Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.270
Filtrar
1.
Nature ; 590(7846): 463-467, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33536618

RESUMO

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.


Assuntos
Antibacterianos/biossíntese , Furanos/metabolismo , Streptomyces coelicolor/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Furanos/química , Hormônios/química , Hormônios/classificação , Hormônios/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/classificação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Relação Estrutura-Atividade
2.
Subcell Biochem ; 96: 177-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252729

RESUMO

DNA binding proteins under starvation (Dps) are proteins belonging to the ferritin family with the capacity for DNA binding, in addition to iron storage and ferroxidation. Present only in the prokaryotes, these multifaceted proteins have been assigned with a number of roles, from pathogenesis to nucleoid condensation and protection. They have a significant role in protecting the cells from free radical assaults, indirectly by sequestration of iron and by directly binding to the DNA. Due to their symmetry, stability and biomineralization capacity, these proteins have ever increasing potential applications in biotechnology and drug delivery. This chapter tries to bring together all these aspects of Dps in the view of current understanding and older perspectives by studies of our group as well as other experts in the field.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Ferro/metabolismo , Células Procarióticas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/classificação , Ferritinas/classificação , Oxirredução
3.
PLoS One ; 15(12): e0240497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33383576

RESUMO

Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-cell wall antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.


Assuntos
Proteínas de Bactérias/genética , Parede Celular/genética , Corynebacterium glutamicum/genética , Galactanos/metabolismo , Genoma Bacteriano , Ácidos Micólicos/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Biologia Computacional/métodos , Corynebacterium glutamicum/metabolismo , Elementos de DNA Transponíveis , Galactanos/genética , Expressão Gênica , Ontologia Genética , Loci Gênicos , Anotação de Sequência Molecular , Mutagênese Insercional , Peptidoglicano/genética , Plasmídeos/química , Plasmídeos/metabolismo
4.
Nucleic Acids Res ; 48(18): 10184-10198, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32894292

RESUMO

H-NS is a nucleoid structuring protein and global repressor of virulence and horizontally-acquired genes in bacteria. H-NS can interact with itself or with homologous proteins, but protein family diversity and regulatory network overlap remain poorly defined. Here, we present a comprehensive phylogenetic analysis that revealed deep-branching clades, dispelling the presumption that H-NS is the progenitor of varied molecular backups. Each clade is composed exclusively of either chromosome-encoded or plasmid-encoded proteins. On chromosomes, stpA and newly discovered hlpP are core genes in specific genera, whereas hfp and newly discovered hlpC are sporadically distributed. Six clades of H-NS plasmid proteins (Hpp) exhibit ancient and dedicated associations with plasmids, including three clades with fidelity for plasmid incompatibility groups H, F or X. A proliferation of H-NS homologs in Erwiniaceae includes the first observation of potentially co-dependent H-NS forms. Conversely, the observed diversification of oligomerization domains may facilitate stable co-existence of divergent homologs in a genome. Transcriptomic and proteomic analysis in Salmonella revealed regulatory crosstalk and hierarchical control of H-NS homologs. We also discovered that H-NS is both a repressor and activator of Salmonella Pathogenicity Island 1 gene expression, and both regulatory modes are restored by Sfh (HppH) in the absence of H-NS.


Assuntos
Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Enterobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Filogenia , Proteômica
5.
J Proteome Res ; 19(11): 4718-4729, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32897080

RESUMO

We present METATRYP version 2 software that identifies shared peptides across the predicted proteomes of organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine METATRYP database with predicted proteomes from environmental sequencing confirms a low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed to facilitate ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa analyzed, sharing <1% peptides with taxa outside of the betacoronavirus group, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.


Assuntos
Organismos Aquáticos/genética , Coronavirus/genética , Metagenômica/métodos , Proteoma , Software , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Betacoronavirus/genética , Análise por Conglomerados , Infecções por Coronavirus/virologia , Humanos , Anotação de Sequência Molecular , Pandemias , Peptídeos/classificação , Peptídeos/genética , Pneumonia Viral/virologia , Proteoma/classificação , Proteoma/genética , Análise de Sequência de Proteína , Transcriptoma/genética , Proteínas Virais/classificação , Proteínas Virais/genética
6.
Science ; 369(6507): 1094-1098, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855335

RESUMO

Bacterial production of gaseous hydrocarbons such as ethylene and methane affects soil environments and atmospheric climate. We demonstrate that biogenic methane and ethylene from terrestrial and freshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway. This pathway, present in numerous species, uses a nitrogenase-like reductase that is distinct from known nitrogenases and nitrogenase-like reductases and specifically functions in C-S bond breakage to reduce ubiquitous and appreciable volatile organic sulfur compounds such as dimethyl sulfide and (2-methylthio)ethanol. Liberated methanethiol serves as the immediate precursor to methionine, while ethylene or methane is released into the environment. Anaerobic ethylene production by this pathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depleted soils. Methane production reveals an additional bacterial pathway distinct from archaeal methanogenesis.


Assuntos
Proteínas de Bactérias/química , Etilenos/biossíntese , Metano/biossíntese , Metionina/biossíntese , Oxirredutases/química , Rhodospirillum rubrum/enzimologia , Anaerobiose , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biocatálise , Vias Biossintéticas , Oxirredutases/classificação , Oxirredutases/genética , Microbiologia do Solo
7.
Sci Rep ; 10(1): 8679, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457360

RESUMO

Cyanobacteria are ubiquitous organisms with a relevant contribution to primary production in all range of habitats. Cyanobacteria are well known for their part in worldwide occurrence of aquatic blooms while producing a myriad of natural compounds, some with toxic potential, but others of high economical impact, as geosmin. We performed an environmental survey of cyanobacterial soil colonies to identify interesting metabolic pathways and adaptation strategies used by these microorganisms and isolated, sequenced and assembled the genome of a cyanobacterium that displayed a distinctive earthy/musty smell, typical of geosmin, confirmed by GC-MS analysis of the culture's volatile extract. Morphological studies pointed to a new Oscillatoriales soil ecotype confirmed by phylogenetic analysis, which we named Microcoleus asticus sp. nov. Our studies of geosmin gene presence in Bacteria, revealed a scattered distribution among Cyanobacteria, Actinobacteria, Delta and Gammaproteobacteria, covering different niches. Careful analysis of the bacterial geosmin gene and gene tree suggests an ancient bacterial origin of the gene, that was probably successively lost in different time frames. The high sequence similarities in the cyanobacterial geosmin gene amidst freshwater and soil strains, reinforce the idea of an evolutionary history of geosmin, that is intimately connected to niche adaptation.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/genética , Naftóis/metabolismo , Actinobacteria/genética , Actinobacteria/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Cianobactérias/química , Cianobactérias/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Genoma Bacteriano , Família Multigênica , Naftóis/análise , Naftóis/isolamento & purificação , Filogenia , Extração em Fase Sólida , Terpenos/análise
8.
Nucleic Acids Res ; 48(10): 5624-5638, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329776

RESUMO

CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/classificação , Proteínas Associadas a CRISPR/genética , Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/classificação , Endodesoxirribonucleases/genética , Células HEK293 , Humanos , Mutação , Filogenia , Prevotella/enzimologia , Biossíntese de Proteínas , Domínios Proteicos , Transcrição Genética
9.
Nucleic Acids Res ; 48(W1): W72-W76, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32282909

RESUMO

Key steps in a computational study of protein function involve analysis of (i) relationships between homologous proteins, (ii) protein domain architecture and (iii) gene neighborhoods the corresponding proteins are encoded in. Each of these steps requires a separate computational task and sets of tools. Currently in order to relate protein features and gene neighborhoods information to phylogeny, researchers need to prepare all the necessary data and combine them by hand, which is time-consuming and error-prone. Here, we present a new platform, TREND (tree-based exploration of neighborhoods and domains), which can perform all the necessary steps in automated fashion and put the derived information into phylogenomic context, thus making evolutionary based protein function analysis more efficient. A rich set of adjustable components allows a user to run the computational steps specific to his task. TREND is freely available at http://trend.zhulinlab.org.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Software , Proteínas Arqueais/classificação , Proteínas de Bactérias/classificação , Genes Arqueais , Genes Bacterianos , Filogenia , Domínios Proteicos , Análise de Sequência de Proteína
10.
PLoS One ; 15(3): e0230031, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163464

RESUMO

We characterised 80 Staphylococcus aureus strains isolated from human patients with SSTIs at a rural hospital in Ethiopia. Susceptibility to antibiotic of all strains was tested. The MLST method was used to type and a phylogenetic analysis was conducted employing the sequences of 7 housekeeping genes. PCR amplification was used to investigate the presence of the following virulence genes in all strains: hla (α-haemolysin), tstH (toxic shock syndrome toxin), luk PV (Panton-Valentine leukocidin), fnbA (fibronectin binding protein A) and mecA (methicillin resistance). Most of the strains were resistant to penicillin and ampicillin, but only 3 strains were resistant to oxacillin, and 1 of them was a true MRSA. The MLST results showed a high diversity of sequence types (ST), 55% of which were new, and ST152 was the most prevalent. A phylogeny study showed that many of the new STs were phylogenetically related to other previously described STs, but bore little relationship to the only ST from Ethiopia described in the database. Virulence gene detection showed a high prevalence of strains encoding the hla, fnbA and pvl genes (98.77%, 96.3% and 72.84%, respectively), a low prevalence of the tst gene (13.58%) and a markedly low prevalence of MRSA (1.25%). S. aureus strains isolated from patients in a rural area in Ethiopia showed low levels of antibiotic resistance, except to penicillin. Moreover, this study reveals new STs in Eastern Africa that are phylogenetically related to other previously described STs, and confirm the high prevalence of the pvl gene and the low prevalence of MRSA on the continent.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Virulência/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Toxinas Bacterianas/classificação , Toxinas Bacterianas/genética , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana/genética , Etiópia , Exotoxinas/classificação , Exotoxinas/genética , Hospitais Rurais , Humanos , Leucocidinas/classificação , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Penicilinas/farmacologia , Filogenia , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade
11.
Photochem Photobiol Sci ; 19(2): 274-280, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32002529

RESUMO

In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we cloned and expressed the putative luxG gene of P. leiognathi YL in the Escherichia coli BL21 strain. The product of luxG is a flavin reductase that consists of 206 amino acids, corresponding to a subunit molecular mass of ∼26 kDa. Phylogenetic analysis demonstrated that P. leiognathi YL LuxG has a rather distant evolutionary relationship with Frase I of Aliivibrio fischeri and Frp of Vibrio harveyi, but a close evolutionary relationship with Fre from Escherichia coli, which are all enzymes related to oxido-reductase. Further comparison shows that the changes in the functionally conserved sites may contribute to the functional divergence of LuxG and Fre. LuxG could supply reduced flavin mononucleotide (FMN) for bacterial luminescence by catalyzing the oxidation of nicotinamide adenine dinucleotide hydrogen (NADH). Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection. The NADH samples with concentrations of 0.1-1 nM were used to validate the linear relationship, and it was found that the logarithmic deviations were less than 3%, which showed more sensitive and stable results than the NADH detection by recombinant E. coli including the exogenously expressed luciferase and intrinsic Fre. Investigation of P. leiognathi YL LuxG would provide a basic understanding of its evolution, and structural and functional properties, which might contribute to the development of a NADH detection kit in the future.


Assuntos
Proteínas de Bactérias/metabolismo , Medições Luminescentes , NAD/análise , Oxirredutases/metabolismo , Photobacterium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/metabolismo , Evolução Molecular , Oxirredutases/classificação , Oxirredutases/genética , Filogenia , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência
12.
Nat Microbiol ; 5(4): 599-609, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31988381

RESUMO

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.


Assuntos
Amoeba/microbiologia , Coevolução Biológica , Interações Hospedeiro-Patógeno/genética , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Macrófagos/microbiologia , Fatores de Virulência/genética , Adaptação Fisiológica , Amoeba/classificação , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Feminino , Especificidade de Hospedeiro , Humanos , Legionella pneumophila/classificação , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Filogenia , Cultura Primária de Células , Seleção Genética , Virulência , Fatores de Virulência/classificação , Fatores de Virulência/metabolismo
13.
PLoS One ; 15(1): e0227500, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923228

RESUMO

A new species of the Campylobacter genus is described, isolated from the preputial mucosa of bulls (Bos taurus). The five isolates obtained exhibit characteristics of Campylobacter, being Gram-negative non-motile straight rods, oxidase positive, catalase negative and microaerophilic. Phenotypic characteristics and nucleotide sequence analysis of 16S rRNA and hsp60 genes demonstrated that these isolates belong to a novel species within the genus Campylobacter. Based on hsp60 gene phylogenetic analysis, the most related species are C. ureolyticus, C. blaseri and C. corcagiensis. The whole genome sequence analysis of isolate FMV-PI01 revealed that the average nucleotide identity with other Campylobacter species was less than 75%, which is far below the cut-off for isolates of the same species. However, whole genome sequence analysis identified coding sequences highly homologous with other Campylobacter spp. These included several virulence factor coding genes related with host cell adhesion and invasion, transporters involved in resistance to antimicrobials, and a type IV secretion system (T4SS), containing virB2-virB11/virD4 genes, highly homologous to the C. fetus subsp. venerealis. The genomic G+C content of isolate FMV-PI01 was 28.3%, which is one of the lowest values reported for species of the genus Campylobacter. For this species the name Campylobacter portucalensis sp. nov. is proposed, with FMV-PI01 (= LMG 31504, = CCUG 73856) as the type strain.


Assuntos
Campylobacter/genética , Pênis/microbiologia , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Campylobacter/classificação , Campylobacter/isolamento & purificação , Campylobacter/metabolismo , Bovinos , Chaperonina 60/classificação , Chaperonina 60/genética , Chaperonina 60/metabolismo , Epitélio/microbiologia , Genótipo , Masculino , Fenótipo , Filogenia , RNA Ribossômico 16S/química , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Sequenciamento Completo do Genoma
14.
Biochimie ; 170: 21-25, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31843578

RESUMO

Gre factors are ubiquitous transcription regulators that stimulate co-transcriptional RNA cleavage by bacterial RNA polymerase (RNAP). Here, we show that the stress-resistant bacterium Deinococcus peraridilitoris encodes four Gre factor homologs, Gfh proteins, that have distinct effects on transcription by RNAP. Two of the factors, Gfh1α and Gfh2ß inhibit transcription initiation, and one of them, Gfh1α can also regulate transcription elongation. We show that this factor strongly stimulates transcriptional pausing and intrinsic termination in the presence of manganese ions but has no effect on RNA cleavage. Thus, some Gfh factors encoded by Deinococci serve as lineage-specific transcription inhibitors that may play a role in stress resistance, while the functions of others remain to be discovered.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Deinococcus/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Transcrição Genética , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Deinococcus/crescimento & desenvolvimento , Deinococcus/metabolismo , Conformação de Ácido Nucleico , Clivagem do RNA , Homologia de Sequência
15.
Environ Microbiol ; 22(1): 270-285, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31657110

RESUMO

Fumarylacetoacetate hydrolase (FAH) superfamily proteins are found ubiquitously in microbial pathways involved in the catabolism of aromatic substances. Although extensive bioinformatic data on these proteins have been acquired, confusion caused by problems with the annotation of these proteins hinders research into determining their physiological functions. Here we classify 606 FAH superfamily proteins using a maximum likelihood (ML) phylogenetic tree, comparative gene-neighbourhood patterns and in vitro enzyme assays. The FAH superfamily proteins used for the analyses are divided into five distinct subfamilies, and two of them, FPH-A and FPH-B, contain the majority of the proteins of undefined function. These subfamilies include clusters designated FPH-I and FPH-II, respectively, which include two distinct types of fumarylpyruvate hydrolase (FPH), an enzyme involved in the final step of the gentisate pathway. We determined the crystal structures of these FPH enzymes at 2.0 Å resolutions and investigate the substrate binding mode by which these types of enzymes can accommodate fumarylpyruvate as a substrate. Consequentially, we identify the molecular signatures of the two types of FPH enzymes among the broadly conserved FAH superfamily proteins. Our studies allowed us to predict the relationship of unknown FAH superfamily proteins using their sequence information.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Hidrolases/química , Hidrolases/classificação , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Filogenia
16.
Appl Environ Microbiol ; 86(2)2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31676477

RESUMO

Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. ß-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of ß-O-4 aryl ether bonds. However, only a very limited number of these enzymes have been described and characterized so far. Using peptide pattern recognition (PPR) as well as phylogenetic analyses, 96 putatively novel ß-etherases have been identified, some even originating from bacteria outside the order Sphingomonadales A set of 13 diverse enzymes was selected for biochemical characterization, and ß-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known ß-etherases. Moreover, conserved sequence motifs specific for either LigE- or LigF-type enzymes were deduced from multiple-sequence alignments and the PPR-derived peptides. In combination with structural information available for the ß-etherases LigE and LigF, insight into the potential structural and/or functional role of conserved residues within these sequence motifs is provided. Phylogenetic analyses further suggest the presence of additional bacterial enzymes with potential ß-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric ß-etherases.IMPORTANCE The use of biomass as a renewable source and replacement for crude oil for the provision of chemicals and fuels is of major importance for current and future societies. Lignin, the most abundant aromatic polymer in nature, holds promise as a renewable starting material for the generation of required aromatic structures. However, a controlled and selective lignin depolymerization to yield desired aromatic structures is a very challenging task. In this regard, bacterial ß-etherases are especially interesting, as they are able to cleave the most abundant bond type in lignin with high selectivity. With this study, we significantly expanded the toolbox of available ß-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further ß-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.


Assuntos
Alphaproteobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Lignina/metabolismo , Oxirredutases/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/classificação , Mineração de Dados , Glutationa/metabolismo , Oxirredutases/classificação
17.
Genomics ; 112(1): 1042-1053, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226484

RESUMO

Brevibacillus laterosporus is a globally ubiquitous, spore forming bacterium, strains of which have shown toxic activity against invertebrates and microbes and several have been patented due to their commercial potential. Relatively little is known about this bacterium. Here, we examined the genomes of six published and five newly determined genomes of B. laterosporus, with an emphasis on the relationships between known and putative toxin encoding genes, as well as the phylogenetic relationships between strains. Phylogenetically, strain relationships are similar using average nucleotide identity (ANI) values and multi-gene approaches, although PacBio sequencing revealed multiple copies of the 16S rDNA gene which lessened utility at the strain level. Based on ANI values, the New Zealand isolates were distant from other isolates and may represent a new species. While all of the genomes examined shared some putative toxicity or virulence related proteins, many specific genes were only present in a subset of strains.


Assuntos
Toxinas Bacterianas/genética , Brevibacillus/classificação , Brevibacillus/genética , Anti-Infecciosos/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Brevibacillus/metabolismo , Brevibacillus/patogenicidade , Genes Bacterianos , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Plasmídeos/genética , Virulência/genética
18.
J Biol Chem ; 295(51): 17724-17737, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33454010

RESUMO

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Bifidobacterium longum/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/genética , Filogenia , Ácido Fítico/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Temperatura
19.
PLoS One ; 14(12): e0220586, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31825979

RESUMO

Carbapenemase-producing organisms (CPOs) are Gram-negative bacteria that are typically resistant to most or all antibiotics and are responsible for a global pandemic of high mortality. Rapid, accurate detection of CPOs and the classification of their carbapenemases are valuable tools for reducing the mortality of the CPO-associated infections, preventing the spread of CPOs, and optimizing use of new ß-lactamase inhibitor combinations such as ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam. The current study evaluated the performance of CPO Complete, a novel, manual, phenotypic carbapenemase detection and classification test. The test was evaluated for sensitivity and specificity against 262 CPO isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii and 67 non-CPO isolates. It was also evaluated for carbapenemase classification accuracy against 205 CPOs that produced a single carbapenemase class. The test exhibited 100% sensitivity 98.5% specificity for carbapenemase detection within 90 minutes and detected 74.1% of carbapenemases within 10 minutes. In the classification evaluation, 99.0% of carbapenemases were correctly classified for isolates that produced a single carbapenemase. The test is technically simple and has potential for adaptation to automated instruments. With lyophilized kit storage at temperatures up to 38°C, the CPO Complete test has the potential to provide rapid, accurate carbapenemase detection and classification in both limited resource and technologically advanced laboratories.


Assuntos
Acinetobacter baumannii/enzimologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/classificação , beta-Lactamases/metabolismo , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Ensaios Enzimáticos , Humanos , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação
20.
Biomed Res Int ; 2019: 5617153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886228

RESUMO

Several computational approaches for predicting subcellular localization have been developed and proposed. These approaches provide diverse performance because of their different combinations of protein features, training datasets, training strategies, and computational machine learning algorithms. In some cases, these tools may yield inconsistent and conflicting prediction results. It is important to consider such conflicting or contradictory predictions from multiple prediction programs during protein annotation, especially in the case of a multiclass classification problem such as subcellular localization. Hence, to address this issue, this work proposes the use of the particle swarm optimization (PSO) algorithm to combine the prediction outputs from multiple different subcellular localization predictors with the aim of integrating diverse prediction models to enhance the final predictions. Herein, we present PSO-LocBact, a consensus classifier based on PSO that can be used to combine the strengths of several preexisting protein localization predictors specially designed for bacteria. Our experimental results indicate that the proposed method can resolve inconsistency problems in subcellular localization prediction for both Gram-negative and Gram-positive bacterial proteins. The average accuracy achieved on each test dataset is over 98%, higher than that achieved with any individual predictor.


Assuntos
Proteínas de Bactérias/classificação , Biologia Computacional/métodos , Espaço Intracelular/química , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Algoritmos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Consenso
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...