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Archives of Microbiology (v.192, #4)
Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish by Cynthia Sequeiros; Marisol Vallejo; Emilio Rogelio Marguet; Nelda Lila Olivera (pp. 237-245).
After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.
Keywords: Lactic acid bacteria; Lactoccocus lactis ; Bacteriocin; Antimicrobial peptide; Lactococcus garvieae ; Fish pathogen
Relationship between endoplasmic reticulum- and Golgi-associated calcium homeostasis and 4-NQO-induced DNA repair in Saccharomyces cerevisiae by Nadine Paese Poletto; João Antonio Pêgas Henriques; Diego Bonatto (pp. 247-257).
Calcium (Ca2+) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca2+ homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca2+ homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca2+ transporter of Golgi complex, interacts with Cod1p, which regulates Ca2+ levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Δ, cod1Δrad4Δ, and cod1Δpmr1Δ strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Δpmr1Δ and cod1Δrad4Δ strains have a strong arrest at G2/M phases of cell cycle after 4-NQO treatment, while pmr1Δrad4Δ have a similar sensitivity and cell cycle arrest profile when compared to rad4Δ after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca2+ transporters affect the repair of 4-NQO-induced DNA damage.
Keywords: Calcium homeostasis; DNA repair; Saccharomyces cerevisiae ; 4-NQO; Systems biology; Unfolded protein response
Encystation process of Giardia lamblia: morphological and regulatory aspects by Joana Bittencourt-Silvestre; Leandro Lemgruber; Wanderley de Souza (pp. 259-265).
One important step in the life cycle of the pathogenic protozoan Giardia lamblia is the transformation of the proliferative form, the trophozoite, into the non-proliferative cyst. This process, known as encystation, can be triggered in vitro. Morphological analysis showed that during trophozoite-cyst transformation, major changes take place: modification of the protozoan shape, internalization of the flagella, fragmentation of the adhesive disk, and appearance of encystation vesicles (ESVs), which later on fuse with the plasma membrane forming the cell wall. Sites of attachment of these vesicles to the inner portion of the protozoan plasma membrane were observed 6 h after the beginning of the encystation process. These sites were only visible when we used high-resolution scanning electron microscopy to study Giardia surface. In order to analyze the involvement of protein kinases and phosphatases on the encystation process, inhibitors of these enzymes were added to the culture medium, and their effect on the differentiation process was determined using light, immunofluorescence, and electron microscopy. Significant inhibition was observed with LY294002, an inhibitor of PI3 kinase; genistein, an inhibitor of tyrosine kinase; and staurosporine, at concentrations, which inhibit protein kinase C. Okadaic acid, an inhibitor or protein phosphatase, and wortmannin, an inhibitor of PI3K, did not interfere with the encystation process. However, they induced the appearance of large and pleomorphic forms where several nuclei and disorganization of the peripheral vesicles were observed.
Keywords: Giardia ; Encystment; Protein kinase; Protozoan differentiation; High-resolution scanning electron microscopy
Alteration of flagellar phenotype of Escherichia coli strain P12b, the standard type strain for flagellar antigen H17, possessing a new non-fliC flagellin gene flnA, and possible loss of original flagellar phenotype and genotype in the course of subculturing through semisolid media by Yuliy A. Ratiner; Leila M. Sihvonen; Yanqun Liu; Lei Wang; Anja Siitonen (pp. 267-278).
A practically important phenomenon, resulting in the loss of the original flagellar phenotype (genotype) of bacteria, is described in the Escherichia coli H17 type strain P12b possessing two distinct genes for H17 and H4 flagellins, respectively. By PCR, sequencing, and phylogenetic investigation, the H17 gene (originally expressed) was considered a new non-fliC flagellin gene and assigned flnA, while the H4 gene (originally cryptic) was reaffirmed as fliC. H17 and H4 flagella differed morphologically. The phenomenon consisted in the replacement of H17 cells by H4 cells during subculturing through certain semisolid media and resulted from the excision of flnA H17 entirely or in part. The substitution rate depended on the density and nutrient composition of media and reached 100% even after a single passage through 0.3% LB agar. Such phenomenon can lead to an unexpected loss of original H17 phenotype. Our review of the literature showed that the loss of the original flagellar genotype (phenotype) of P12b has occurred in some laboratories while the authors continued to consider their cultures H17. We showed how to distinguish these alternative flagellin genotypes using popular fliC primers. Attention was also paid to possible discrepancies between serological and molecular results in flagellar typing of E. coli.
Keywords: Escherichia coli ; Non-fliC flagellin genes; Alteration of flagellar phenotype and genotype; Flagellar typing
Isolation and characterization of the equol-producing bacterium Slackia sp. strain NATTS by Hirokazu Tsuji; Kaoru Moriyama; Koji Nomoto; Naoto Miyanaga; Hideyuki Akaza (pp. 279-287).
Several kinds of carbohydrates such as sorbose, adonitol, and melezitose were found to enhance equol production from daidzein in an in vitro human fecal culture. Sorbose, one of the most effective carbohydrates, was used as a carbohydrate source for isolating the NATTS strain, which was a Gram-positive, non-spore-forming rod bacterium with high ability to convert daidzein to equol isolated from the 7th maintenance culture. The strain was found to belong to the genus Slackia family Coriobacteriaceae by 16S rRNA sequence-based analysis, and the prevalence of the Slackia sp. in Japanese adults was examined by reverse transcription-quantitative PCR (RT-qPCR), which was found to be 40% at a mean population level of 106 cells per gram of feces.
Keywords: Isoflavone; Daidzein; Equol; Intestinal bacteria; Carbohydrates; Slackia
S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces by Xin Qing Zhao; Bertolt Gust; Lutz Heide (pp. 289-297).
The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.
Keywords: S-Adenosylmethionine (SAM); Streptomyces ; Novobiocin; SAM synthetase (EC 2.5.1.6); Adenosyl homocysteinase (EC 3.3.1.1); Adenosine kinase (EC 2.7.1.20)
Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage by L. Cao-Hoang; F. Dumont; P. A. Marechal; P. Gervais (pp. 299-305).
The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.
Keywords: Cold shock; Chilling rate; Membrane permeabilization; Cell viability
Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines by Rodrigo Makowiecky Stuart; Aline Silva Romão; Aline Aparecida Pizzirani-Kleiner; João Lúcio Azevedo; Welington Luiz Araújo (pp. 307-313).
The diversity of endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane plants and its isoline was evaluated by cultivation followed by amplified rDNA restriction analysis (ARDRA) of randomly selected strains. Transgenic and non-transgenic cultivars and their crop management (herbicide application or manual weed control) were used to assess the possible non-target effects of genetically modified sugarcane on the fungal endophytic community. A total of 14 ARDRA haplotypes were identified in the endophytic community of sugarcane. Internal transcribed spacer (ITS) sequencing revealed a rich community represented by 12 different families from the Ascomycota phylum. Some isolates had a high sequence similarity with genera that are common endophytes in tropical climates, such as Cladosporium, Epicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of molecular variance indicated that fluctuations in fungal population were related to both transgenic plants and herbicide application. While herbicide applications quickly induced transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. These results represent the first draft on composition of endophytic filamentous fungi associated with sugarcane plants. They are an important step in understanding the possible effects of transgenic plants and their crop management on the fungal endophytic community.
Keywords: Endophytic fungi; Transgenic sugarcane; Imidazolinone; ARDRA; AMOVA