Lipophilic quinolone derivatives: Synthesis and in vitro antibacterial evaluation.

This paper reports on the design of a series of 10 novel lipophilic piperazinyl derivatives of the 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, their synthesis, their characterisation by 1H, 13C and 19F NMR, IR spectroscopy and HRMS, as well as their biological activity against bacteria of medical interest. Among these derivatives, 2 were as potent as the parent quinolone against Neisseriagonorrhoeae whereas all the compounds displayed lower activity than the parent quinolone against other bacteria of medical interest. Our results showing that the increased lipophilicity was deleterious for antibacterial activity may help to design new quinolone derivatives in the future, especially lipophilic quinolones which have been poorly investigated previously.

The BCC7 Protein Contributes to the Toxoplasma Basal Pole by Interfacing between the MyoC Motor and the IMC Membrane Network.

T. gondii is a eukaryotic parasite that has evolved a stage called tachyzoite which multiplies in host cells by producing two daughter cells internally. These nascent tachyzoites bud off their mother and repeat the division process until the expanding progenies escape to settle and multiply in other host cells. Over these intra- and extra-cellular phases, the tachyzoite maintains an essential apicobasal polarity that emerges through a unique bidirectional budding process of the elongating cells. This process requires the assembly of several molecular complexes that, at the nascent pole, encompass structural and myosin motor elements. To characterize a recently identified basal pole marker named BCC7 with respect to the posterior myosin J and myosin C motors, we used conventional biochemistry as well as advanced proteomic and in silico analysis in conjunction with live and super resolution microscopy of transgenic fluorescent tachyzoites. We document that BCC7 forms a ribbed ring below which myosin C motor entities distribute regularly. In addition, we identified-among 13 BCC7 putative partners-two novel and five known members of the inner membrane complex (IMC) family which ends at the apical side of the ring. Therefore, BCC7 could assist the stabilization of the IMC plaques and contribute to the parasite biomechanical properties.

Circular code motifs in the ribosome: a missing link in the evolution of translation?

The origin of the genetic code remains enigmatic five decades after it was elucidated, although there is growing evidence that the code coevolved progressively with the ribosome. A number of primordial codes were proposed as ancestors of the modern genetic code, including comma-free codes such as the RRY, RNY, or GNC codes (R = G or A, Y = C or T, N = any nucleotide), and the X circular code, an error-correcting code that also allows identification and maintenance of the reading frame. It was demonstrated previously that motifs of the X circular code are significantly enriched in the protein-coding genes of most organisms, from bacteria to eukaryotes. Here, we show that imprints of this code also exist in the ribosomal RNA (rRNA). In a large-scale study involving 133 organisms representative of the three domains of life, we identified 32 universal X motifs that are conserved in the rRNA of >90% of the organisms. Intriguingly, most of the universal X motifs are located in rRNA regions involved in important ribosome functions, notably in the peptidyl transferase center and the decoding center that form the original "proto-ribosome." Building on the existing accretion models for ribosome evolution, we propose that error-correcting circular codes represented an important step in the emergence of the modern genetic code. Thus, circular codes would have allowed the simultaneous coding of amino acids and synchronization of the reading frame in primitive translation systems, prior to the emergence of more sophisticated start codon recognition and translation initiation mechanisms.

Characterization of accessory genes in coronavirus genomes.

BACKGROUND: The Covid19 infection is caused by the SARS-CoV-2 virus, a novel member of the coronavirus (CoV) family. CoV genomes code for a ORF1a / ORF1ab polyprotein and four structural proteins widely studied as major drug targets. The genomes also contain a variable number of open reading frames (ORFs) coding for accessory proteins that are not essential for virus replication, but appear to have a role in pathogenesis. The accessory proteins have been less well characterized and are difficult to predict by classical bioinformatics methods. METHODS: We propose a computational tool GOFIX to characterize potential ORFs in virus genomes. In particular, ORF coding potential is estimated by searching for enrichment in motifs of the X circular code, that is known to be over-represented in the reading frames of viral genes. RESULTS: We applied GOFIX to study the SARS-CoV-2 and related genomes including SARS-CoV and SARS-like viruses from bat, civet and pangolin hosts, focusing on the accessory proteins. Our analysis provides evidence supporting the presence of overlapping ORFs 7b, 9b and 9c in all the genomes and thus helps to resolve some differences in current genome annotations. In contrast, we predict that ORF3b is not functional in all genomes. Novel putative ORFs were also predicted, including a truncated form of the ORF10 previously identified in SARS-CoV-2 and a little known ORF overlapping the Spike protein in Civet-CoV and SARS-CoV. CONCLUSIONS: Our findings contribute to characterizing sequence properties of accessory genes of SARS coronaviruses, and especially the newly acquired genes making use of overlapping reading frames.

The stress sigma factor of RNA polymerase RpoS/σS is a solvent-exposed open molecule in solution.

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.

Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC50) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.

Description of compensatory gyrA mutations restoring fluoroquinolone susceptibility in Mycobacterium tuberculosis.

OBJECTIVES: Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility. METHODS: Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied. RESULTS: IC50s of mutant gyrase complexes bearing GyrA D94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s. CONCLUSIONS: We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria.

Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.

Structural and functional features of Crl proteins and identification of conserved surface residues required for interaction with the RpoS/σS subunit of RNA polymerase.

In many γ-proteobacteria, the RpoS/σS sigma factor associates with the core RNAP (RNA polymerase) to modify global gene transcription in stationary phase and under stress conditions. The small regulatory protein Crl stimulates the association of σS with the core RNAP in Escherichia coli and Salmonella enterica serovar Typhimurium, through direct and specific interaction with σS. The structural determinants of Crl involved in σS binding are unknown. In the present paper we report the X-ray crystal structure of the Proteus mirabilis Crl protein (CrlPM) and a structural model for Salmonella Typhimurium Crl (CrlSTM). Using a combination of in vivo and in vitro assays, we demonstrated that CrlSTM and CrlPM are structurally similar and perform the same biological function. In the Crl structure, a cavity enclosed by flexible arms contains two patches of conserved and exposed residues required for σS binding. Among these, charged residues that are likely to be involved in electrostatic interactions driving Crl-σS complex formation were identified. CrlSTM and CrlPM interact with domain 2 of σS with the same binding properties as with full-length σS. These results suggest that Crl family members share a common mechanism of σS binding in which the flexible arms of Crl might play a dynamic role.

Mycobacterium tuberculosis DNA gyrase ATPase domain structures suggest a dissociative mechanism that explains how ATP hydrolysis is coupled to domain motion.

DNA gyrase, a type II topoisomerase, regulates DNA topology by creating a double-stranded break in one DNA duplex and transporting another DNA duplex [T-DNA (transported DNA)] through this break. The ATPase domains dimerize, in the presence of ATP, to trap the T-DNA segment. Hydrolysis of only one of the two ATPs, and release of the resulting Pi, is rate-limiting in DNA strand passage. A long unresolved puzzle is how the non-hydrolysable ATP analogue AMP-PNP (adenosine 5'-[ß,γ-imido]triphosphate) can catalyse one round of DNA strand passage without Pi release. In the present paper we discuss two crystal structures of the Mycobacterium tuberculosis DNA gyrase ATPase domain: one complexed with AMP-PCP (adenosine 5'-[ß,γ-methylene]triphosphate) was unexpectedly monomeric, the other, an AMP-PNP complex, crystallized as a dimer. In the AMP-PNP structure, the unprotonated nitrogen (P-N=P imino) accepts hydrogen bonds from a well-ordered 'ATP lid', which is known to be required for dimerization. The equivalent CH2 group, in AMP-PCP, cannot accept hydrogen bonds, leaving the 'ATP lid' region disordered. Further analysis suggested that AMP-PNP can be converted from the imino (P-N=P) form into the imido form (P-NH-P) during the catalytic cycle. A main-chain NH is proposed to move to either protonate AMP-P-N=P to AMP-P-NH-P, or to protonate ATP to initiate ATP hydrolysis. This suggests a novel dissociative mechanism for ATP hydrolysis that could be applicable not only to GHKL phosphotransferases, but also to unrelated ATPases and GTPases such as Ras. On the basis of the domain orientation in our AMP-PCP structure we propose a mechanochemical scheme to explain how ATP hydrolysis is coupled to domain motion.

Mycobacterium tuberculosis DNA gyrase possesses two functional GyrA-boxes.

In contrast with most bacteria which possess two type II topoisomerases (topoisomerase IV and DNA gyrase), Mycobacterium tuberculosis possesses only one, DNA gyrase, which is functionally a hybrid enzyme. Functional differences between the two type IIA topoisomerases are thought to be specified by a CTD (C-terminal DNA-binding domain), which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. tuberculosis DNA gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. Although the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogues, it harbours a second key motif similar in all respects to that of the conserved GyrA-box sequence motif. Biochemical assays showed that the GyrA-box is responsible for DNA supercoiling, whereas the second GyrA-box-l (GyrA-box-like motif) is responsible for the enhanced decatenation activity, suggesting that the mechanistic originality of M. tuberculosis DNA gyrase depends largely on the particular DNA path around the CTD allowed for by the presence of GyrA-box-l. The results of the present study also provide, through phylogenetic exploration of the entire Corynebacterineae suborder, a new and broader insight into the functional diversity of bacterial type IIA topoisomerases.

Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase ATPase domain.

Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47(C1) and Mtb-GyrB47(C2), have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P21 and diffracted to resolutions of 2.9 and 3.3 Å, respectively.

CeGAL: Redefining a Widespread Fungal-Specific Transcription Factor Family Using an In Silico Error-Tracking Approach.

In fungi, the most abundant transcription factor (TF) class contains a fungal-specific 'GAL4-like' Zn2C6 DNA binding domain (DBD), while the second class contains another fungal-specific domain, known as 'fungal_trans' or middle homology domain (MHD), whose function remains largely uncharacterized. Remarkably, almost a third of MHD-containing TFs in public sequence databases apparently lack DNA binding activity, since they are not predicted to contain a DBD. Here, we reassess the domain organization of these 'MHD-only' proteins using an in silico error-tracking approach. In a large-scale analysis of ~17,000 MHD-only TF sequences present in all fungal phyla except Microsporidia and Cryptomycota, we show that the vast majority (>90%) result from genome annotation errors and we are able to predict a new DBD sequence for 14,261 of them. Most of these sequences correspond to a Zn2C6 domain (82%), with a small proportion of C2H2 domains (4%) found only in Dikarya. Our results contradict previous findings that the MHD-only TF are widespread in fungi. In contrast, we show that they are exceptional cases, and that the fungal-specific Zn2C6-MHD domain pair represents the canonical domain signature defining the most predominant fungal TF family. We call this family CeGAL, after the highly characterized members: Cep3, whose 3D structure is determined, and GAL4, a eukaryotic TF archetype. We believe that this will not only improve the annotation and classification of the Zn2C6 TF but will also provide critical guidance for future fungal gene regulatory network analyses.

Extending the definition of the GyrB quinolone resistance-determining region in Mycobacterium tuberculosis DNA gyrase for assessing fluoroquinolone resistance in M. tuberculosis.

Fluoroquinolone (FQ) resistance is emerging in Mycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target in M. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified in M. tuberculosis clinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system.

Fluoroquinolone resistance in Mycobacterium tuberculosis has become increasingly important. A review of mutations in DNA gyrase, the fluoroquinolone target, is needed to improve the molecular detection of resistance. We performed a systematic review of studies reporting mutations in DNA gyrase genes in clinical M. tuberculosis isolates. From 42 studies that met inclusion criteria, 1220 fluoroquinolone-resistant M. tuberculosis isolates underwent sequencing of the quinolone resistance-determining region (QRDR) of gyrA; 780 (64%) had mutations. The QRDR of gyrB was sequenced in 534 resistant isolates; 17 (3%) had mutations. Mutations at gyrA codons 90, 91 or 94 were present in 654/1220 (54%) resistant isolates. Four different GyrB numbering systems were reported, resulting in mutation location discrepancies. We propose a consensus numbering system. Most fluoroquinolone-resistant M. tuberculosis isolates had mutations in DNA gyrase, but a substantial proportion did not. The proposed consensus numbering system can improve molecular detection of resistance and identification of novel mutations.

Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase CTD.

Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolone in the treatment of tuberculosis. The C-terminal domain (CTD) of the DNA gyrase A subunit possesses a unique feature, the ability to wrap DNA in a chiral manner, that plays an essential role during the catalytic cycle. A construct of 36 kDa corresponding to this domain has been overproduced, purified and crystallized. Diffraction data were collected to 1.55 Å resolution. Cleavage of the N-terminal His tag was crucial for obtaining crystals. The crystals belonged to space group P2(1)2(1)2(1), with one molecule in the asymmetric unit and a low solvent content (33%). This is the first report of the crystallization and preliminary X-ray diffraction studies of a DNA gyrase CTD from a species that contains one unique type II topoisomerase.

Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states.

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

DNA gyrase inhibition assays are necessary to demonstrate fluoroquinolone resistance secondary to gyrB mutations in Mycobacterium tuberculosis.

The main mechanism of fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis is mutation in DNA gyrase (GyrA(2)GyrB(2)), especially in gyrA. However, the discovery of unknown mutations in gyrB whose implication in FQ resistance is unclear has become more frequent. We investigated the impact on FQ susceptibility of eight gyrB mutations in M. tuberculosis clinical strains, three of which were previously identified in an FQ-resistant strain. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. We demonstrated that the eight substitutions in GyrB (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A), recently identified in FQ-resistant clinical strains or encountered in M. tuberculosis strains isolated in France, are not implicated in FQ resistance. These results underline that, as opposed to phenotypic FQ susceptibility testing, the DNA gyrase inhibition assay is the only way to prove the role of a DNA gyrase mutation in FQ resistance. Therefore, the use of FQ in the treatment of tuberculosis (TB) patients should not be ruled out only on the basis of the presence of mutations in gyrB.

Conformational transition of DNA bound to Hfq probed by infrared spectroscopy.

Hfq is a bacterial protein involved in RNA metabolism. Besides this, Hfq's role in DNA restructuring has also been suggested. Since this mechanism remains unclear, we examined the DNA conformation upon Hfq binding by combining vibrational spectroscopy and neutron scattering. Our analysis reveals that Hfq, which preferentially interacts with deoxyadenosine rich sequences, induces partial opening of dA-dT sequences accompanied by sugar repuckering of the dA strand and hence results in a heteronomous A/B duplex. Sugar repuckering is probably correlated with a global dehydration of the complex. By taking into account Hfq's preferential binding to A-tracts, which are commonly found in promoters, potential biological implications of Hfq binding to DNA are discussed.