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1.
J Sci Food Agric ; 102(1): 85-94, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031874

RESUMO

BACKGROUND: Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. RESULTS: The encoding gene of cellobiose 2-epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by-products, because of high kcat (197.6 s-1 ) and preferable thermostability. The Rt8-CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L-1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a ß-galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d-tagatose was successfully produced from lactose through the co-expression of l-arabinose isomerase and ß-galactosidase. CONCLUSION: Our study shed light on the efficient production of epilactose using a food-grade host expressing a novel CE enzyme. Moreover, an efficient and low-cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d-tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Celobiose/metabolismo , Dissacarídeos/biossíntese , Racemases e Epimerases/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/genética , Estabilidade Enzimática , Expressão Gênica , Temperatura Alta , Lactose/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Braz J Biol ; 83: e250550, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34730714

RESUMO

Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.


Assuntos
Benzaldeídos , Aromatizantes , Alcaligenes faecalis/metabolismo , Bacillus subtilis/metabolismo , Benzaldeídos/metabolismo , Meios de Cultura , Enterococcus faecium/metabolismo , Fermentação , Aromatizantes/metabolismo , Microbiologia Industrial , Pseudomonas fluorescens/metabolismo
3.
Enzyme Microb Technol ; 150: 109863, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489022

RESUMO

Protein-glutaminase (EC 3.5.1.44, PG) converts protein glutamine residues in proteins and peptides into glutamic acid residue, and markedly improves the solubility, emulsification, and foaming properties of food proteins. However, the source bacteria, Chryseobacterium proteolyticum, have low enzyme production ability, inefficient genetic operation, and high production cost. Therefore, it is critical to establish an efficient expression system for active PG. Here, combinatorial engineering was developed for high-yield production of PG in Bacillus subtilis. First, we evaluated different B. subtilis strains for PG self-activation. Then, combinatorial optimization involving promoters, signal peptides, and culture medium was applied to produce active recombinant PG in a B. subtilis expression system. Through combinatorial engineering, PG enzyme activity reached 3.23 U/mL in shaken-flask cultures. Active PG with the yield of 7.07 U/mL was obtained at 40 h by the PSecA-YdeJ combination in fed-batch fermentation, which is the highest yield of PG in existing reports.


Assuntos
Bacillus subtilis , Proteínas de Bactérias/biossíntese , Chryseobacterium , Glutaminase/biossíntese , Bacillus subtilis/metabolismo , Chryseobacterium/enzimologia , Fermentação , Engenharia de Proteínas , Sinais Direcionadores de Proteínas
4.
Nat Commun ; 12(1): 5429, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521822

RESUMO

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Código Genético , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/classificação , Aminoacil-tRNA Sintetases/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Códon , Citocinese/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo
5.
Biochemistry (Mosc) ; 86(8): 942-951, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488571

RESUMO

Translation of the genetic information into proteins, performed by the ribosome, is a key cellular process in all organisms. Translation usually proceeds smoothly, but, unfortunately, undesirable events can lead to stalling of translating ribosomes. To rescue these faulty arrested ribosomes, bacterial cells possess three well-characterized quality control systems, tmRNA, ArfA, and ArfB. Recently, an additional ribosome rescue mechanism has been discovered in Bacillus subtilis. In contrast to the "canonical" systems targeting the 70S bacterial ribosome, this latter mechanism operates by first splitting the ribosome into the small (30S) and large (50S) subunits to then clearing the resultant jammed large subunit from the incomplete nascent polypeptide. Here, I will discuss the recent microbiological, biochemical, and structural data regarding functioning of this novel rescue system.


Assuntos
Biossíntese de Proteínas , RNA Bacteriano/química , Ribossomos/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Bioquímica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Conformação de Ácido Nucleico , Peptídeos/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
6.
Int J Mol Sci ; 22(17)2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34502476

RESUMO

Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H2O2. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2'-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Técnicas Biossensoriais , Microrganismos Geneticamente Modificados , Plasmídeos , Regiões Promotoras Genéticas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
7.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502250

RESUMO

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation.


Assuntos
Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteoma/análise , Proteoma/metabolismo , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologia , Bacillus subtilis/citologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
8.
J Agric Food Chem ; 69(40): 11835-11846, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34590486

RESUMO

BsCsn46A, a GH46 family chitosanase from Bacillus subtilis, has great potential for industrial chitooligosaccharide production due to its high activity and stability. In this study, a special amino acid Pro121 was identified not fit in the helix structure, which was located in the opposite side of the active center in BsCsn46A, by the PoPMuSiC algorithm. Then, saturation mutagenesis was performed to explore the role of the site amino acid 121. Compared with the wild type, the specific activity of P121N, P121C, and P121V was increased by 1.69-, 1.97-, and 2.15-fold, respectively. In particular, the specific activity of P121N was increased without loss of thermostability, indicating that replacing the structural stiffness of proline in the helical structure could significantly improve the chitosanase activity. The Km values of P121N, P121C, and P121V decreased significantly, indicating that the affinity between the enzyme-substrate complex was enhanced. Through molecular docking, it was found that the increase of hydrogen bonds and van der Waals force between the enzyme-substrate complex and the removal of unfavorable bonds might be the main reason for the change of enzyme properties. In addition, the optimal temperature of the three mutants changed from 60 to 55 °C. These results indicate that the site 121 plays a critical role in the catalytic activity and enzymatic properties of chitosanase. To our knowledge, the results provide novel data on chitosanase activity and identify an excellent candidate of industrial chitosanase.


Assuntos
Bacillus subtilis , Glicosídeo Hidrolases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Mutagênese , Temperatura
9.
J Proteome Res ; 20(10): 4886-4892, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34473931

RESUMO

Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signaling functions, indicating importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment, whereas automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high-throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems level, we here established an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO2 and Fe(III)-NTA-IMAC cartridge-based enrichment and the most advantageous, species-specific loading amounts for Streptococcus pyogenes, Listeria monocytogenes, and Bacillus subtilis. For higher sample amounts (≥250 µg), we observed superior performance of the Fe(III)-NTA cartridges, whereas for lower sample amounts (≤100 µg), TiO2-based enrichment is equally efficient. Both cartridges largely enriched the same set of phosphopeptides, suggesting no improvement of peptide yield by the complementary use of the two cartridges. Our data represent, to the best of our knowledge, the largest phosphoproteome identified in a single study for each of these bacteria.


Assuntos
Cromatografia de Afinidade , Fosfopeptídeos , Bacillus subtilis/metabolismo , Listeria monocytogenes/metabolismo , Fosfopeptídeos/metabolismo , Fosforilação , Proteoma/metabolismo , Streptococcus pyogenes/metabolismo , Titânio
10.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576222

RESUMO

Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism Bacillus subtilis can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate B. subtilis persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both B. subtilis vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant B. subtilis spores are not susceptible to the antimicrobial peptides.


Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Separação Celular , Tolerância a Medicamentos , Escherichia coli/efeitos dos fármacos , Citometria de Fluxo , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase
11.
Biochemistry ; 60(37): 2781-2794, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34472844

RESUMO

RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis. We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg2+ and Sr2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg2+. Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.


Assuntos
Cátions Bivalentes/metabolismo , Conformação de Ácido Nucleico/efeitos dos fármacos , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sítios de Ligação/genética , Cátions Bivalentes/química , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/química , Riboswitch/fisiologia , Transcrição Genética/genética
12.
Nat Microbiol ; 6(9): 1175-1187, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373624

RESUMO

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.


Assuntos
Proteínas de Bactérias/metabolismo , Ciclo Celular , Proteínas do Citoesqueleto/metabolismo , Replicação do DNA , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Streptococcus pneumoniae/genética
13.
Mol Cell ; 81(16): 3310-3322.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416138

RESUMO

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.


Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismo
14.
J Biol Chem ; 297(3): 101078, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34400169

RESUMO

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram-positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through the ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized Domain of Unknown Function 1775 (DUF1775) containing YcnI. DUF1775 domains are found across bacterial phylogeny, and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance and inductively coupled plasma-MS, found that this protein can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper-binding site featuring a unique monohistidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.


Assuntos
Bacillus subtilis/metabolismo , Cobre/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Quelantes/metabolismo , Cristalografia por Raios X/métodos , Regulação Bacteriana da Expressão Gênica/genética , Homeostase , Ligantes , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Óperon/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo
15.
Microb Physiol ; 31(3): 260-279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34438398

RESUMO

Fast adaptation to environmental changes ensures bacterial survival, and proteolysis represents a key cellular process in adaptation. The Clp protease system is a multi-component machinery responsible for protein homoeostasis, protein quality control, and targeted proteolysis of transcriptional regulators in prokaryotic cells and prokaryote-derived organelles of eukaryotic cells. A functional Clp protease complex consists of the tetradecameric proteolytic core ClpP and a hexameric ATP-consuming Clp-ATPase, several of which can associate with the same proteolytic core. Clp-ATPases confer substrate specificity by recognising specific degradation tags, and further selectivity is conferred by adaptor proteins, together allowing for a fine-tuned degradation process embedded in elaborate regulatory networks. This review focuses on the contribution of the Clp protease system to prokaryotic survival and summarises the current state of knowledge for exemplary bacteria in an increasing degree of interaction with eukaryotic cells. Starting from free-living bacteria as exemplified by a non-pathogenic and a pathogenic member of the Firmicutes, i.e., Bacillus subtilis and Staphylococcus aureus, respectively, we turn our attention to facultative and obligate intracellular bacterial pathogens, i.e., Mycobacterium tuberculosis, Listeria monocytogenes, and Chlamydia trachomatis, and conclude with mitochondria. Under stress conditions, the Clp protease system exerts its pivotal role in the degradation of damaged proteins and controls the timing and extent of the heat-shock response by regulatory proteolysis. Key regulators of developmental programmes like natural competence, motility, and sporulation are also under Clp proteolytic control. In many pathogenic species, the Clp system is required for the expression of virulence factors and essential for colonising the host. In accordance with its evolutionary origin, the human mitochondrial Clp protease strongly resembles its bacterial counterparts, taking a central role in protein quality control and homoeostasis, energy metabolism, and apoptosis in eukaryotic cells, and several cancer cell types depend on it for proliferation.


Assuntos
Bacillus subtilis , Endopeptidase Clp , Bacillus subtilis/metabolismo , Endopeptidase Clp/genética , Homeostase , Humanos , Mitocôndrias/metabolismo , Staphylococcus aureus/metabolismo
16.
Mol Cell ; 81(17): 3623-3636.e6, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34270916

RESUMO

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.


Assuntos
Bacillus subtilis/citologia , Citidina Trifosfato/metabolismo , Pirofosfatases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Cromossomos Bacterianos/genética , Citidina Trifosfato/fisiologia , Proteínas do Citoesqueleto/genética , Pirofosfatases/fisiologia
17.
Sci Rep ; 11(1): 13731, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215768

RESUMO

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.


Assuntos
Álcool Desidrogenase/genética , Bacillus subtilis/genética , Etanol/metabolismo , Engenharia Metabólica , Piruvato Descarboxilase/genética , Bacillus subtilis/metabolismo , Biomassa , Etanol/química , Fermentação/genética , Hidrólise , Ácido Láctico/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Zymomonas/enzimologia , Zymomonas/genética
18.
PLoS One ; 16(7): e0254796, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34297729

RESUMO

Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.


Assuntos
Antibacterianos/farmacologia , Daptomicina/farmacologia , Farmacorresistência Bacteriana , Enterococcus faecalis/efeitos dos fármacos , Fosfomicina/farmacologia , Peptidoglicano/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Sinergismo Farmacológico , Enterococcus faecalis/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
19.
Biomolecules ; 11(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201916

RESUMO

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Transporte Proteico/fisiologia , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Histidina Quinase/química , Ligação de Hidrogênio
20.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207153

RESUMO

Aerobic denitrification is considered as a promising biological method to eliminate the nitrate contaminants in waterbodies. However, the molecular mechanism of this process varies in different functional bacteria. In this study, the nitrogen removal characteristics for a newly isolated aerobic denitrifier Bacillus subtilis JD-014 were investigated, and the potential functional genes involved in the aerobic denitrification process were further screened through transcriptome analysis. JD-014 exhibited efficient denitrification performance when having sodium succinate as the carbon source with the range of nitrate concentration between 50 and 300 mg/L. Following the transcriptome data, most of the up-regulated differentially expressed genes (DEGs) were associated with cell motility, carbohydrate metabolism, and energy metabolism. Moreover, gene nirsir annotated as sulfite reductase was screened out and further identified as a regulator participating in the nitrogen removal process within JD-014. The findings in present study provide meaningful information in terms of a comprehensive understanding of genetic regulation of nitrogen metabolism, especially for Bacillus strains.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Carbono/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Ontologia Genética , Nitratos/metabolismo , Nitrificação , Reprodutibilidade dos Testes
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