RESUMO
The extracellular polymeric substances (EPS) have shown free radical scavenging and antitumor activity against both breast and colon cell lines. In this regard, actinobacteria have become an increasingly popular sources of EPS. Therefore, in this study four Streptomyces strains isolated from contaminated soil (M7, A5, A14 and MC1) were evaluated for determining its biofilm-forming capacity including under pesticide stress. In addition, chemical composition of EPS and its cytotoxic effects over 4T1 breast cancer cell and Caco-2 human tumor colon cells were evaluated. The results demonstrated that Streptomyces sp. A5 had the highest capability to develop biofilm more than other strains tested, even under pesticide stress. Moreover, this strain produced EPS with a total protein/total polysaccharide rate of 1.59 ± 0.05. On the other hand, cytotoxicity assays of EPS showed that Streptomyces sp. A5 display a higher toxic effect against 4T1 Breast cancer cells (96.2 ± 13.5 %), Caco-2 (73.9 ± 6.4 %) and low toxicity (29.9 % ± 9.1 %) against non-transformed intestinal cells (IEC-18). Data do not show cytotoxic effect relationship with biofilm-forming capabilities of strains, nor the chemical composition of EPS matrix. The gene that codes for polysaccharide deacetylase, parB-like and transRDD proteins, were identified. These results contribute to the knowledge about the variability of chemical composition and potential cytotoxic properties of EPS produced by Streptomyces biofilms. It proposes interesting future challenges for linking Streptomyces-based pesticide remediation technology with the development of new antitumor drugs.
Assuntos
Matriz Extracelular de Substâncias Poliméricas , Streptomyces , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/parasitologia , Humanos , Streptomyces/químicaRESUMO
The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB'. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB', several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB' and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB'. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per µg of DNA; in the new hosts, the plasmid was relatively stable as 29-53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.
Assuntos
Lactobacillus/genética , Plasmídeos/genética , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Sequência de Bases/genética , Replicação do DNA/genética , DNA Bacteriano/genética , Vetores Genéticos/genética , Origem de Replicação/genética , Replicon/genética , Análise de Sequência de DNA/métodos , Transposases/genéticaRESUMO
Highly contaminated γ-hexachlorocyclohexane (lindane) areas were reported worldwide. Low aqueous solubility and high hydrophobicity make lindane particularly resistant to microbial degradation. Physiological and genetic Streptomyces features make this genus more appropriate for bioremediation compared with others. Complete degradation of lindane was only proposed in the genus Sphingobium although the metabolic context of the degradation was not considered. Streptomyces sp.M7 has demonstrated ability to remove lindane from culture media and soils. In this study, we used MS-based label-free quantitative proteomic, RT-qPCR and exhaustive bioinformatic analysis to understand lindane degradation and its metabolic context in Streptomyces sp. M7. We identified the proteins involved in the up-stream degradation pathway. In addition, results demonstrated that mineralization of lindane is feasible since proteins from an unusual down-stream degradation pathway were also identified. Degradative steps were supported by an active catabolism that supplied energy and reducing equivalents in the form of NADPH. To our knowledge, this is the first study in which degradation steps of an organochlorine compound and metabolic context are elucidate in a biotechnological genus as Streptomyces. These results serve as basement to study other degradative actinobacteria and to improve the degradation processes of Streptomyces sp. M7.
Assuntos
Hexaclorocicloexano/metabolismo , Redes e Vias Metabólicas , Proteoma/metabolismo , Proteômica/métodos , Streptomyces/genética , Streptomyces/metabolismo , Transcriptoma , Biodegradação Ambiental , Proteoma/análiseRESUMO
In the last few decades, highly toxic organic compounds like the organochlorine pesticide (OP) hexachlorocyclohexane (HCH) have been released into the environment. All HCH isomers are acutely toxic to mammals. Although nowadays its use is restricted or completely banned in most countries, it continues posing serious environmental and health concerns. Since HCH toxicity is well known, it is imperative to develop methods to remove it from the environment. Bioremediation technologies, which use microorganisms and/or plants to degrade toxic contaminants, have become the focus of interest. Microorganisms play a significant role in the transformation and degradation of xenobiotic compounds. Many Gram-negative bacteria have been reported to have metabolic abilities to attack HCH. For instance, several Sphingomonas strains have been reported to degrade the pesticide. On the other hand, among Gram-positive microorganisms, actinobacteria have a great potential for biodegradation of organic and inorganic toxic compounds. This review compiles and updates the information available on bacterial removal of HCH, particularly by Streptomyces strains, a prolific genus of actinobacteria. A brief account on the persistence and deleterious effects of these pollutant chemical is also given.
Assuntos
Bactérias/metabolismo , Hexaclorocicloexano/metabolismo , Actinobacteria/metabolismo , Biodegradação Ambiental , Microbiologia Ambiental , Poluentes Ambientais/metabolismo , Bactérias Gram-Negativas/metabolismo , Hexaclorocicloexano/química , Redes e Vias Metabólicas , Plantas/metabolismo , Plantas/microbiologiaRESUMO
Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705alpha and Lac705beta. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705alpha/Lac705beta peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705beta peptide alone. Furthermore, Lac705beta increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705alpha protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Deltapsi and DeltapH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705alpha peptide is responsible for receptor recognition, and the Lac705beta peptide is the active component on the cell membrane of CRL691 cells.