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1.
Nat Commun ; 11(1): 1791, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286293

RESUMO

Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.


Assuntos
Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Segregação de Cromossomos , GMP Cíclico/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Myxococcus xanthus/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Cromossomos Bacterianos/metabolismo , GMP Cíclico/metabolismo , DNA Bacteriano/metabolismo , Loci Gênicos , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , Transcrição Genética
2.
Nat Commun ; 11(1): 1485, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198399

RESUMO

Higher-order chromosome folding and segregation are tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remain poorly characterized, especially in non-model species. Here, we investigate the role of DNA-partitioning protein ParB and SMC condensin complexes in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for reliable chromosome segregation. We use chromatin immunoprecipitation and photoactivated single-molecule localization microscopy to show the formation of distinct, parS-dependent ParB-nucleoprotein subclusters. We further show that SMC/ScpAB complexes, loaded via ParB at parS sites, mediate chromosomal inter-arm contacts (as previously shown in Bacillus subtilis). However, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding; instead, this complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. Our results complement current models of ParB-SMC/ScpAB crosstalk and show that some condensin complexes evolved functions that are apparently uncoupled from chromosome folding.


Assuntos
Adenosina Trifosfatases/metabolismo , Estruturas Cromossômicas/química , Estruturas Cromossômicas/metabolismo , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , Corynebacterium glutamicum/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Bacillus subtilis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/genética , DNA Primase/genética , DNA Primase/metabolismo , DNA Bacteriano , Nucleoproteínas/metabolismo , Origem de Replicação
3.
Proc Natl Acad Sci U S A ; 117(12): 6752-6761, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32144140

RESUMO

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome.


Assuntos
Antineoplásicos/farmacologia , Catecóis/farmacologia , Reprogramação Celular , Cupriavidus necator/genética , Escherichia coli/genética , Pseudomonas putida/genética , Biologia Sintética/métodos , Proliferação de Células , Cromossomos Bacterianos , Cupriavidus necator/metabolismo , Sistemas de Liberação de Medicamentos , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Engenharia Genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Pseudomonas putida/metabolismo , Células Tumorais Cultivadas
4.
Mol Genet Genomics ; 295(4): 891-909, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32189066

RESUMO

Wolbachia is an obligate intracellular Gram-negative alpha-proteobacterium that has diverse effects on reproduction of arthropod hosts, including cytoplasmic incompatibility, male killing, feminization, and parthenogenesis. Some of these effects have important potential for control of insect pests, including mosquitoes that vector pathogens of humans. In mosquitoes, and in most other arthropods, elimination of Wolbachia by antibiotic treatment has no effect on host survival and reverses the Wolbachia-associated phenotype. Elimination of Wolbachia strain wFol, which enables parthenogenetic reproduction of the Collembolan, Folsomia candida, would result in population extinction. However, F. candida adults remain viable and resume reproduction when antibiotics are removed, suggesting that wFol survives antibiotic treatment in a quiescent persister state similar to that induced by chromosomally encoded toxin-antitoxin (TA) modules in free-living bacteria. Computational approaches were used to document the presence of antitoxin genes upstream of Wolbachia RelE/ParE, Fic, and AbiEii toxin genes. Moreover, this analysis revealed that Wolbachia RatA toxin is encoded by a single copy gene associated with an ssrS noncoding RNA gene. Documentation of potentially functional TA modules expands our understanding of the metabolic capabilities of Wolbachia, and provides an explanation for variable and sometimes contradictory results of antibiotic treatments. The presence of chromosomal TA modules in Wolbachia genomes suggests that wFol, and potentially other strains of Wolbachia, can enter a quiescent persister state.


Assuntos
Partenogênese/genética , Reprodução/genética , Sistemas Toxina-Antitoxina/genética , Wolbachia/genética , Animais , Cromossomos Bacterianos/genética , Culicidae/microbiologia , DNA Topoisomerase IV/genética , Genoma Bacteriano/genética , Humanos , Masculino , Controle de Pragas , Simbiose/genética , Wolbachia/patogenicidade
5.
PLoS Genet ; 16(3): e1008615, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130223

RESUMO

The relative linear order of most genes on bacterial chromosomes is not conserved over evolutionary timescales. One explanation is that selection is weak, allowing recombination to randomize gene order by genetic drift. However, most chromosomal rearrangements are deleterious to fitness. In contrast, we propose the hypothesis that rearrangements in gene order are more likely the result of selection during niche adaptation (SNAP). Partial chromosomal duplications occur very frequently by recombination between direct repeat sequences. Duplicated regions may contain tens to hundreds of genes and segregate quickly unless maintained by selection. Bacteria exposed to non-lethal selections (for example, a requirement to grow on a poor nutrient) can adapt by maintaining a duplication that includes a gene that improves relative fitness. Further improvements in fitness result from the loss or inactivation of non-selected genes within each copy of the duplication. When genes that are essential in single copy are lost from different copies of the duplication, segregation is prevented even if the original selection is lifted. Functional gene loss continues until a new genetic equilibrium is reached. The outcome is a rearranged gene order. Mathematical modelling shows that this process of positive selection to adapt to a new niche can rapidly drive rearrangements in gene order to fixation. Signature features (duplication formation and divergence) of the SNAP model were identified in natural isolates from multiple species showing that the initial two steps in the SNAP process can occur with a remarkably high frequency. Further bioinformatic and experimental analyses are required to test if and to which extend the SNAP process acts on bacterial genomes.


Assuntos
Aclimatação/genética , Cromossomos Bacterianos/genética , Duplicação Gênica/genética , Rearranjo Gênico/genética , Seleção Genética/genética , Aberrações Cromossômicas , Evolução Molecular , Frequência do Gene/genética , Ordem dos Genes/genética , Genoma Bacteriano/genética , Modelos Teóricos , Filogenia
6.
Nucleic Acids Res ; 48(5): 2199-2208, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009151

RESUMO

Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box. While the mechanism of transcription repression by Zur is well-studied, little is known on how derepression by Zur could be facilitated. Using single-molecule/single-cell measurements, we find that in live Escherichia coli cells, Zur's unbinding rate from DNA is sensitive to Zur protein concentration in a first-of-its-kind biphasic manner, initially impeded and then facilitated with increasing Zur concentration. These results challenge conventional models of protein unbinding being unimolecular processes and independent of protein concentration. The facilitated unbinding component likely occurs via a ternary complex formation mechanism. The impeded unbinding component likely results from Zur oligomerization on chromosome involving inter-protein salt-bridges. Unexpectedly, a non-repressor form of Zur is found to bind chromosome tightly, likely at non-consensus sequence sites. These unusual behaviors could provide functional advantages in Zur's facile switching between repression and derepression.


Assuntos
DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Genética , Sítios de Ligação , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Ligação Proteica , Multimerização Proteica , Análise de Célula Única , Zinco/metabolismo
7.
PLoS Genet ; 16(1): e1008473, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31895943

RESUMO

To prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum.


Assuntos
Divisão Celular , Cromossomos Bacterianos/genética , Reparo de DNA por Recombinação , Quebras de DNA de Cadeia Dupla , Escherichia coli
8.
Nucleic Acids Res ; 48(4): 2035-2049, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31950157

RESUMO

Negative supercoiling by DNA gyrase is essential for maintaining chromosomal compaction, transcriptional programming, and genetic integrity in bacteria. Questions remain as to how gyrases from different species have evolved profound differences in their kinetics, efficiency, and extent of negative supercoiling. To explore this issue, we analyzed homology-directed mutations in the C-terminal, DNA-wrapping domain of the GyrA subunit of Escherichia coli gyrase (the 'CTD'). The addition or removal of select, conserved basic residues markedly impacts both nucleotide-dependent DNA wrapping and supercoiling by the enzyme. Weakening CTD-DNA interactions slows supercoiling, impairs DNA-dependent ATP hydrolysis, and limits the extent of DNA supercoiling, while simultaneously enhancing decatenation and supercoil relaxation. Conversely, strengthening DNA wrapping does not result in a more extensively supercoiled DNA product, but partially uncouples ATP turnover from strand passage, manifesting in futile cycling. Our findings indicate that the catalytic cycle of E. coli gyrase operates at high thermodynamic efficiency, and that the stability of DNA wrapping by the CTD provides one limit to DNA supercoil introduction, beyond which strand passage competes with ATP-dependent supercoil relaxation. These results highlight a means by which gyrase can evolve distinct homeostatic supercoiling setpoints in a species-specific manner.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Girase/genética , DNA Bacteriano/genética , DNA Super-Helicoidal/química , Trifosfato de Adenosina/química , Catálise , Cromossomos Bacterianos/genética , DNA Girase/química , DNA Bacteriano/química , DNA Super-Helicoidal/genética , Escherichia coli/enzimologia , Modelos Moleculares , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos/genética
9.
BMC Genomics ; 21(1): 16, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906865

RESUMO

BACKGROUND: The genus Borrelia comprises spirochaetal bacteria maintained in natural transmission cycles by tick vectors and vertebrate reservoir hosts. The main groups are represented by a species complex including the causative agents of Lyme borreliosis and relapsing fever group Borrelia. Borrelia miyamotoi belongs to the relapsing fever group of spirochetes and forms distinct populations in North America, Asia, and Europe. As all Borrelia species B. miyamotoi possess an unusual and complex genome consisting of a linear chromosome and a number of linear and circular plasmids. The species is considered an emerging human pathogen and an increasing number of human cases are being described in the Northern hemisphere. The aim of this study was to produce a high quality reference genome that will facilitate future studies into genetic differences between different populations and the genome plasticity of B. miyamotoi. RESULTS: We used multiple available sequencing methods, including Pacific Bioscience single-molecule real-time technology (SMRT) and Oxford Nanopore technology (ONT) supplemented with highly accurate Illumina sequences, to explore the suitability for whole genome assembly of the Russian B. miyamotoi isolate, Izh-4. Plasmids were typed according to their potential plasmid partitioning genes (PF32, 49, 50, 57/62). Comparing and combining results of both long-read (SMRT and ONT) and short-read methods (Illumina), we determined that the genome of the isolate Izh-4 consisted of one linear chromosome, 12 linear and two circular plasmids. Whilst the majority of plasmids had corresponding contigs in the Asian B. miyamotoi isolate FR64b, there were only four that matched plasmids of the North American isolate CT13-2396, indicating differences between B. miyamotoi populations. Several plasmids, e.g. lp41, lp29, lp23, and lp24, were found to carry variable major proteins. Amongst those were variable large proteins (Vlp) subtype Vlp-α, Vlp-γ, Vlp-δ and also Vlp-ß. Phylogenetic analysis of common plasmids types showed the uniqueness in Russian/Asian isolates of B. miyamotoi compared to other isolates. CONCLUSIONS: We here describe the genome of a Russian B. miyamotoi clinical isolate, providing a solid basis for future comparative genomics of B. miyamotoi isolates. This will be a great impetus for further basic, molecular and epidemiological research on this emerging tick-borne pathogen.


Assuntos
Borrelia/genética , Genoma Bacteriano/genética , Genômica/métodos , Plasmídeos/genética , Sequenciamento Completo do Genoma/métodos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Borrelia/classificação , Borrelia/patogenicidade , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Humanos , Ixodes/microbiologia , Doença de Lyme/microbiologia , Filogenia , Febre Recorrente/microbiologia , Especificidade da Espécie
10.
Nucleic Acids Res ; 48(1): 249-263, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31667508

RESUMO

Phage-inducible chromosomal island-like elements (PLEs) are bacteriophage satellites found in Vibrio cholerae. PLEs parasitize the lytic phage ICP1, excising from the bacterial chromosome, replicating, and mobilizing to new host cells following cell lysis. PLEs protect their host cell populations by completely restricting the production of ICP1 progeny. Previously, it was found that ICP1 replication was reduced during PLE(+) infection. Despite robust replication of the PLE genome, relatively few transducing units are produced. We investigated if PLE DNA replication itself is antagonistic to ICP1 replication. Here we identify key constituents of PLE replication and assess their role in interference of ICP1. PLE encodes a RepA_N initiation factor that is sufficient to drive replication from the PLE origin of replication during ICP1 infection. In contrast to previously characterized bacteriophage satellites, expression of the PLE initiation factor was not sufficient for PLE replication in the absence of phage. Replication of PLE was necessary for interference of ICP1 DNA replication, but replication of a minimalized PLE replicon was not sufficient for ICP1 DNA replication interference. Despite restoration of ICP1 DNA replication, non-replicating PLE remained broadly inhibitory against ICP1. These results suggest that PLE DNA replication is one of multiple mechanisms contributing to ICP1 restriction.


Assuntos
Bacteriófagos/genética , DNA Helicases/genética , DNA Bacteriano/genética , Vibrio cholerae/genética , Replicação Viral/genética , Bacteriófagos/metabolismo , Cromossomos Bacterianos/química , Cromossomos Bacterianos/imunologia , Cromossomos Bacterianos/virologia , DNA Helicases/imunologia , Replicação do DNA , DNA Bacteriano/imunologia , Lisogenia/genética , Origem de Replicação , Vibrio cholerae/imunologia , Vibrio cholerae/virologia
11.
Int J Infect Dis ; 90: 181-187, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31682959

RESUMO

OBJECTIVES: To determine the molecular characteristics of a sequence type 338 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain and the relationship among MRSA strains from various lineages and areas. METHODS: Whole-genome sequencing, genomic comparison, antimicrobial susceptibility testing, and hemolysis analysis were performed to identify the resistance determinants and virulence factors of strain ZY05 and the relationships among CC59 clones. RESULTS: MRSA strain ZY05 was resistant to tetracycline, erythromycin, and clindamycin, and the resistance genes erm(B) and tet(K) were detected in the genome. ZY05 harbors the genomic islands νSaα, νSaß, νSaγ, and ΦSa2, the pathogenicity island νSa1, and virulence factors such as Panton-Valentine leukocidin, phenol-soluble modulins, alpha-hemolysin, enterotoxin B, enterotoxin K, and enterotoxin Q, which are the same as those present in ST59 strains. In addition, the virulence potential of ST338 did not differ from that of ST59. This strain contains the staphylococcal cassette chromosome mec (SCCmec) type VT, a distinct SCCmec type previously reported from Taiwan. The results of core genome multilocus sequence typing (cgMLST) analysis showed that the gene distances between ST59 and ST338 were close among CC59 isolates, while strains from Taiwan were identical to isolates from the Chinese mainland with respect to these two sequence types. CONCLUSIONS: The ST338 strain ZY05, which has a close genetic relationship to ST59 strains, is multidrug-resistant and highly virulent. Strains of two identical lineages, ST59 and ST338, from Taiwan and the Chinese mainland may have the same genetic background.


Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Adulto , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , China , Cromossomos Bacterianos/genética , Exotoxinas/genética , Exotoxinas/metabolismo , Genoma Bacteriano , Ilhas Genômicas , Humanos , Leucocidinas/genética , Leucocidinas/metabolismo , Masculino , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
12.
Nucleic Acids Res ; 48(1): 200-211, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665475

RESUMO

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Escherichia coli/metabolismo , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação , Termodinâmica
13.
Nat Rev Genet ; 21(4): 227-242, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31767998

RESUMO

Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.


Assuntos
Cromossomos Bacterianos , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano
14.
Int J Syst Evol Microbiol ; 70(2): 849-856, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31793856

RESUMO

Borrelia species are vector-borne parasitic bacteria with unusual, highly fragmented genomes that include a linear chromosome and linear as well as circular plasmids that differ numerically between and within various species. Strain CA690T, which was cultivated from a questing Ixodes spinipalpis nymph in the San Francisco Bay area, CA, was determined to be genetically distinct from all other described species belonging to the Borrelia burgdorferi sensu lato complex. The genome, including plasmids, was assembled using a hybrid assembly of short Illumina reads and long reads obtained via Oxford Nanopore Technology. We found that strain CA690T has a main linear chromosome containing 902176 bp with a blast identity ≤91 % compared with other Borrelia species chromosomes and five linear and two circular plasmids. A phylogeny based on 37 single-copy genes of the main linear chromosome and rooted with the relapsing fever species Borrelia duttonii strain Ly revealed that strain CA690T had a sister-group relationship with, and occupied a basal position to, species occurring in North America. We propose to name this species Borrelia maritima sp. nov. The type strain, CA690T, has been deposited in two national culture collections, DSMZ (=107169) and ATCC (=TSD-160).


Assuntos
Grupo Borrelia Burgdorferi/classificação , Ixodes/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Grupo Borrelia Burgdorferi/isolamento & purificação , California , Cromossomos Bacterianos , DNA Bacteriano/genética , Plasmídeos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
15.
Int J Infect Dis ; 91: 218-222, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31843670

RESUMO

OBJECTIVES: The aim was to investigate methicillin-resistant Staphylococcus aureus (MRSA) carriage rates and to characterize the staphylococcal cassette chromosome mec (SCCmec) among Syrian refugees and the host community in Duhok, Iraq. METHODS: A total of 492 host community and 355 Syrian refugee subjects were recruited. Participant bio-information was collected using a study questionnaire. MRSA carriage was identified as recommended in the Clinical and Laboratory Standards Institute guidelines. PCR was performed for typing the mecA gene and SCCmec groups. RESULTS: In the host community, 76/492 (15.4%) carried MRSA, whereas 49/355 (13.8%) Syrian refugees carried MRSA (p = 0.505). Refugees were classified according to their arrival date; 36/278 (13%) existing refugees and 13/77 (16.9%) new refugee arrivals were identified as MRSA carriers (p = 0.375). Regarding the risk factors influencing MRSA spread, no association was found between MRSA prevalence and risk factors. The most common types of SCCmec in both communities were types IVa and V: 67.1% and 5.26%, and 49% and 2.04%, respectively. CONCLUSIONS: It is inferred that the effect of MRSA in refugees on the host community will be negligible. However, the refugee carriage rate is higher than that found in Western countries, the final destination of refugees. Therefore, screening for MRSA is mandated and the rapid detection of carriers would prevent its spread.


Assuntos
Portador Sadio , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Refugiados , Adolescente , Cromossomos Bacterianos , Feminino , Humanos , Iraque , Masculino , Staphylococcus aureus Resistente à Meticilina/genética , Síria/etnologia , Adulto Jovem
16.
J Biotechnol ; 307: 63-68, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31678458

RESUMO

Extensively modulating gene expression to achieve optimal flux is a critical step in metabolic engineering. Gene expression is usually modulated at the transcriptional level by controlling the strength of a promoter. However, this type of modulation is often hampered by its inability to fully sample the complete continuum of transcriptional control. In Escherichia coli, this limitation can be solved by constructing promoters with a wide range of strengths. In this study, a highly efficient method was developed to modulate a particular chromosomal gene of E. coli at a wide range of expression levels. This was achieved by combining highly efficient single-stranded oligonucleotide-mediated recombination and a stringent counter selection system kil. Using this strategy, a chromosomal library, with a range from 0.3% to 388% relative to the wild lac promoter, was easily obtained. The strength of our chromosomal promoter library was approximately 5-60 times wider in range than those of libraries reported before.


Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Regiões Promotoras Genéticas/genética , DNA de Cadeia Simples , Escherichia coli/metabolismo , Biblioteca Gênica , Oligonucleotídeos/genética , Recombinação Genética
17.
Nucleic Acids Res ; 48(5): 2412-2423, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31875223

RESUMO

Exquisite control of the DnaA initiator is critical to ensure that bacteria initiate chromosome replication in a cell cycle-coordinated manner. In many bacteria, the DnaA-related and replisome-associated Hda/HdaA protein interacts with DnaA to trigger the Regulatory Inactivation of DnaA (RIDA) and prevent over-initiation events. In the Caulobacter crescentus Alphaproteobacterium, the RIDA process also targets DnaA for its rapid proteolysis by Lon. The impact of the RIDA process on adaptation of bacteria to changing environments remains unexplored. Here, we identify a novel and conserved DnaA-related protein, named HdaB, and show that homologs from three different Alphaproteobacteria can inhibit the RIDA process, leading to over-initiation and cell death when expressed in actively growing C. crescentus cells. We further show that HdaB interacts with HdaA in vivo, most likely titrating HdaA away from DnaA. Strikingly, we find that HdaB accumulates mainly during stationary phase and that it shortens the lag phase upon exit from stationary phase. Altogether, these findings suggest that expression of hdaB during stationary phase prepares cells to restart the replication of their chromosome as soon as conditions improve, a situation often met by free-living or facultative intracellular Alphaproteobacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Sequência Conservada , Replicação do DNA , Caulobacter crescentus/citologia , Caulobacter crescentus/crescimento & desenvolvimento , Morte Celular , Divisão Celular , Cromossomos Bacterianos/metabolismo , Modelos Biológicos , Mutação/genética , Ligação Proteica
18.
Nat Commun ; 10(1): 5288, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754112

RESUMO

Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.


Assuntos
Bacteriófagos/imunologia , Cromossomos Bacterianos/imunologia , Listeria monocytogenes/imunologia , Prófagos/imunologia , Sequência de Aminoácidos , Bacteriocinas/genética , Bacteriocinas/imunologia , Bacteriólise/imunologia , Bacteriófagos/genética , Bacteriófagos/fisiologia , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/virologia , Genoma Bacteriano/genética , Genoma Bacteriano/imunologia , Genoma Viral/genética , Genoma Viral/imunologia , Interações Hospedeiro-Patógeno/imunologia , Listeria monocytogenes/genética , Listeria monocytogenes/virologia , Lisogenia/genética , Lisogenia/imunologia , Metaloproteases/genética , Metaloproteases/imunologia , Prófagos/genética , Prófagos/fisiologia , Homologia de Sequência de Aminoácidos , Ativação Viral/genética , Ativação Viral/imunologia
19.
mBio ; 10(6)2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719174

RESUMO

The candidate phyla radiation (CPR) comprises a large monophyletic group of bacterial lineages known almost exclusively based on genomes obtained using cultivation-independent methods. Within the CPR, Gracilibacteria (BD1-5) are particularly poorly understood due to undersampling and the inherent fragmented nature of available genomes. Here, we report the first closed, curated genome of a gracilibacterium from an enrichment experiment inoculated from the Gulf of Mexico and designed to investigate hydrocarbon degradation. The gracilibacterium rose in abundance after the community switched to dominance by Colwellia Notably, we predict that this gracilibacterium completely lacks glycolysis, the pentose phosphate and Entner-Doudoroff pathways. It appears to acquire pyruvate, acetyl coenzyme A (acetyl-CoA), and oxaloacetate via degradation of externally derived citrate, malate, and amino acids and may use compound interconversion and oxidoreductases to generate and recycle reductive power. The initial genome assembly was fragmented in an unusual gene that is hypervariable within a repeat region. Such extreme local variation is rare but characteristic of genes that confer traits under pressure to diversify within a population. Notably, the four major repeated 9-mer nucleotide sequences all generate a proline-threonine-aspartic acid (PTD) repeat. The genome of an abundant Colwellia psychrerythraea population has a large extracellular protein that also contains the repeated PTD motif. Although we do not know the host for the BD1-5 cell, the high relative abundance of the C. psychrerythraea population and the shared surface protein repeat may indicate an association between these bacteria.IMPORTANCE CPR bacteria are generally predicted to be symbionts due to their extensive biosynthetic deficits. Although monophyletic, they are not monolithic in terms of their lifestyles. The organism described here appears to have evolved an unusual metabolic platform not reliant on glucose or pentose sugars. Its biology appears to be centered around bacterial host-derived compounds and/or cell detritus. Amino acids likely provide building blocks for nucleic acids, peptidoglycan, and protein synthesis. We resolved an unusual repeat region that would be invisible without genome curation. The nucleotide sequence is apparently under strong diversifying selection, but the amino acid sequence is under stabilizing selection. The amino acid repeat also occurs in a surface protein of a coexisting bacterium, suggesting colocation and possibly interdependence.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Biodegradação Ambiental , Metabolismo Energético , Variação Genética , Genoma Bacteriano , Bactérias/classificação , Composição de Bases , Biocombustíveis/microbiologia , Cromossomos Bacterianos , Genômica , Glicólise , Hidrocarbonetos/metabolismo , Filogenia
20.
Elife ; 82019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31692448

RESUMO

Nucleosomes restrict DNA accessibility throughout eukaryotic genomes, with repercussions for replication, transcription, and other DNA-templated processes. How this globally restrictive organization emerged during evolution remains poorly understood. Here, to better understand the challenges associated with establishing globally restrictive chromatin, we express histones in a naive system that has not evolved to deal with nucleosomal structures: Escherichia coli. We find that histone proteins from the archaeon Methanothermus fervidus assemble on the E. coli chromosome in vivo and protect DNA from micrococcal nuclease digestion, allowing us to map binding footprints genome-wide. We show that higher nucleosome occupancy at promoters is associated with lower transcript levels, consistent with local repressive effects. Surprisingly, however, this sudden enforced chromatinization has only mild repercussions for growth unless cells experience topological stress. Our results suggest that histones can become established as ubiquitous chromatin proteins without interfering critically with key DNA-templated processes.


Assuntos
Cromossomos Bacterianos/metabolismo , Escherichia coli/metabolismo , Histonas/metabolismo , Methanobacteriales/enzimologia , Nucleossomos/metabolismo , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Expressão Gênica , Histonas/genética , Proteínas Recombinantes/genética
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