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1.
PLoS One ; 15(7): e0233945, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701964

RESUMO

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.


Assuntos
Brevibacterium/metabolismo , Queijo/microbiologia , Etanolamina/metabolismo , Microbiologia de Alimentos , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Propilenoglicol/metabolismo , Psychrobacter/metabolismo , Transcriptoma , Aclimatação , Ágar , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Técnicas de Cocultura , Meios de Cultura , Transporte de Elétrons/genética , Fermentação/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Plasmídeos , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Virulência/genética
2.
Int J Food Microbiol ; 328: 108668, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32474228

RESUMO

Biofilms are comprised of microorganisms embedded in a self-produced matrix that normally adhere to a surface. In the food processing environment they are suggested to be a source of contamination leading to food spoilage or the transmission of food-borne pathogens. To date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without measuring the associated biofilm matrix components. Here, we assessed the presence of biofilms within a meat processing environment, processing pork, poultry and beef, by the detection of microorganisms and at least two biofilm matrix components. Sampling included 47 food contact surfaces and 61 non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. The 108 samples were analysed for the presence of microorganisms by cultivation and targeted quantitative real-time PCR based on 16S rRNA. Furthermore, the presence of the major matrix components carbohydrates, extracellular DNA and proteins was evaluated. Overall, we identified ten biofilm hotspots, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these biofilm positive samples, we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix (present in 80 % of biofilms), Pseudomonas and Psychrobacter (isolated from 70 % biofilms). From each biofilm we isolated bacteria from four to twelve different genera, indicating the presence of multi-species biofilms. This work ultimately determined the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Especially the identification of biofilms in water hoses and associated parts highlights the need of a frequent monitoring at these sites. The knowledge gained about the presence and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.


Assuntos
Brochothrix/isolamento & purificação , Manipulação de Alimentos , Carne/microbiologia , Pseudomonas/isolamento & purificação , Psychrobacter/isolamento & purificação , Animais , Áustria , Biofilmes/classificação , Biofilmes/crescimento & desenvolvimento , Bovinos , Desinfecção/métodos , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Aves Domésticas/microbiologia , RNA Ribossômico 16S/análise
3.
Int J Food Microbiol ; 327: 108658, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32464431

RESUMO

The total cultivable microbiota of the ice-stored European sea bass (Dicentrarchus labrax), the most important commercial fish species of the Mediterranean aquaculture, was determined using 16S rRNA gene sequence analysis. High Resolution Melting (HRM) curve profiles and sequencing analysis (V3-V4 region of the 16S rRNA gene) were used respectively for the differentiation and identification of the collected isolates from six time intervals (day 0, 4, 8, 12, 14 and 16) while fish were stored in ice. HRM analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles. The isolates with HRM profiles which were >91% similar within each group were found to belong to the same species using sequencing analysis. Thus, the ten groups consist of representatives of Psychrobacter glacincola, Ps. alimentarius, Ps. cryohalolentis, Ps. maritimus, Ps. fozii, Pseudomonas sp., Paeniglutamicibacter sp., Carnobacterium sp., Leucobacter aridicolis and Bacillus thuringiensis. Based on this approach, Ps. cryohalolentis was found to be the most dominant phylotype at the beginning of fish shelf-life compared to other species. The abundance of this bacterium decreased throughout storage, while Ps. glacincola increased and dominated at the time of the sensory minimum acceptability (day 14) and rejection (day 16). To conclude, HRM could be used for the rapid determination of sea bass microbiota, using the representatives of each group as reference bacterial strains, in order for scientists to solve rapidly stakeholders problems related with microbial quality or safety of fish.


Assuntos
Aquicultura/métodos , Bactérias/classificação , Bactérias/genética , Bass/microbiologia , Manipulação de Alimentos , RNA Ribossômico 16S/genética , Animais , Bactérias/isolamento & purificação , Gelo , Desnaturação de Ácido Nucleico , Pseudomonas/isolamento & purificação , Psychrobacter/genética
4.
Extremophiles ; 24(4): 537-549, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32418069

RESUMO

(7R,8S)-diaminopelargonic acid transaminase from the cold-adapted Gram-negative bacterium Psychrobacter cryohalolentis (Pcryo361) is able to react with unnatural substrates including (S)-( +)-1-phenylethylamine, aldehydes and α-diketones. Additionally, Pcryo361 is active at 0-50 °C and retains up to 10% of the maximum activity at 0 °C. Here, we report a detailed study on the stability and low temperature activity of Pcryo361. At the optimal pH for (S)-amine activity (pH 10.0), the enzyme was stable at 0-10 °C and no decrease in the enzyme activity was observed within 24 h in a slightly alkaline medium, pH 8.0, at 35 °C. Pcryo361 was solvent stable and was activated in 10% DMSO and DMFA at 35 °C. An analysis of the efficiency of catalysis of Pcryo361 at 35 °C and 10 °C showed that the specificity towards (S)-( +)-1-phenylethylamine dropped at 10 °C; however, the specificity towards 2,3-butanedione remained unchanged. Inhibition analysis showed that Pcryo361 activity was not inhibited by acetophenone but inhibited by amines (products of aldehyde amination). The observed pH stability and low temperature activity of Pcryo361 with activated keto substrates are attractive features in the field of development of stereoselective amination at low temperatures.


Assuntos
Psychrobacter , Aminas , Sequência de Aminoácidos , Temperatura Baixa , Concentração de Íons de Hidrogênio , Transaminases
5.
Extremophiles ; 24(1): 63-70, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31309337

RESUMO

In this paper, the structure of the capsular polysaccharide isolated from the psychrotolerant bacterium Psychrobacter arcticus 273-4 is reported. The polymer was purified by gel filtration chromatography and the structure was elucidated by means of one- and two-dimensional NMR spectroscopy, in combination with chemical analyses. The polysaccharide consists of a trisaccharidic repeating unit containing two residues of glucose and a residue of a N,N-diacetyl-pseudaminic acid.


Assuntos
Psychrobacter , Parede Celular , Espectroscopia de Ressonância Magnética , Polissacarídeos
6.
Int J Syst Evol Microbiol ; 70(1): 211-219, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31617840

RESUMO

One slightly beige-white pigmented, Gram-stain-negative, rod-shaped bacterium, strain I-STPP5bT, was isolated from the trachea of a Gentoo penguin chick individual (Pygoscelin papua) investigated in Fildes Bay, Chilean Antarctic (62° 12' S, 58° 57' W). I-STPP5bT consists of a 3.4 Mb chromosome with a DNA G+C content of 44.4 mol%. Of the 3056 predicted genes, 1206 were annotated as hypothetical proteins and 51 were tRNAs. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the isolate shared a 16S rRNA gene sequence identity to the type strains of Psychrobacter phenylpyruvicus (98.8 %), Psychrobacter arenosus and Psychrobacter pasteurii (both 98.3 %), Psychrobacter piechaudii (98.2 %) and Psychrobacter sanguinis (98.1 %), but 16S rRNA gene sequence similarities to all other Psychrobacter species were ≤98.0 %. Partial gyrB nucleotide and amino acid sequence similarities among strain STPP5bT and the next related type strains were all below 81.8 and 92.9%, respectively. DNA-DNA hybridisation (DDH) with P. phenylpyruvicus LMG 5372T, P. arenosus DSM 15389T and P. sanguinis DSM 23635T also showed low values (all below 30 %). The main cellular fatty acids of the strain were C18 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c. Based on phylogenetic, chemotaxonomic, genomic and phenotypic analyses we propose a new species of the genus Psychrobacter, with the name Psychrobacter pygoscelis sp. nov. and strain I-STPP5bT (=CIP 111410T= CCM 8799T=LMG 30301T) as type strain.


Assuntos
Filogenia , Psychrobacter/classificação , Spheniscidae/microbiologia , Traqueia/microbiologia , Animais , Regiões Antárticas , Técnicas de Tipagem Bacteriana , Composição de Bases , Chile , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Psychrobacter/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
7.
Enzyme Microb Technol ; 131: 109434, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615682

RESUMO

Widely used in multiple industrial processes, nitro-aromatic compounds, especially nitrobenzene, in low temperature environment are considered as heavy pollutants. Among the disposal methods, the bioreduction method has attracted much attention. In this study, a novel cold-adapted nitroreductase gene (psntr) was cloned from Antarctic sea-ice bacteria Psychrobacter sp. ANT206. The psntr gene was 813 bp in length and encoded a protein with flavin mononucleotide (FMN) binding sites. Homology modeling was performed to obtain structural information such as the longer loops and reduced amount of hydrogen bonds, which might be related to the high catalytic efficiency of PsNTR at low temperature. The psntr gene was successfully cloned in cold-shock pCold I vector and transformed to the expression host Escherichia coli (E. coli) BL21 with the induction by isopropyl ß-D-thiogalactoside (IPTG) at low temperature (16 °C) for 24 h. The recombinant PsNTR (rPsNTR) was purified using Ni-NTA with the specific activity of 51.59 µmol/min/mg. Interestingly, rPsNTR displayed the highest activity at 25 °C and still maintained 46.9% of the activity at 0 °C. rPsNTR also exhibited the highest activity (136.4%) at 1.0 M NaCl with incredible salt tolerance. The kinetic parameters and substrates specificity analysis demonstrated that rPsNTR could reduce various nitro-aromatic compounds. Moreover, the result of the reduction capability revealed that the recombinant E. coli exhibited a maximum nitrobenzene reduction rate of 3.03 mM/h at 16 °C. These findings revealed that the characteristics of rPsNTR might make it an excellent candidate for the bioreduction of various nitro-aromatic compounds in the low temperature and high-salt wastewater.


Assuntos
Temperatura Baixa , Nitrobenzenos/metabolismo , Nitrorredutases/isolamento & purificação , Nitrorredutases/metabolismo , Psychrobacter/enzimologia , Biotransformação , Clonagem Molecular , Biologia Computacional , Poluentes Ambientais/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Nitrorredutases/química , Nitrorredutases/genética , Oxirredução , Conformação Proteica , Psychrobacter/genética , Cloreto de Sódio/metabolismo
8.
Curr Microbiol ; 76(12): 1435-1442, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31494741

RESUMO

Bacteria under stress increase the proportion of dormant cells to ensure their survival. Cold and osmotic stress are similar, because in both the availability of water is reduced. Glycine betaine (GB) is one of the most common osmoprotectants in bacteria and possesses cryoprotectant properties. Our aim was to determine whether GB modifies the proportion of dormant Deinococcus sp. UDEC-P1 and Psychrobacter sp. UDEC-A5 cells exposed to osmotic stress. Both bacterial strains were incubated in the presence of up to 1 M NaCl with or without GB. Active and dormant cells were evaluated by both spectrophotometric and flow cytometry analysis. Without GB, Deinococcus sp. UDEC-P1 grew in the presence of 0.05 M NaCl, but with 5 mM GB grew at 0.1 M NaCl. Psychrobacter sp. UDEC-A5 grew in the presence of up to 0.25 M NaCl, but with 5 mM GB grew at 0.5 M NaCl. Under osmotic stress, the proportion of dormant cells of Deinococcus sp. UDEC-P1 and Psychrobacter sp. UDEC-A5 increased significantly (about eightfold and fivefold, respectively). The addition of GB (5 mM) exerted a different effect on the two strains, since it avoided the entrance into the dormancy of Psychrobacter sp. UDEC-A5 cells, but not of Deinococcus sp. UDEC-P1 cells. Our results suggest that the effect of GB on bacterial metabolism is strain dependent. For bacteria in which GB avoids dormancy, such as Psychrobacter sp. UDEC-A5, it could be a "double-edged sword" by reducing the "seed bank" available to recover the active population when favorable conditions return.


Assuntos
Betaína/metabolismo , Deinococcus/crescimento & desenvolvimento , Psychrobacter/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Deinococcus/fisiologia , Pressão Osmótica , Psychrobacter/fisiologia , Cloreto de Sódio/metabolismo , Estresse Fisiológico
9.
Molecules ; 24(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207974

RESUMO

A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and encoded a protein (PsRNR) of 770 amino acids. Homology modeling indicated that PsRNR had reduced hydrogen bonds and salt bridges, which might be the main reason for the catalytic efficiency at low temperatures. A site directed mutation exhibited that His 667 in the active site was absolutely crucial for the enzyme catalysis. The recombinant PsRNR (rPsRNR) showed maximum activity at 30 °C and had thermal instability, suggesting that rPsRNR was a cold-adapted enzyme. Interestingly, rPsRNR displayed remarkable salt tolerance, remaining stable at 0.5-3.0 M NaCl. Furthermore, rPsRNR had a higher kcat value, contributing to its efficient catalytic activity at a low temperature. Overall, cold-adapted RNase R in this study was an excellent candidate for antimicrobial treatment.


Assuntos
Adaptação Biológica , Temperatura Baixa , Microbiologia Ambiental , Camada de Gelo/microbiologia , Psychrobacter/fisiologia , Ribonucleases/metabolismo , Tolerância ao Sal , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ativação Enzimática , Cinética , Modelos Biológicos , Conformação Molecular , Estrutura Molecular , Psychrobacter/isolamento & purificação , Ribonucleases/genética
10.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 324-331, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045561

RESUMO

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.


Assuntos
Proteínas de Bactérias/química , Hidrocarbonetos Halogenados/química , Hidrolases/química , Psychrobacter/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hidrocarbonetos Halogenados/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Psychrobacter/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica
11.
Nat Commun ; 10(1): 1691, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979882

RESUMO

On coral reefs, microorganisms are essential for recycling nutrients to primary producers through the remineralization of benthic-derived organic matter. Diel investigations of reef processes are required to holistically understand the functional roles of microbial players in these ecosystems. Here we report a metagenomic analysis characterizing microbial communities in the water column overlying 16 remote forereef sites over a diel cycle. Our results show that microbial community composition is more dissimilar between day and night samples collected from the same site than between day or night samples collected across geographically distant reefs. Diel community differentiation is largely driven by the flux of Psychrobacter sp., which is two-orders of magnitude more abundant during the day. Nighttime communities are enriched with species of Roseobacter, Halomonas, and Alteromonas encoding a greater variety of pathways for carbohydrate catabolism, further illustrating temporal patterns of energetic provisioning between different marine microbes. Dynamic diel fluctuations of microbial populations could also support the efficient trophic transfer of energy posited in coral reef food webs.


Assuntos
Recifes de Corais , Microbiota , Fotoperíodo , Alteromonas , Ecossistema , Monitoramento Ambiental , Halomonas , Compostos Orgânicos/química , Oceano Pacífico , Psychrobacter , RNA Ribossômico/química , Roseobacter
12.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022896

RESUMO

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.


Assuntos
Plasmídeos/genética , Psychrobacter/genética , Regiões Árticas , Aderência Bacteriana , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Carnitina/metabolismo , Elementos de DNA Transponíveis , Fenótipo , Plasmídeos/metabolismo , Psychrobacter/fisiologia
13.
Dev Comp Immunol ; 96: 9-17, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30790604

RESUMO

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Peixes/farmacologia , Psychrobacter/efeitos dos fármacos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Mutação , Psychrobacter/fisiologia , Coelhos , Testes de Toxicidade
14.
Int J Food Microbiol ; 293: 102-113, 2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30677559

RESUMO

Thawed hake (Merluccius capensis and M. paradoxus) and plaice (Pleuronectes platessa) fillets were used as a model to evaluate the effect of storage temperature (0 or 10 °C) and biological variability (fish species, lot to lot) on bacterial growth kinetics and microbial successions. Both culture dependent methods (plate counts on non-selective and selective media) and culture independent methods (qPCR and 16S rRNA gene metabarcoding) were used. Bacterial counts exceeded 107 cfu/g within 2-3 days at 10 °C and 7-8 days at 0 °C. Plate counts on three media (Plate Count Agar +0.5% NaCl, Iron Agar Lyngby and Pseudomonas Selective medium) and 16S rRNA gene counts estimated by qPCR were highly correlated. Growth was modelled using the D-model and specific growth rate ranged between 0.97 and 1.24 d-1 and 3.54 and 5.90 d-1 at 0 and 10 °C, respectively. The initial composition of the microbiota showed lot-to-lot variation, but significant differences between the two fish species were detected. Alpha diversity significantly decreased during storage. When bacterial counts exceeded 107 cfu/g, the microbiota was dominated by members of the genera Pseudomonas, Psychrobacter, Acinetobacter, Serratia, Flavobacterium, Acinetobacter, Carnobacterium, Brochothrix and Vagococcus. However, Photobacterium and Shewanella, two genera frequently associated with fish spoilage, were either absent or minor components of the microbiota. As expected, storage temperature significantly affected the abundance of several species. The inference of microbial association networks with three different approaches (an ensemble approach using the CoNet app, Sparse Correlations for Compositional data, and SParse InversE Covariance Estimation for Ecological Association Inference) allowed the detection of both a core microbiota, which was present throughout storage, and a number of taxa, which became dominant at the end of spoilage and were characterized by a disproportionate amount of negative interactions.


Assuntos
Contaminação de Alimentos/análise , Armazenamento de Alimentos , RNA Ribossômico 16S/isolamento & purificação , Alimentos Marinhos/microbiologia , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Animais , Carga Bacteriana , Brochothrix/genética , Brochothrix/isolamento & purificação , Carnobacterium/genética , Carnobacterium/isolamento & purificação , Temperatura Baixa , Contagem de Colônia Microbiana , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Peixes , Microbiologia de Alimentos , Consórcios Microbianos , Photobacterium/genética , Photobacterium/isolamento & purificação , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Psychrobacter/genética , Psychrobacter/isolamento & purificação , RNA Ribossômico 16S/genética , Shewanella/genética , Shewanella/isolamento & purificação
15.
Int J Food Microbiol ; 293: 87-93, 2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30677560

RESUMO

The effect of air packaging (AP), vacuum packaging (VP) and modified atmosphere packaging (MAP, 75% CO2/25% N2) on the composition of the microbial community of lightly salted grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C was studied. Physicochemical characteristics (total volatile basic nitrogen (TVB-N) value, pH value and biogenic amines (BA)) were also monitored. Based on sensory evaluation, the shelf life of fillets under AP, VP, and MAP was 8, 16 and 24 days, respectively. High-throughput sequencing showed that Acinetobacter and Pseudomonas dominated the microflora of fresh grass carp. At the sensory rejection time, Pseudomonas and Psychrobacter dominated the AP products. Lactococcus became the predominant genus of both VP and MAP fillets, and there was no obvious difference in the composition of the dominant microbiota between these two treatments at the end of the shelf life.


Assuntos
Carpas/microbiologia , Embalagem de Alimentos , Conservação de Alimentos , Vácuo , Acinetobacter/isolamento & purificação , Adulto , Animais , Atmosfera , Aminas Biogênicas/análise , DNA Bacteriano/isolamento & purificação , Feminino , Armazenamento de Alimentos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Concentração de Íons de Hidrogênio , Lactococcus/isolamento & purificação , Masculino , Microbiota/efeitos dos fármacos , Pseudomonas/isolamento & purificação , Psychrobacter/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificação , Cloreto de Sódio , Paladar , Adulto Jovem
16.
Microb Biotechnol ; 12(5): 1049-1063, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-29105344

RESUMO

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell-cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth-limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture-based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and ParacoccusJM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.


Assuntos
Acil-Butirolactonas/metabolismo , Produtos Biológicos/metabolismo , Paracoccus/efeitos dos fármacos , Poríferos/microbiologia , Pseudoalteromonas/efeitos dos fármacos , Psychrobacter/metabolismo , Percepção de Quorum/efeitos dos fármacos , Animais , Psychrobacter/isolamento & purificação , Virulência/efeitos dos fármacos
17.
Int J Biol Macromol ; 129: 1047-1055, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30240713

RESUMO

Peroxiredoxin (Prx, EC 1.11.1.15) is a family of the thiol-dependent antioxidant enzyme. In this study, a cold-adapted Prx gene from Antarctic psychrophilic bacterium Psychrobacter sp. ANT206 (PsPrx) consisted of an open reading frame (ORF) of 567 bp was cloned. Amino acid sequence analysis revealed that PsPrx contained one catalytic site (Thr45, Cys48 and Arg121) and could be categorized as a typical 2-Cys Prx. Compared with the mesophilic StPrx, PsPrx with a reduced amount of hydrogen bonds and salt bridges and other characteristics, may be responsible for its enzymatic stability and flexibility at low temperature. The recombinant PsPrx (rPsPrx) was purified to homogeneity by Ni-NTA and its enzymatic characterization was described. Interestingly, rPsPrx exhibited the maximum activity at 30 °C and remained 42.6% of its maximum activity at 0 °C. rPsPrx was a salt-tolerance enzyme that showed 42.2% of its maximum activity under 2.5 M NaCl. The kinetic parameters of different substrates revealed that it could efficiently catalyze the peroxides, especially H2O2 and t-BOOH (tert­butyl hydroperoxide). Moreover, rPsPrx exhibited the ability to protect super-coiled DNA from oxidative damage. These results indicated that rPsPrx has special catalytic properties and may be a promising candidate for food and industrial applications.


Assuntos
Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Psychrobacter/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Temperatura Baixa , DNA Bacteriano/metabolismo , Escherichia coli/genética , Expressão Gênica , Cinética , Modelos Moleculares , Peroxirredoxinas/química , Multimerização Proteica , Estrutura Quaternária de Proteína , Psychrobacter/genética , Homologia de Sequência de Aminoácidos
18.
Arch Microbiol ; 201(5): 559-569, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30448872

RESUMO

Cold-active bacteria are currently of great interest in biotechnology, and their genomic and physiological features have been extensively studied. One of the model psychrotolerant bacteria are Psychrobacter spp. Analysis of Arctic psychrophilic Psychrobacter sp. DAB_AL32B genome content provided an insight into its overall stress response, and genes conferring protection against various life-limiting factors (i.e., low temperature, increased ultraviolet radiation, oxidative stress and osmotic pressure) were recognized and described. Moreover, it was revealed that the strain carries a large plasmid pP32BP2. Its replication system was used for the construction of two novel shuttle vectors (pPS-NR-Psychrobacter-Escherichia coli-specific plasmid and pPS-BR-Psychrobacter-various Proteobacteria-specific plasmid) of an increased carrying capacity, which may be used for genetic engineering of Psychrobacter spp.


Assuntos
Vetores Genéticos/genética , Genoma Bacteriano/genética , Plasmídeos/genética , Psychrobacter/genética , Regiões Árticas , Temperatura Baixa , Conservação dos Recursos Naturais , Escherichia coli/genética , Genômica/métodos , Raios Ultravioleta
19.
J Org Chem ; 83(23): 14323-14337, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30388010

RESUMO

Lipopolysaccharides (LPSs) play key roles in humoral immunity. Recently, the LPS structure of the Psychrobacter cryohalolentis K5T strain was reported. Due to the presence of unnatural amino sugars and branched linkages, its structure is unique. Herein we report the total synthesis of an LPS analogue of P. cryohalolentis K5T. After overcoming the issues like ring conformation changes and elimination of triflate, we were able to develop a strategy for the synthesis of the newly reported 2,3,4-triacetamido-2,3,4-trideoxy-l-arabinose derivative. Coupling of different donors with suitable acceptors from the nonreducing end to the reducing end and further functional group modifications delivered the protected LPS hexasaccharide repeating unit. After functional group modifications, we were unable to oxidize the hindered primary hydroxyl group to synthesize the target molecule. Alternatively, removal of the permanent protecting groups afforded the LPS hexasaccharide repeating unit analogue of Psychrobacter cryohalolentis K5T.


Assuntos
Amino Açúcares/síntese química , Polissacarídeos/síntese química , Polissacarídeos/metabolismo , Psychrobacter/metabolismo , Configuração de Carboidratos
20.
Biochemistry ; 57(49): 6757-6761, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30472832

RESUMO

The temperature dependence of psychrophilic and mesophilic ( R)-3-hydroxybutyrate dehydrogenase steady-state rates yields nonlinear and linear Eyring plots, respectively. Solvent viscosity effects and multiple- and single-turnover pre-steady-state kinetics demonstrate that while product release is rate-limiting at high temperatures for the psychrophilic enzyme, either interconversion between enzyme-substrate and enzyme-product complexes or a step prior to it limits the rate at low temperatures. Unexpectedly, a similar change in the rate-limiting step is observed with the mesophilic enzyme, where a step prior to chemistry becomes rate-limiting at low temperatures. This observation may have implications for past and future interpretations of temperature-rate profiles.


Assuntos
Hidroxibutirato Desidrogenase/química , Hidroxibutirato Desidrogenase/metabolismo , Acetoacetatos/metabolismo , Acinetobacter baumannii/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Cinética , Modelos Lineares , Modelos Biológicos , Psychrobacter/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solventes , Especificidade por Substrato , Temperatura , Valeratos/metabolismo , Viscosidade
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