RESUMO
For millennia, humanity has relied on plants for its medicines, and modern pharmacology continues to reexamine and mine plant metabolites for novel compounds and to guide improvements in biological activity, bioavailability, and chemical stability. The critical problem of antibiotic resistance and increasing exposure to viral and parasitic diseases has spurred renewed interest into drug treatments for infectious diseases. In this context, an urgent revival of natural product discovery is globally underway with special attention directed toward the numerous and chemically diverse plant defensive compounds such as phytoalexins and phytoanticipins that combat herbivores, microbial pathogens, or competing plants. Moreover, advancements in "omics," chemistry, and heterologous expression systems have facilitated the purification and characterization of plant metabolites and the identification of possible therapeutic targets. In this review, we describe several important amino acid-derived classes of plant defensive compounds, including antimicrobial peptides (e.g., defensins, thionins, and knottins), alkaloids, nonproteogenic amino acids, and phenylpropanoids as potential drug leads, examining their mechanisms of action, therapeutic targets, and structure-function relationships. Given their potent antibacterial, antifungal, antiparasitic, and antiviral properties, which can be superior to existing drugs, phytoalexins and phytoanticipins are an excellent resource to facilitate the rational design and development of antimicrobial drugs.
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Aminoácidos/metabolismo , Anti-Infecciosos/farmacologia , Plantas/metabolismo , Desenvolvimento de Medicamentos , Compostos Fitoquímicos/farmacologiaRESUMO
While humans lack the biosynthetic pathways for meso-diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum (VsDapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso-diaminopimelate. Kinetic studies show that VsDapL follows a Michaelis-Menten mechanism, with a KMapp of 4.0 mM toward its substrate l,l-diaminopimelate. The kcat (0.46 s-1) and the kcat/KM (115 s-1 M-1) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that VsDapL exists in a monomer-dimer self-association, with a KD2-1 of 7.4 µM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in VsDapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.
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Antibacterianos/química , Inibidores Enzimáticos/química , Transaminases/antagonistas & inibidores , Transaminases/química , Verrucomicrobia/enzimologia , Relação Estrutura-Atividade , Transaminases/genética , Verrucomicrobia/genéticaRESUMO
meso-Diaminopimelate decarboxylase catalyzes the decarboxylation of meso-diaminopimelate, the final reaction in the diaminopimelate l-lysine biosynthetic pathway. It is the only known pyridoxal-5-phosphate-dependent decarboxylase that catalyzes the removal of a carboxyl group from a d-stereocenter. Currently, only prokaryotic orthologs have been kinetically and structurally characterized. Here, using complementation and kinetic analyses of enzymes recombinantly expressed in Escherichia coli, we have functionally tested two putative eukaryotic meso-diaminopimelate decarboxylase isoforms from the plant species Arabidopsis thaliana We confirm they are both functional meso-diaminopimelate decarboxylases, although with lower activities than those previously reported for bacterial orthologs. We also report in-depth X-ray crystallographic structural analyses of each isoform at 1.9 and 2.4 Å resolution. We have captured the enzyme structure of one isoform in an asymmetric configuration, with one ligand-bound monomer and the other in an apo-form. Analytical ultracentrifugation and small-angle X-ray scattering solution studies reveal that A. thaliana meso-diaminopimelate decarboxylase adopts a homodimeric assembly. On the basis of our structural analyses, we suggest a mechanism whereby molecular interactions within the active site transduce conformational changes to the active-site loop. These conformational differences are likely to influence catalytic activity in a way that could allow for d-stereocenter selectivity of the substrate meso-diaminopimelate to facilitate the synthesis of l-lysine. In summary, the A. thaliana gene loci At3g14390 and At5g11880 encode functional. meso-diaminopimelate decarboxylase enzymes whose structures provide clues to the stereochemical control of the decarboxylation reaction catalyzed by these eukaryotic proteins.
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Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Carboxiliases/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carboxiliases/genética , Domínio Catalítico , Cristalografia por Raios X , Domínios ProteicosRESUMO
The rising concern over drug-resistant microorganisms has increased the need for rapid and portable detection systems. However, the traditional methods for the analysis of microorganisms can be both resource and time intensive. This contribution presents an alternative approach for the characterization of microorganisms using a microscale electrokinetic technique. The present study aims to develop and validate a library with a novel parameter referred to as the electrokinetic equilibrium condition for each strain, which will allow for fast identification of the studied bacterial and yeast cells in electrokinetic (EK) microfluidic devices. To create the library, experiments with six organisms of interest were conducted using insulator-based EK devices with circle-shaped posts. The organisms included one yeast strain, Saccharomyces cerevisiae; one salmonella strain, Salmonella enterica; two species from the same genus, Bacillus cereus and Bacillus subtilis; and two Escherichia coli strains. The results from these experiments were then analyzed with a mathematical model in COMSOL Multiphysics®, which yielded the electrokinetic equilibrium condition for each distinct strain. Lastly, to validate the applicability EK library, the COMSOL model was used to estimate the trapping conditions needed in a device with oval-shaped posts for each organism, and these values were then compared with experimentally obtained values. The results suggest the library can be used to estimate trapping voltages with a maximum relative error of 12%. While the proposed electrokinetic technique is still a novel approach and the analysis of additional microorganisms would be needed to expand the library, this contribution further supports the potential of microscale electrokinetics as a technique for the rapid and robust characterization of microbes. Graphical abstract.
Assuntos
Eletroforese/métodos , Bactérias/classificação , Bactérias/efeitos dos fármacos , Resistência Microbiana a Medicamentos , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Since the elucidation of the genetic code almost 50 years ago, many nonrandom aspects of its codon organization remain only partly resolved. Here, we investigate the recent hypothesis of 'dual-use' codons which proposes that in addition to allowing adjustment of codon optimization to tRNA abundance, the degeneracy in the triplet-based genetic code also multiplexes information regarding DNA's helical shape and protein-binding dynamics while avoiding interference with other protein-level characteristics determined by amino acid properties. How such structural optimization of the code within eukaryotic chromatin could have arisen from an RNA world is a mystery, but would imply some preadaptation in an RNA context. We analyzed synonymous (protein-silent) and nonsynonymous (protein-altering) mutational impacts on molecular dynamics in 13823 identically degenerate alternative codon reorganizations, defined by codon transitions in 7680 GPU-accelerated molecular dynamic simulations of implicitly and explicitly solvated double-stranded aRNA and bDNA structures. When compared to all possible alternative codon assignments, the standard genetic code minimized the impact of synonymous mutations on the random atomic fluctuations and correlations of carbon backbone vector trajectories while facilitating the specific movements that contribute to DNA polymer flexibility. This trend was notably stronger in the context of RNA supporting the idea that dual-use codon optimization and informational multiplexing in DNA resulted from the preadaptation of the RNA duplex to resist changes to thermostability. The nonrandom and divergent molecular dynamics of synonymous mutations also imply that the triplet-based code may have resulted from adaptive functional expansion enabling a primordial doublet code to multiplex gene regulatory information via the shape and charge of the minor groove.
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Códon/genética , Códon/fisiologia , Mutação Silenciosa/fisiologia , Aminoácidos/genética , Animais , Cromatina/genética , Simulação por Computador , DNA/genética , DNA/metabolismo , Evolução Molecular , Código Genético , Humanos , Simulação de Dinâmica Molecular , Mutação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA de Transferência/genética , Mutação Silenciosa/genéticaRESUMO
Enzymes are usually comprised of multiple subunits and more often than not they are made up of identical subunits. In this review we examine lysine biosynthesis and focus on the enzyme dihydrodipicolinate synthase in terms of its structure, function and the evolution of its varied number of subunits (quaternary structure). Dihydrodipicolinate synthase is the first committed step in the biosynthesis of lysine, which occurs naturally in plants, bacteria, archaea and fungi, but is not synthesized in mammals. In bacteria, there have been four separate pathways identified from tetrahydrodipicolinate to meso-diaminopimelate, which is the immediate precursor to lysine. Dihydrodipicolinate synthases from many bacterial and plant species have been structurally characterised and the results show considerable variability with respect to their quaternary structure, hinting at their evolution. The oligomeric state of the enzyme plays a key role, both in catalysis and in the allosteric regulation of the enzyme by lysine. While most bacteria and plants have tetrameric enzymes, where the structure of the dimeric building blocks is conserved, the arrangement of the dimers differs. We also review a key development in the field, namely the discovery of a human dihydrodipicolinate synthase-like enzyme, now known as 4-hydroxy-2-oxoglutarate aldolase . This discovery complicates the rationale underpinning drug development against bacterial dihydrodipicolinate synthases, since genetic errors in 4-hydroxy-2-oxoglutarate aldolase cause the disease Primary Hyperoxaluria Type 3 and therefore compounds that are geared towards the inhibition of bacterial dihydrodipicolinate synthase may be toxic to mammalian cells.
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Evolução Molecular , Hidroliases/química , Hidroliases/metabolismo , Animais , Humanos , Lisina/metabolismoRESUMO
Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ÏKZ, have long genomes packaged into large, atypical virions. Many aspects of ÏKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ÏKZ and related phages, â¼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase ß subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" ß' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution. IMPORTANCE: In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ÏKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ÏKZ.
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Genes Essenciais/genética , Fagos de Salmonella/genética , Proteínas Virais/genética , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral/genética , Vírion/genéticaRESUMO
While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an 'accessory' during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context.
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Códon , DNA/química , Proteínas de Bactérias/genética , Composição de Bases , DNA de Algas/química , DNA Arqueal/química , DNA Bacteriano/química , Genoma , Mutação , Seleção Genética , Transaminases/genéticaRESUMO
L,L-Diaminopimelate aminotransferase (DapL) is an enzyme required for the biosynthesis of meso-diaminopimelate (m-DAP) and L-lysine (Lys) in some bacteria and photosynthetic organisms. m-DAP and Lys are both involved in the synthesis of peptidoglycan (PG) and protein synthesis. DapL is found in specific eubacterial and archaeal lineages, in particular in several groups of pathogenic bacteria such as Leptospira interrogans (LiDapL), the soil/water bacterium Verrucomicrobium spinosum (VsDapL) and the alga Chlamydomonas reinhardtii (CrDapL). Here we present the first comprehensive inhibition study comparing the kinetic activity of DapL orthologs using previously active small molecule inhibitors formerly identified in a screen with the DapL of Arabidopsis thaliana (AtDapL), a flowering plant. Each inhibitor is derived from one of four classes with different central structural moieties: a hydrazide, a rhodanine, a barbiturate, or a thiobarbituate functionality. The results show that all five compounds tested were effective at inhibiting the DapL orthologs. LiDapL and AtDapL showed similar patterns of inhibition across the inhibitor series, whereas the VsDapL and CrDapL inhibition patterns were different from that of LiDapL and AtDapL. CrDapL was found to be insensitive to the hydrazide (IC50 >200 µM). VsDapL was found to be the most sensitive to the barbiturate and thiobarbiturate containing inhibitors (IC50 â¼5 µM). Taken together, the data shows that the homologs have differing sensitivities to the inhibitors with IC50 values ranging from 4.7 to 250 µM. In an attempt to understand the basis for these differences the four enzymes were modeled based on the known structure of AtDapL. Overall, it was found that the enzyme active sites were conserved, although the second shell of residues close to the active site were not. We conclude from this that the altered binding patterns seen in the inhibition studies may be a consequence of the inhibitors forming additional interactions with residues proximal to the active site, or that the inhibitors may not act by binding to the active site. Compounds that are specific for DapL could be potential biocides (antibiotic, herbicide or algaecide) that are nontoxic to animals since animals do not contain the enzymes necessary for PG or Lys synthesis. This study provides important information to expand our current understanding of the structure/activity relationship of DapL and putative inhibitors that are potentially useful for the design and or discovery of novel biocides.
Assuntos
Ácido Diaminopimélico/química , Chumbo/farmacologia , Transaminases/química , Sequência de Aminoácidos , Ácido Diaminopimélico/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptidoglicano , Conformação Proteica , Relação Estrutura-Atividade , Transaminases/metabolismoRESUMO
The fight against bacterial antibiotic resistance must be given critical attention to avert the current and emerging crisis of treating bacterial infections due to the inefficacy of clinically relevant antibiotics. Intrinsic genetic mutations and transferrable antibiotic resistance genes (ARGs) are at the core of the development of antibiotic resistance. However, traditional alignment methods for detecting ARGs have limitations. Artificial intelligence (AI) methods and approaches can potentially augment the detection of ARGs and identify antibiotic targets and antagonistic bactericidal and bacteriostatic molecules that are or can be developed as antibiotics. This review delves into the literature regarding the various AI methods and approaches for identifying and annotating ARGs, highlighting their potential and limitations. Specifically, we discuss methods for (1) direct identification and classification of ARGs from genome DNA sequences, (2) direct identification and classification from plasmid sequences, and (3) identification of putative ARGs from feature selection.
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We present the whole-genome sequence of four bacterial endophytes associated with German hardneck garlic cloves (Allium sativum L.). Among them, Agrobacterium fabrum and Pantoea agglomerans are associated with plant protection, while Rahnella perminowiae and Stenotrophomonas lactitubi are pathogens. These data will facilitate the identification of genes to improve garlic.
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Our study presents the whole-genome sequences and annotation of five bacteria isolates, each demonstrating distinct antibiotic resistance. These isolates include Bacillus paranthracis RIT 841, Atlantibacter hermanii RIT 842, Pantoea leporis RIT 844, Enterococcus casseliflavus RIT 845, and Pseudomonas alkylphenolica RIT 846, underscoring the importance of understanding antimicrobial resistance.
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Enzymes are nature's catalysts, mediating chemical processes in living systems. The study of enzyme function and mechanism includes defining the maximum catalytic rate and affinity for substrate/s (among other factors), referred to as enzyme kinetics. Enzyme kinetics is a staple of biochemistry curricula and other disciplines, from molecular and cellular biology to pharmacology. However, because enzyme kinetics involves concepts rarely employed in other areas of biology, it can be challenging for students and researchers. Traditional graphical analysis was replaced by computational analysis, requiring another skill not core to many life sciences curricula. Computational analysis can be time-consuming and difficult in free software (e.g., R) or require costly software (e.g., GraphPad Prism). We present Enzyme Kinetics Analysis (EKA), a web-tool to augment teaching and learning and streamline EKA. EKA is an interactive and free tool for analyzing enzyme kinetic data and improving student learning through simulation, built using R and RStudio's ShinyApps. EKA provides kinetic models (Michaelis-Menten, Hill, simple reversible inhibition models, ternary-complex, and ping-pong) for users to fit experimental data, providing graphical results and statistics. Additionally, EKA enables users to input parameters and create data and graphs, to visualize changes to parameters (e.g., K M or number of measurements). This function is designed for students learning kinetics but also for researchers to design experiments. EKA (enzyme-kinetics.shinyapps.io/enzkinet_webpage/) provides a simple, interactive interface for teachers, students, and researchers to explore enzyme kinetics. It gives researchers the ability to design experiments and analyze data without specific software requirements.
Assuntos
Enzimas , Software , Cinética , Enzimas/metabolismo , Humanos , Bioquímica/educação , Internet , Estudantes , Ensino , CurrículoRESUMO
Antimicrobial resistance (AMR) is a global threat to human health since infections caused by antimicrobial-resistant bacteria are life-threatening conditions with minimal treatment options. Bacteria become resistant when they develop the ability to overcome the compounds that are meant to kill them, i.e., antibiotics. The increasing number of resistant pathogens worldwide is contrasted by the slow progress in the discovery and production of new antibiotics. About 700,000 global deaths per year are estimated as a result of drug-resistant infections, which could escalate to nearly 10 million by 2050 if we fail to address the AMR challenge. In this study, we collected and isolated bacteria from the environment to screen for antibiotic resistance. We identified several bacteria that showed resistance to multiple clinically relevant antibiotics when tested in antibiotic susceptibility disk assays. We also found that two strains, identified as Pantoea rodasii RIT 836 and Pseudomonas endophytica RIT 838 via whole genome sequencing and annotation, produce bactericidal compounds against both Gram-positive and Gram-negative bacteria in disc-diffusion inhibitory assays. We mined the two strains' whole-genome sequences to gain more information and insights into the antibiotic resistance and production by these bacteria. Subsequently, we aim to isolate, identify, and further characterize the novel antibiotic compounds detected in our assays and bioinformatics analysis.
Assuntos
Antibacterianos , Anti-Infecciosos , Pantoea , Humanos , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Bactérias Gram-Positivas , Pseudomonas/genética , Sequenciamento Completo do GenomaRESUMO
The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.
Assuntos
Deriva Genética , Genoma Bacteriano , Lisina , Simbiose , Lisina/biossíntese , Lisina/metabolismo , Lisina/genética , Hidroliases/genética , Hidroliases/química , Hidroliases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , AnimaisRESUMO
Antimicrobial resistance (AMR) is one of the most alarming global public health challenges of the 21st century. Over 3 million antimicrobial-resistant infections occur in the United States annually, with nearly 50,000 cases being fatal. Innovations in drug discovery methods and platforms are crucial to identify novel antibiotics to combat AMR. We present the isolation and characterization of potentially novel antibiotic lead compounds produced by the cross-feeding of two rhizosphere bacteria, Acinetobacter sp. RIT 592 and Exiguobacterium sp. RIT 594. We used solid-phase extraction (SPE) followed by liquid chromatography (LC) to enrich antibiotic extracts and subsequently mass spectrometry (MS) analysis of collected fractions for compound structure identification and characterization. The MS data were processed through the Global Natural Product Social Molecular Networking (GNPS) database. The supernatant from RIT 592 induced RIT 594 to produce a cocktail of antimicrobial compounds active against Gram-positive and negative bacteria. The GNPS analysis indicated compounds with known antimicrobial activity in the bioactive samples, including oligopeptides and their derivatives. This work emphasizes the utility of microbial community-based platforms to discover novel clinically relevant secondary metabolites. Future work includes further structural characterization and antibiotic activity evaluation of the individual compounds against pathogenic multidrug-resistant (MDR) bacteria.
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BACKGROUND: Bacteria belonging to the genus Novosphingobium are known to be metabolically versatile and occupy different ecological niches. In the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. In this study, we analyzed the whole genome sequencing data of six bacteria in the Novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. Additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity (AAI) and genomic signatures. RESULTS: The taxonomic clustering of Novosphingobium strains is generally influenced by their isolation source. AAI and genomic signature provide strong support the classification of Novosphingobium sp. PP1Y as Novosphingobium pentaromaticivorans PP1Y. The identification and subsequent functional annotation of the unique core genome in the marine Novosphingobium bacteria show that ectoine synthesis may be the main contributing factor in salt water adaptation. Genes coding for the synthesis and receptor of the cell-cell signaling molecules, of the N-acyl-homoserine lactones (AHL) class are identified. Notably, a solo luxR homolog was found in strain PP1Y that may have been recently acquired via horizontal gene transfer as evident by the presence of multiple mobile elements upstream of the gene. Additionally, phylogenetic tree analysis and sequence comparison with functionally validated aromatic ring hydroxylating dioxygenases (ARDO) revealed the presence of several ARDOs (oxygenase) in Novosphingobium bacteria with the majority of them belonging to the Groups II and III of the enzyme. CONCLUSIONS: The combination of prior knowledge on the distinctive phenotypes of Novosphingobium strains and meta-analysis of their whole genomes enables the identification of several genes that are relevant in industrial applications and bioremediation. The results from such targeted but comprehensive comparative genomics analysis have the potential to contribute to the understanding of adaptation, cell-cell communication and bioremediation properties of bacteria belonging to the genus Novosphingobium.
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Adaptação Fisiológica/genética , Alphaproteobacteria/citologia , Alphaproteobacteria/fisiologia , Genômica , Oceanos e Mares , Transdução de Sinais/genética , Adaptação Fisiológica/efeitos dos fármacos , Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Sequência Conservada , Dioxigenases/genética , Transferência Genética Horizontal/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Fenótipo , Filogenia , Percepção de Quorum/genética , Sais/farmacologia , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21â (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70â Å resolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.
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Proteínas de Bactérias/química , Staphylococcus aureus Resistente à Meticilina/química , Oxo-Ácido-Liases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: Antibiotic resistant infections have become a global health crisis causing 1.2 million deaths worldwide in 2019 [1]. In a previous study, we identified a bacterium from a rare genus, Yimella, and found in an initial antibiotic screening that they produce broad-spectrum bactericidal compounds [2]. Herein, we focus on the characterization of these potential novel antimicrobial compounds produced by Yimella sp. RIT 621. RESULTS: We used solid-phase extraction and C18 reverse-phase chromatography to isolate the antibiotic-active compounds found in organic extracts from liquid cultures of Yimella sp. RIT 621. We tracked the antimicrobial activity by testing the extracts in disc diffusion inhibitory assays and observed its increase after each purification stage.