well-known DERAs., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

(2 S ,3 S )-4-Fluorovaline (FVal) is an analogue of valine, where a single CH 3 group is substituted by a CH 2 F group. In the absence of valine, E. coli valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of E. coli peptidyl-prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a 13 C-labeled CH 2 F group. Although the melting temperature is lower by about 11 °C relative to the wild-type protein, the three-dimensional protein structure is almost completely conserved, as shown by X-ray crystallography. The CH 2 F groups invariably populate staggered rotamers. Most CH 2 F groups populate two different rotamers. The increased space requirement of fluorine versus hydrogen does not prohibit rotamers that position fluorine next to a backbone carbonyl carbon. 19 F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted 19 F resonances correlate with large 3 J HF coupling constants, confirming the impact of the γ- gauche effect on the signal dispersion. The present work is the second experimental verification of the effect and extends its validity to fluorovaline. The abundance of valine in proteins and structural conservation with FVal renders this valine analogue attractive for probing proteins by 19 F NMR spectroscopy.

We report identification of 5 patients with infections caused by NDM-5-producing Escherichia coli harboring PBP3 mutations that showed reduced susceptibility to aztreonam-avibactam and cefiderocol. Durlobactam, a novel diazabicyclooctane β-lactamase inhibitor, demonstrated minimum inhibitory concentrations ranging from 0.5 to 2 µg/mL supporting future investigations into a potential role in clinical management., Competing Interests: Potential conflicts of interest. S. L. A. reports serving on advisory boards for Entasis, Melinta, Shionogi, and Basilea and receiving consulting fees from GlaxoSmithKline. J. M. P. reports grant support from Merck and serving as a consultant for Merck, Shionogi, Entasis, Abbvie, and GSK. R. K. S. has served as a consultant for Merck, Melinta, Entasis, Abbvie, GlaxoSmithKline, Pfizer, Shionogi, Venatorx, and Roche, and has received investigator-initiated research funding from Merck, Melinta, Shionogi, Roche, and Venatorx. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Introduction: Extended-spectrum β-lactamase (ESBL)-producing pathogenic E. coli is a global public health issue, especially in sub-Saharan African countries such as Cameroon. It contributes to increase significantly hospital length of stay, morbidity, mortality and economic costs because of treatment failures. This study aims at determining the resistance background and virulence profiles of ESBL-E. coli isolates among childhood diarrhoea during the cholera outbreak occuring in Yaoundé, Cameroon., Materials and Methods: During a four-month periods, from March 1st to June 30th, 2023,  a total of 84 stool samples were collected from 90 under five children presenting clinical signs of gastroenteritis and attending four hospitals in Yaoundé, Cameroon. Bacterial identification was done using API20E and antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. After extraction, genomic DNA was subjected to conventional and multiplex polymerase chain reaction methods (PCRs) for detection of resistance and virulence genes. Statistical analysis was performed using Epi info™ (7.2.5.0). Statistical significance was considered at a p-value < 0.05., Results: Out of 150 patients contacted, 90 patients were enrolled, 84 samples were collected, 52.38%(44/84) and 3.57%(03/84) were confirmed as extended-spectrum β-lactamase and carbapenemase-producing E. coli respectively. The risk factors were analyzed, and children who drank natural fruit juice (OR: 0.4, p-value: 0.03) were found to be significantly associated with ESBL-producing E. coli. The ESBL-producing E. coli isolates showed a high level of resistance to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefepime, colistin, and tetracycline. The bla CTX-M was more prevalent ß-lactamase resistance gene. The tetracycline resistance genes tet(A) and tet(B) were also detected. The most important virulence genes detected were FimH (81.81%) and papA (79.54%)., Conclusion: These findings suggest implementing routine surveillance and screening for antimicrobial resistance among children under five. Antimicrobial stewardship strategies (ASP) need to be implemented to curb the emergence and dissemination of ESBL-producing E. coli. In addition, a national surveillance program for antimicrobial resistance needs to be implemented at local and regional levels in order to reduce morbidity in Cameroon., (© 2024. The Author(s).)

The preparation of pure metabolites of bioactive compounds, particularly (poly)phenols, is essential for the accurate determination of their pharmacological profiles in vivo . Since the extraction of these metabolites from biological material is tedious and impractical, they can be synthesized enzymatically in vitro by bacterial PAPS-independent aryl sulfotransferases (ASTs). However, only a few ASTs have been studied and used for (poly)phenol sulfation. This study introduces new fully characterized recombinant ASTs selected according to their similarity to the previously characterized ASTs. These enzymes, produced in Escherichia coli , were purified, biochemically characterized, and screened for the sulfation of nine flavonoids and two phenolic acids using p- nitrophenyl sulfate. All tested compounds were proved to be substrates for the new ASTs, with kaempferol and luteolin being the best converted acceptors. ASTs from Desulfofalx alkaliphile ( Dal AST) and Campylobacter fetus ( Cf AST) showed the highest efficiency in the sulfation of tested polyphenols. To demonstrate the efficiency of the present sulfation approach, a series of new authentic metabolite standards, regioisomers of kaempferol sulfate, were enzymatically produced, isolated, and structurally characterized.

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s -1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered., Competing Interests: JB, AL consultant for Nitrase Therapeutics, LI No competing interests declared, (© 2024, Banwait et al.)

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli pose a serious threat to human health because of their resistance to the most commonly prescribed antibiotics: penicillins and cephalosporins. In this study, we provide a genomic and metagenomic context for the determinant beta-lactam resistance genes of ESBL-positive E. coli isolated from various wastewater treatment utilities in Oregon, USA. Class A beta-lactamase genes on chromosomes ( bla CTX-M, bla TEM) were clustered with antibiotic resistance genes associated with other classes of antibiotics (sulfonamides and aminoglycosides) along with insertional elements. ESBL genes such as bla CTX-M, bla TEM, and bla SHV were also detected on conjugable plasmids of IncF and IncI incompatibility types. One novel IncF plasmid (pSHV2A&#95;ESBLF) was identified, which carried a multidrug resistance genotype ( bla SHV-2A, aadA22 , aac 3, aph 6, tet A, and sul 1) in addition to a mer (mercury resistance) operon, colicin, and aerobactin genes. Shotgun metagenomic analysis of the ESBL-producing E. coli -originating wastewater samples showed the presence of class A beta-lactamases; however, the ESBL genes identified in the E. coli genomes were below the detection limits. Other ESBL-associated genes (i.e., bla OXA.11, bla FOX.7, and bla GES.17) were identified in the wastewater samples, and their occurrences were correlated with the core microbial genera (e.g., Paraprevotella ). In the E. coli genomes and wastewater samples, tetracycline, aminoglycoside, and beta-lactam resistance determinants frequently co-occurred. The combination of whole-genome and metagenomic analysis provides a holistic description of ESBL-producing organisms and genes in wastewater systems.IMPORTANCEUsing a hybrid sequencing and assembly strategy (short- and long-read sequencing), we identified the distribution of ARGs and virulence factors harbored on plasmids and chromosomes. We further characterized plasmids' incompatibility types and the co-occurrences of ARGs and virulence factors on plasmids and chromosomes. We investigated the transferability of plasmid-mediated beta-lactams via conjugation. Finally, using shotgun metagenomic analysis of the ESBL-producing Escherichia coli -originated wastewater samples, we described the microbial community, the resistome composition, and the potential associations with plasmid-mediated beta-lactam genes and other ARGs., Competing Interests: The authors declare no conflict of interest.

Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a ] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b ]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase ( Rp EH), obtaining mutant Escherichia coli / Rpeh H336W/L360F with a 6.4-fold enhanced enantiomeric ratio ( E ) from 5.5 to 35.4. This enabled a gram-scale resolution of rac - 1a by E. coli / Rpeh H336W/L360F , producing ( S )- 1a (98.2% ee s ) and ( R )- 1b (75.0% ee p ) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral ( S )-flutriafol (98.2% ee ) and ( R )-flutriafol (75.0% ee ) were easily synthesized by a one-step chemocatalytic process from ( S )- 1a and a two-step chemocatalytic process from ( R )- 1b , respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of Rp EH toward bulky 2,2-bisaryl-substituted oxirane 1a .

L-lactate modifies proteins through lactylation 1 , but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular L-lactate sensors required for L-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to L-lactate with micromolar affinity and they directly catalyse L-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to L-lactate, AARS2 associates with cyclic GMP-AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of L-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular L-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination, and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the Escherichia coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and dsDNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and ssDNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

β-nicotinamide mononucleotide (β-NMN) is a key precursor of nicotinamide adenine dinucleotide, and becomes attractive in the nutrition and health care fields, but its enzymatic synthesis is expensive. In this study, a six-enzyme cascade catalytic system was constructed to produce β-NMN. Using D-ribose and nicotinamide as substrates, the β-NMN yield reached 97.5 % catalyzed by purified enzymes. Then, after knocking out the genes encoding proteins that consume β-NMN in E. coli BL21(DE3), the similar β-NMN yield, 97.2 %, using the crude enzymes could be also obtained. After that, β-NMN synthesis was performed under increased substrate concentration, and 'modular' crude enzymes cascade catalytic reaction system was proposed to reduce the inhibition of polyphosphate on ribose-phosphate diphosphokinase activity, and the β-NMN yield reached 78.4 % at 10 mM D-ribose, which is 1.82 times of that in 'one-pot' reaction and represents the highest β-NMN preparation level with phosphoribosylpyrophosphate as the core reported till now., (Copyright © 2024 Elsevier Inc. All rights reserved.)

(S)-equol, the most influential metabolite of daidzein in vivo, has aroused great attention due to the excellent biological activities. Although existing studies have accomplished the construction of its heterologous synthetic pathway in the context of anaerobicity and inefficiency of natural strains, the low productivity of (S)-equol limits its industrial application. Here, rational design strategies based on decreasing the pocket steric hindrance and fine-tuning the pocket microenvironment to systematically redesign the binding pocket of enzyme were developed and processed to the rate-limiting enzyme dihydrodaidzein reductase in (S)-equol synthesis. After iterative combinatorial mutagenesis, an effective mutant S118G/T169A capable of significantly increasing (S)-equol yield was obtained. Computational analyses illustrated that the main reason of the increased activity relied on the decreased critical distance and more stable interacting conformation. Then, the reaction optimization was performed, and the recombinant Escherichia coli whole-cell biocatalyst harboring S118G/T169A enabled the efficient conversion of 2 mM daidzein to (S)-equol, achieving conversion rate of 84.5 %, which was 2.9 times higher than that of the parental strain expressing wide type dihydrodaidzein reductase. This study provides an effective idea and a feasible method for enzyme modification and whole-cell catalytic synthesis of (S)-equol, and will greatly accelerate the process of industrial production., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Perillyl alcohol (POH) is a secondary metabolite of plants. POH and its derivatives are known to be effective as an anticancer treatment. In this study, oxidative derivatives of POH, which are difficult to synthesize chemically, were synthesized using the engineered bacterial cytochrome P450 BM3 (CYP102A1) as a biocatalyst. The activity of wild-type (WT) CYP102A1 and 29 engineered enzymes toward POH was screened using a high-performance liquid chromatography. They produced one major product. Among them, the engineered CYP102A1 M601 mutant with seven mutations (R47L/F81I/F87V/E143G/L150F/L188Q/E267V) showed the highest conversion, 6.4-fold higher than the WT. Structure modeling using AlphFold2 and PyMoL suggests that mutations near the water channel may be responsible for the increased catalytic activity of the M601 mutant. The major product was identified as a POH-8,9-epoxide by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis. The optimal temperature and pH for the product formation were 35 °C and pH 7.4, respectively. The k cat and K m of M601 were 540 min -1 and 2.77 mM, respectively. To improve POH-8,9-epoxide production, substrate concentration and reaction time were optimized. The optimal condition for POH-8,9-epoxide production by M601 was 5.0 mM POH, pH 7.4, 35 ℃, and 6 h reaction, which produced the highest concentration of 1.72 mM. Therefore, the biosynthesis of POH-8,9-epoxide using M601 as a biocatalyst is suggested to be an efficient and sustainable synthetic process that can be applied to chemical and pharmaceutical industries., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The overuse of antimicrobials in livestock has contributed to the emergence and selection of clinically relevant multidrug-resistant bacteria. In Brazil, there is no conclusive information on the occurrence of Escherichia coli producing extended-spectrum β-lactamase (ESβL) in cattle breeding, which is an important sector of agribusiness in this country. Herein, we investigated the presence of ESβL-positive E. coli strains in dairy cattle from a commercial farm with routine practice of therapeutic cephalosporins. Ninety-five rectal swab samples were collected from healthy dairy calves and cows under treatment with ceftiofur. Samples were screened for the presence of ESβL producers, and positive isolates were identified by MALDI-TOF, with subsequent screening for genes encoding ESβL variants by PCR and sequencing. The presence of ESβL (CTX-M-15)-producing E. coli was confirmed in calves, and lactating and dry cows. Most ESβL strains with genetic homologies ≥ 90% were grouped into two major PFGE clusters, confirming the suscessful expansion of clonally related lineages in animals from different lactating cycles, on the same property. Four representatives CTX-M-15-positive E. coli strains had their genomes sequenced, belonging to the clonal complex (CC) 23 and sequence type (ST) 90. A phylogeographical landscape of ST90 was performed revealing a global One Health linkage. Our results highlight the intestinal microbiota of dairy cattle as a hotspot for the spread of critical priority ESβL-producing E. coli and demonstrate that ST90 is an international clone genomically adapted to human and animal hosts, which deserve additional investigation to determine its zoonotic potential and impact in food chain., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl-l-tyrosine (DDT) inhibits both Escherichia coli TS (EcTS) and Lactobacillus casei TS (LcTS). The available X-ray structure of the DDT:dUMP:EcTS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to EcTS and LcTS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β 2 and α 2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β 2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β 2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b 558 , b 595 and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d 2+ in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d 2+ α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (K d ) of the cyanide complex of heme d 2+ is ∼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d 2+ but not to b 558 2+ or b 595 2+ . This agrees with the published structural data that the enzyme's active site is not a di-heme site. The observed rate of binding (k obs ) increases as the concentration of cyanide is increased, giving a second-order rate constant (k on ) of ∼0.1 M -1  s -1 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The alarming rise in the rate of antibiotic resistance is a matter of significant concern. DNA gyrase B (GyrB), a critical bacterial enzyme involved in DNA replication, transcription, and recombination, has emerged as a promising target for antibacterial agents. Inhibition of GyrB disrupts bacterial DNA replication, leading to cell death, making it an attractive candidate for antibiotic development. Although several classes of antibiotics targeting GyrB are currently in clinical use, the emergence of antibiotic resistance necessitates the exploration of novel inhibitors. In this study, we aimed to identify potential Escherichia coli GyrB inhibitors from a database of phytoconstituents sourced from Indian medicinal plants. Utilizing virtual screening, we performed a rigorous search to identify compounds with the most promising inhibitory properties against GyrB. Two compounds, namely Zizogenin and Cucurbitacin S, were identified based on their favorable drug likeliness and pharmacokinetic profiles. Employing advanced computational techniques, we analyzed the binding interactions of Zizogenin and Cucurbitacin S with the ATP-binding site of GyrB through molecular docking simulations. Both compounds exhibited robust binding interactions, evidenced by their high docking energy scores. To assess the stability of these interactions, we conducted extensive 100 ns molecular dynamics (MD) simulations, which confirmed the stability of Zizogenin and Cucurbitacin S when bound to GyrB. In conclusion, our study highlights Zizogenin and Cucurbitacin S as promising candidates for potential antibacterial agents targeting GyrB. Experimental validation of these compounds is warranted to further explore their efficacy and potential as novel antibiotics to combat antibiotic-resistant bacteria.Communicated by Ramaswamy H. Sarma.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race 1-5 . While the mechanisms of many bacterial antiphage defence systems are known 1 , how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Here, we report a novel endonuclease and N 6 -adenine DNA methyltransferase (m 6 A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3&#95;c0261 and UP3&#95;c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3&#95;c0261 and UP3&#95;c0262 genes were individually expressed and purified in Escherichia coli. The UP3&#95;c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5' extension. The UP3&#95;c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3&#95;c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3&#95;c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3&#95;c0262 with HPLC-MS/MS and MS/MS showed that UP3&#95;c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3&#95;c0261 and UP3&#95;c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The widespread use of non-naturally degradable plastics is causing increasingly serious harm to the environment. Reducing plastic pollutants has become the core of ecological and environment management. Biological methods such as enzymes demonstrate advantages in depolymerizing plastics with mild reaction conditions and recycling of depolymerization products. However, there are few reports on the biological depolymerization of polyamide plastics. In this study, by using 4-nitropropionanilide as the model substrate, we screened against our plastic depolymerase library and obtained a Meiothermus ruber- derived enzyme (MrABH) that can hydrolyze the polyamide bond. We expressed this enzyme in Escherichia coli and purified the protein by affinity chromatography. Furthermore, we investigated the catalytic properties, enzymatic properties, and catalytic products of this enzyme with polyamide as the substrate. MrABH had good stability at pH 8.0-10.0, with the optimal performance at pH 9.0 and 30 ℃. The catalytic performance of this enzyme for ester bonds and amide bonds was similar. MrABH can catalyze the depolymerization of PA6 and PA66 to produce monomers and oligomers, demonstrating the potential to be used in the depolymerization and recycling of polyamide.

Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C-O cleavage. This "anhydroglycolysis" pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.

Cerium photoredox catalysis has emerged as a powerful strategy to activate molecules under mild conditions. Radical intermediates are formed using visible light and simple complexes of the earth-abundant lanthanide. Here, we report an artificial photoenzyme enabling this chemistry inside a protein. We utilize a de novo designed protein scaffold that tightly binds lanthanide ions in its central cavity. Upon visible-light irradiation, the cerium-dependent enzyme catalyzes the radical C-C bond cleavage of 1,2-diols in aqueous solution. Protein engineering led to variants with improved photostability and metal binding behavior. The photoenzyme cleaves a range of aromatic and aliphatic substrates, including lignin surrogates. Surface display of the protein scaffold on Escherichia coli facilitates whole-cell photobiocatalysis. Furthermore, we show that also natural lanthanide-binding proteins are suitable for this approach. Our study thus demonstrates a new-to-nature enzymatic photoredox activity with broad catalytic potential.

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this study, we present an extensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Urinary tract infections (UTIs) are the second most common infection, affecting 150 million people each year worldwide. Enterobacteriaceae species expressing extended-spectrum β-lactamases (ESBLs) are on the rise across the globe and are becoming a severe problem in the therapeutic management of clinical cases of urinary tract infection. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. This study aimed to investigate the prevalence of Enterobacteriaceae isolates exhibiting ESBL and their selective oral antibiogram profile at the district general hospital, Polonnaruwa., Results: A total of 4386 urine specimens received to the Microbiology Laboratory during the study period. Among them, 1081 (24.6%) showed positive results for urine culture while 200/1081 specimens showed ESBL isolates. Out of the selected 200 specimen's majority (67.5%) of samples received from the In-Patient Department. There were 200 patients and reported that 115 (57.5%) were females and 85 (42.5%) were males. The majority (51%) of the patients belong to the age group of 55-74 years. Among the ESBLs positive specimens, the majority 74.5% (n = 149) identified organisms were E. coli followed by Klebsiella spp.17.5% (n = 35), Enterobacteriaceae 7% (n = 14) and only1% (n = 2) isolate of Proteus spp. Mecillinam (87.92%) and Nitrofurantoin (83.2%) showed higher effectiveness against E. coli. Nitrofurantoin showed the highest effectiveness against Klebsiella spp. (40%), other Enterobacteriaceae spp. (100%). Proteus spp. showed 100% effectiveness and resistance respectively against Ciprofloxacin, Cotrimoxazole and Nitrofurantoin., Conclusion: The most predominant ESBLs producing uro-pathogen was the E. coli in the study setting and E. coli had higher sensitivity rate against Mecillinam. Among currently used oral antibiotics Nitrofurantoin was the best choice for UTIs caused by ESBL producers., (© 2024. The Author(s).)

Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency ( k cat /K m ), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP + ) with nicotinamide adenine dinucleotide (NAD + ) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L -1 d -1 . This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis., Importance: Hydroxysteroid dehydrogenases have emerged as indispensable tools in the synthesis of steroids, bile acids, and other steroid derivatives for the pharmaceutical and chemical industries. In this study, a novel approach was developed to simultaneously improve the stability and activity of a hydroxysteroid dehydrogenase by combining B-factor analysis and computer-aided prediction. This semi-rational method was demonstrated to be highly effective for enzyme engineering. In addition, NAD kinase was introduced to convert NAD + to NADP + for effective coenzyme regeneration in the whole-cell multienzyme-catalyzed system. This strategy reduces the significant economic costs associated with externally supplemented cofactors in NADP-dependent biosynthetic pathways., Competing Interests: The authors declare no conflict of interest.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli . This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H ( Ec FDH-H). Using an Ec FDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active Ec FDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using Ec FDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 Ec FDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO 2 and N 2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications., Competing Interests: The authors declare no conflict of interest.

This research explores the antimicrobial resistance (AMR) profiles and prevalence of extended-spectrum beta-lactamase (ESBL) and non-ESBL-producing Escherichia coli in Ojerame Dam and Ovokoto Spring, Edo State, Nigeria. Over 12 months, water was systematically sampled to accommodate seasonal variations and analyzed by employing an ESBL-selective medium for bacterial species. Additionally, bacterial isolates underwent identification and characterization using polymerase chain reaction (PCR) and disk diffusion methods to evaluate their susceptibility to antimicrobials. Results indicated significant prevalence of ESBL-producing E. coli, which exhibited complete resistance to common antimicrobials like ceftriaxone, ceftazidime, cefotaxime, and ampicillin while demonstrating 100% sensitivity to ertapenem, imipenem, meropenem, and nitrofurantoin. Non-ESBL-producing E. coli were resistant to ampicillin but sensitive to other antimicrobials mentioned earlier. Furthermore, both ESBL and non-ESBL-producing E. coli displayed multidrug resistance to varying degrees. Specific ESBL genes, including bla TEM , bla CTX-M-1 , and bla CTX-M-15 , were identified, alongside resistance genes like tetA, tetM, sul1, sul2, sul3, qnrA, qnrB, and qnrS in E. coli. This study pioneers the documentation of ESBL-producing E. coli in surface water in the region. This signals impending health risks associated with water being a reservoir of resistant genes while emphasizing the urgency for further research and public awareness concerning the quality of surface water., (© 2024. The Author(s).)

Background: Extended-spectrum β-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire., Results: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum β-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen., Conclusions: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses., (© 2024. The Author(s).)

A series of arylsulfones and heteroarylsulfones have previously been demonstrated to dysregulate the conserved bacterial ClpP protease, causing the unspecific degradation of essential cellular housekeeping proteins and ultimately resulting in cell death. A cocrystal structure of a 2-β-sulfonylamide analog, ACP1-06, with Escherichia coli ClpP showed that its 2-pyridyl sulfonyl substituent adopts two orientations in the binding site related through a sulfone bond rotation. From this, a new bis -aryl phosphine oxide scaffold, designated as ACP6, was designed based on a "conformation merging" approach of the dual orientation of the ACP1-06 sulfone. One analog, ACP6-12, exhibited over a 10-fold increase in activity over the parent ACP1-06 compound, and a cocrystal X-ray structure with ClpP confirmed its predicted binding conformation. This allowed for a comparative analysis of how different ligand classes bind to the hydrophobic binding site. The study highlights the successful application of structure-based rational design of novel phosphine oxide-based antibiotics.