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1.
Nat Commun ; 12(1): 5221, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471117

RESUMO

Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather 'open' to a 'closed' conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.


Assuntos
Antibacterianos/metabolismo , Cromossomos Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Estruturas Cromossômicas , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Família Multigênica , Transcriptoma
2.
Nat Commun ; 12(1): 5216, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471137

RESUMO

Bacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.


Assuntos
Bactérias/metabolismo , Biomarcadores/metabolismo , Técnicas Biossensoriais , Proteínas de Transporte/metabolismo , Patologia Molecular/métodos , Bactérias/genética , Ácidos e Sais Biliares/sangue , Técnicas Biossensoriais/métodos , Proteínas de Transporte/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Transplante de Fígado , Engenharia Metabólica/métodos , Sensibilidade e Especificidade , Alinhamento de Sequência , Vibrio , Vibrioses/diagnóstico
3.
Nat Commun ; 12(1): 5222, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471115

RESUMO

Bacteria of the genus Streptomyces have a linear chromosome, with a core region and two 'arms'. During their complex life cycle, these bacteria develop multi-genomic hyphae that differentiate into chains of exospores that carry a single copy of the genome. Sporulation-associated cell division requires chromosome segregation and compaction. Here, we show that the arms of Streptomyces venezuelae chromosomes are spatially separated at entry to sporulation, but during sporogenic cell division they are closely aligned with the core region. Arm proximity is imposed by segregation protein ParB and condensin SMC. Moreover, the chromosomal terminal regions are organized into distinct domains by the Streptomyces-specific HU-family protein HupS. Thus, as seen in eukaryotes, there is substantial chromosomal remodelling during the Streptomyces life cycle, with the chromosome undergoing rearrangements from an 'open' to a 'closed' conformation.


Assuntos
Cromossomos Bacterianos/fisiologia , Streptomyces/genética , Streptomyces/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Hifas/genética
4.
ACS Infect Dis ; 7(9): 2666-2685, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34503335

RESUMO

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA PpqsA promoter controlled by PqsR with an IC50 of 1 µM. Using isothermal titration calorimetry, a Kd of 10 nM for the PqsR ligand binding domain (PqsRLBD) was determined for 61. Furthermore, the crystal structure of 61 with PqsRLBD was attained with a resolution of 2.65 Å. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.


Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Proteínas de Bactérias/metabolismo , Biofilmes , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/metabolismo
5.
Nat Commun ; 12(1): 4929, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389727

RESUMO

Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.


Assuntos
Escherichia coli/genética , Glicólise/genética , Engenharia Metabólica/métodos , Metabolômica/métodos , Proteômica/métodos , Transcrição Genética , Algoritmos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Carotenoides/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Nucleic Acids Res ; 49(15): 8757-8776, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379789

RESUMO

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.


Assuntos
Genes Bacterianos , Óperon , Biossíntese de Proteínas , RNA de Transferência/genética , Estresse Fisiológico/genética , Anabaena/genética , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Viabilidade Microbiana/genética , RNA de Transferência/metabolismo , Sequências Reguladoras de Ácido Nucleico
7.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
8.
Front Cell Infect Microbiol ; 11: 692224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368013

RESUMO

The chlamydial small non coding RNA, IhtA, regulates the expression of both HctA and DdbA, the uncharacterized product of the C. trachomatis L2 CTL0322 gene. HctA is a small, highly basic, DNA binding protein that is expressed late in development and mediates the condensation of the genome during RB to EB differentiation. DdbA is conserved throughout the chlamydial lineage, and is predicted to express a small, basic, cytoplasmic protein. As it is common for sRNAs to regulate multiple mRNAs within the same physiological pathway, we hypothesize that DdbA, like HctA, is involved in RB to EB differentiation. Here, we show that DdbA is a DNA binding protein, however unlike HctA, DdbA does not contribute to genome condensation but instead likely has nuclease activity. Using a DdbA temperature sensitive mutant, we show that DdbAts creates inclusions indistinguishable from WT L2 in size and that early RB replication is likewise similar at the nonpermissive temperature. However, the number of DdbAts infectious progeny is dramatically lower than WT L2 overall, although production of EBs is initiated at a similar time. The expression of a late gene reporter construct followed live at 40°C indicates that late gene expression is severely compromised in the DdbAts strain. Viability assays, both in host cells and in axenic media indicate that the DdbAts strain is defective in the maintenance of EB infectivity. Additionally, using Whole Genome Sequencing we demonstrate that chromosome condensation is temporally separated from DNA replication during the RB to EB transition. Although DdbA does not appear to be directly involved in this process, our data suggest it is a DNA binding protein that is important in the production and maintenance of infectivity of the EB, perhaps by contributing to the remodeling of the EB chromosome.


Assuntos
Chlamydia trachomatis , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Proteínas de Ligação a DNA/genética , Corpos de Inclusão/metabolismo
9.
Front Cell Infect Microbiol ; 11: 698146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368016

RESUMO

L-arabinose inducible promoters are commonly used in gene expression analysis. However, nutrient source and availability also play a role in biofilm formation; therefore, L-arabinose metabolism could impact biofilm development. In this study we examined the impact of L-arabinose on Salmonella enterica serovar Typhimurium (S. Typhimurium) biofilm formation. Using mutants impaired for the transport and metabolism of L-arabinose, we showed that L-arabinose metabolism negatively impacts S. Typhimurium biofilm formation in vitro. When L-arabinose metabolism is abrogated, biofilm formation returned to baseline levels. However, without the ability to import extracellular L-arabinose, biofilm formation significantly increased. Using RNA-Seq we identified several gene families involved in these different phenotypes including curli expression, amino acid synthesis, and L-arabinose metabolism. Several individual candidate genes were tested for their involvement in the L-arabinose-mediated biofilm phenotypes, but most played no significant role. Interestingly, in the presence of L-arabinose the diguanylate cyclase gene adrA was downregulated in wild type S. Typhimurium. Meanwhile cyaA, encoding an adenylate cyclase, was downregulated in an L-arabinose transport mutant. Using an IPTG-inducible plasmid to deplete c-di-GMP via vieA expression, we were able to abolish the increased biofilm phenotype seen in the transport mutant. However, the mechanism by which the L-arabinose import mutant forms significantly larger biofilms remains to be determined. Regardless, these data suggest that L-arabinose metabolism influences intracellular c-di-GMP levels and therefore biofilm formation. These findings are important when considering the use of an L-arabinose inducible promoter in biofilm conditions.


Assuntos
Arabinose , Proteínas de Bactérias , Biofilmes , Salmonella typhimurium , Arabinose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
10.
Curr Microbiol ; 78(10): 3696-3707, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34426858

RESUMO

We previously reported that the two-component system MacRS regulates morphogenesis and production of the blue-pigmented antibiotic actinorhodin (ACT) in Streptomyces coelicolor. In this study, the role of MacRS was further extended to include control of the production of the red-pigmented antibiotic undecylprodigiosin (RED) and the calcium-dependent antibiotic (CDA), and control of other important cellular activities. Our data indicated that disruption of the MacRS TCS reduced production not only of ACT but also of RED and CDA. RNA-Seq analysis revealed that genes involved in both secondary metabolism and primary metabolism are differentially expressed in the MacRS deletion mutant ΔmacRS. Moreover, we found that genes of the Zur regulon are also markedly downregulated in ΔmacRS, suggesting a role for macRS in zinc homeostasis. In addition to previously identified MacR sites with strong matches to the MacR consensus recognition sequence, a genome-wide search revealed over one hundred less-stringent matches, including potential sites upstream of absR1, crgA, and smeA. Electrophoretic mobility shift assays demonstrated that MacR binds some of these sites in vitro. Although there is no strong MacR site upstream of the ACT regulatory gene actII-orf4 (sco5085), we showed that an engineered MacR site enhanced ACT production, providing an approach for modulating production of useful compounds. Altogether, our work suggests an important role for MacRS in a range of cellular activities in Streptomyces and its potential application in strain engineering.


Assuntos
Streptomyces coelicolor , Antraquinonas , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
11.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445643

RESUMO

The development and spread of antibiotics and biocides resistance is a significant global challenge. To find a solution for this emerging problem, the discovery of novel bacterial cellular targets and the critical pathways associated with antimicrobial resistance is needed. In the present study, we investigated the role of the two most critical envelope stress response regulators, RpoE and CpxR, on the physiology and susceptibility of growing Salmonella enterica serovar enteritidis cells using the polycationic antimicrobial agent, chlorhexidine (CHX). It was shown that deletion of the cpxR gene significantly increased the susceptibility of this organism, whereas deletion of the rpoE gene had no effect on the pathogen's susceptibility to this antiseptic. It has been shown that a lack of the CpxR regulator induces multifaceted stress responses not only in the envelope but also in the cytosol, further affecting the key biomolecules, including DNA, RNA, and proteins. We showed that alterations in cellular trafficking and most of the stress responses are associated with a dysfunctional CpxR regulator during exponential growth phase, indicating that these physiological changes are intrinsically associated with the lack of the CpxR regulator. In contrast, induction of type II toxin-antitoxin systems and decrease of abundances of enzymes and proteins associated with the recycling of muropeptides and resistance to polymixin and cationic antimicrobial peptides were specific responses of the ∆cpxR mutant to the CHX treatment. Overall, our study provides insight into the effects of CpxR on the physiology of S. Enteritidis cells during the exponential growth phase and CHX treatment, which may point to potential cellular targets for the development of an effective antimicrobial agent.


Assuntos
Anti-Infecciosos Locais/farmacologia , Proteínas de Bactérias/metabolismo , Clorexidina/farmacologia , Regulação Bacteriana da Expressão Gênica , Salmonella enteritidis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Mutação , Proteoma/análise , Proteoma/metabolismo , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/metabolismo
12.
Microb Pathog ; 159: 105119, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34339796

RESUMO

Staphylococcus aureus is an eminent and opportunistic human pathogen that can colonize in the intestines, skin tissue and perineal regions of the host and cause severe infectious diseases. The presence of complex regulatory network and existence of virulent gene expression along with tuning metabolism enables the S. aureus to adopt the diversity of environments. Two component system (TCS) is a widely distributed mechanism in S. aureus that permit it for changing gene expression profile in response of environment stimuli. TCS usually consist of transmembrane histidine kinase (HK) and cytosolic response regulator. S. aureus contains totally 16 conserved pairs of two component systems, involving in different signaling mechanisms. There is a connection among these regulatory circuits and they can easily have effect on each other's expression. This review has discussed five major types of TCS in S. aureus and covers the recent knowledge of their virulence gene expression. We can get more understanding towards staphylococcal pathogenicity by getting insights about gene regulatory pathways via TCS, which can further provide implications in vaccine formation and new ways for drug design to combat serious infections caused by S. aureus in humans.


Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Virulência
13.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445336

RESUMO

Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.


Assuntos
Pseudomonas aeruginosa/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Interferência de RNA/fisiologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo
14.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445389

RESUMO

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.


Assuntos
Peroxidases/genética , Peroxidases/metabolismo , Streptomyces/enzimologia , Sequenciamento Completo do Genoma/métodos , Motivos de Aminoácidos , Sequência de Aminoácidos , Antraquinonas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genoma Bacteriano , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peroxidases/química , Conformação Proteica , Streptomyces/genética , Termodinâmica
15.
Int J Med Microbiol ; 311(6): 151525, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34340061

RESUMO

Recently, multiple bifunctional RNAs have been discovered, which can both be translated into proteins and play regulatory roles. hns encodes the global gene silencing factor H-NS, which is widespread in Gram-negative bacteria. This study reported that hns mRNA of Salmonella enterica serovar Typhi (S. Typhi) was a bifunctional RNA that could act as an antisense RNA downregulating the expression of galU, the coding gene of uridine triphosphate-glucose-1-phosphate uridylyltransferase, and attenuating bacterial motility. galU, which is located at the opposite strand of hns, was identified to have a long 3'-untranslated region that overlapped with hns and could be processed to produce short RNA fragments. The overexpression of hns mRNA inhibited the expression of galU. The deletion of galU attenuated the motility of S. Typhi, while the complementation of galU nearly restored the phenotype. Overexpressing hns mRNA in the wild-type strain of S. Typhi inhibited the motility and the expression of flagellar genes, while overexpressing hns mRNA in the galU-deletion mutant did not influence bacterial motility. In conclusion, hns mRNA has been identified to be a new bifunctional RNA that attenuates the motility of S. Typhi by downregulating the expression of galU.


Assuntos
Salmonella typhi , UTP-Glucose-1-Fosfato Uridililtransferase , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , RNA Mensageiro/genética , Salmonella typhi/genética
16.
Adv Protein Chem Struct Biol ; 127: 343-364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34340773

RESUMO

BACKGROUND AND AIM: The persistence of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (MTB) continue to pose a significant challenge to the treatment and control of tuberculosis infections worldwide. XDR-MTB strains exhibit resistance against first-line anti-TB drugs, fluoroquinolones, and second-line injectable drugs. The mechanisms of drug resistance of MTB remains poorly understood. Our study aims at identifying the differentially expressed genes (DEGs), associated gene networks, and signaling cascades involved in rendering this pathogen resistant to multiple drugs, namely, isoniazid, rifampicin, and capreomycin. METHODS: We used the microarray dataset GSE53843. The GEO2R tool was used to prioritize the most significant DEGs (top 250) of each drug exposure sample between XDR strains and non-resistant strains. The validation of the 250 DEGs was performed using volcano plots. Protein-protein interaction networks of the DEGs were created using STRING and Cytoscape tools, which helped decipher the relationship between these genes. The significant DEGs were functionally annotated using DAVID and ClueGO. The concomitant biological processes (BP) and molecular functions (MF) were represented as dot plots. RESULTS AND CONCLUSION: We identified relevant molecular pathways and biological processes, such as cell wall biogenesis, lipid metabolic process, ion transport, phosphopantetheine binding, and triglyceride lipase activity. These processes indicated the involvement of multiple interconnected mechanisms in drug resistance. Our study highlighted the impact of cell wall permeability, with the dysregulation of the mur family of proteins, as essential factors in the inference of resistance. Additionally, upregulation of genes responsible for ion transport such as ctpF, arsC, and nark3, emphasizes the importance of transport channels and efflux pumps in potentially driving out stress-inducing compounds. This study investigated the upregulation of the Lip family of proteins, which play a crucial role in triglyceride lipase activity. Thereby illuminating the potential role of drug-induced dormancy and subsequent resistance in the mycobacterial strains. Multiple mechanisms such as carboxylic acid metabolic process, NAD biosynthetic process, triglyceride lipase activity, phosphopantetheine binding, organic acid biosynthetic process, and growth of symbiont in host cell were observed to partake in resistance of XDR-MTB. This study ultimately provides a platform for important mapping targets for potential therapeutics against XDR-MTB.


Assuntos
Proteínas de Bactérias , Farmacorresistência Bacteriana/genética , Tuberculose Extensivamente Resistente a Medicamentos , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis , Biologia de Sistemas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tuberculose Extensivamente Resistente a Medicamentos/genética , Tuberculose Extensivamente Resistente a Medicamentos/metabolismo , Humanos
17.
Commun Biol ; 4(1): 957, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381156

RESUMO

Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO2 and renewable electricity1-6. Theoretically EET could do this with an efficiency comparable to H2-oxidation7,8 but without the need for a volatile intermediate and the problems it causes for scale up9. However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-110-14, though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation15.


Assuntos
Regulação Bacteriana da Expressão Gênica , Shewanella/genética , Transdução de Sinais , Transporte de Elétrons/genética
18.
Nat Commun ; 12(1): 4976, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404790

RESUMO

To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL--aidB+-ryfAM--gadAH-, producing 30.0 g L-1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.


Assuntos
Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Ácidos Graxos não Esterificados/biossíntese , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Transcriptoma
19.
Nat Commun ; 12(1): 5150, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446715

RESUMO

Recent studies have revealed the prevalence and biological significance of guanidine metabolism in nature. However, the metabolic pathways used by microbes to degrade guanidine or mitigate its toxicity have not been widely studied. Here, via comparative proteomics and subsequent experimental validation, we demonstrate that Sll1077, previously annotated as an agmatinase enzyme in the model cyanobacterium Synechocystis sp. PCC 6803, is more likely a guanidinase as it can break down guanidine rather than agmatine into urea and ammonium. The model cyanobacterium Synechococcus elongatus PCC 7942 strain engineered to express the bacterial ethylene-forming enzyme (EFE) exhibits unstable ethylene production due to toxicity and genomic instability induced by accumulation of the EFE-byproduct guanidine. Co-expression of EFE and Sll1077 significantly enhances genomic stability and enables the resulting strain to achieve sustained high-level ethylene production. These findings expand our knowledge of natural guanidine degradation pathways and demonstrate their biotechnological application to support ethylene bioproduction.


Assuntos
Proteínas de Bactérias/metabolismo , Etilenos/biossíntese , Instabilidade Genômica , Guanidina/metabolismo , Synechococcus/genética , Synechococcus/metabolismo , Synechocystis/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Synechocystis/genética
20.
Mol Oral Microbiol ; 36(5): 278-290, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34351080

RESUMO

Zinc (Zn2+ ) is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the survival and virulence of bacteria. Thus, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent exogenous zinc introduction, present a serious challenge for bacteria colonizing the oral cavity. However, the regulation strategies to keep intracellular Zn2+ homeostasis in Streptococcus mutans, an important causative pathogen of dental caries, are unknown. Because zinc uptake is primarily mediated by an ATP-binding ABC transporter AdcABC in Streptococcus strains, we examined the function of AdcABC and transcription factor AdcR in S. mutans in this study. The results demonstrated that deletion of either adcA or adcCB gene impaired the growth but enhanced the extracellular polymeric matrix production in S. mutans, both of which could be relieved after excessive Zn2+ supplementation. Using RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction examination, LacZ-reporter studies, and electrophoretic mobility shift assay, we showed that a MarR (multiple antibiotic resistance regulator) family transcription factor, AdcR, negatively regulates the expression of the genes adcR, adcC, adcB, and adcA by acting on the adcRCB and adcA promoters in response to Zn2+ concentration in their environmental niches. The deletion of adcR increases the sensitivity of S. mutans to excessive Zn2+ supply. Taken together, our findings suggest that Adc regulon, which consists of a Zn2+ uptake transporter AdcCBA and a Zn2+ -responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans.


Assuntos
Cárie Dentária , Regulon , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Regulon/genética , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Zinco/metabolismo
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