Folia Microbiologica

An overview of 25 years of research on Thermococcus kodakarensis , a genetically versatile model organism for archaeal research Abstract Almost 25 years have passed since the discovery of a planktonic, heterotrophic, hyperthermophilic archaeon named Thermococcus kodakarensis KOD1, previously known as Pyrococcus sp. KOD1, by Imanaka and coworkers. T. kodakarensis is one of the most studied archaeon in terms of metabolic pathways, available genomic resources, established genetic engineering techniques, reporter constructs, in vitro transcription/translation machinery, and gene expression/gene knockout systems. In addition to all these, ease of growth using various carbon sources makes it a facile archaeal model organism. Here, in this review, an attempt is made to reflect what we have learnt from this hyperthermophilic archaeon.

A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds Abstract Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.

Introducing a thermotolerant Gluconobacter japonicus strain, potentially useful for coenzyme Q 10 production Abstract In this report, Gluconobacter strains were screened for coenzyme Q10 (CoQ10) production. A thermotolerant strain, Gluconobacter japonicus FM10, was eventually employed for CoQ10 production optimization. To do so, a two-step optimization strategy was used. The first step focused on biomass increase and the second step focused on increase in CoQ10 production. Factors including temperature, pH, carbon, and nitrogen sources were optimized at the first step, and temperature, pH, and aeration were optimized at the second step. The batch culture fermentation was used with the optimized factors of the first phase (30 °C, pH 6.5, D-sorbitol, and yeast extract-peptone as the carbon and nitrogen sources). After 18 h, the temperature, pH, and aeration were shifted to the optimized values of the second step (36 °C, pH 7, and no aeration). By this strategy, the dry cell mass (17.1 g/L) and CoQ10 (23.2 mg/L) were obtained after 20 h, which the latter was 2.3 times higher than that of the first step of optimization. Among the conditions tested, carbon source was the most important factor on the cell growth at the first step while no aeration was the key factor for CoQ10 production in the second step of optimization.

Heat-killed Candida albicans augments synthetic bacterial component-induced proinflammatory cytokine production Abstract Candida albicans can enhance the invasion of oral epithelial cells by Porphyromonas gingivalis, although the fungus is not a periodontal pathogen. In this study, we investigated whether C. albicans augments proinflammatory cytokine production by mouse macrophage-like J774.1 cells incubated with synthetic bacterial components. Mouse macrophage-like J774.1 cells, mouse primary splenocytes, human THP-1 cells, and A549 cells were pretreated with or without heat-killed C. albicans (HKCA) or substitutes for C. albicans cell wall components in 96-well flat-bottomed plates. Cells were then washed and incubated with Pam3CSK4, a Toll-like receptor (TLR) 2 ligand, or lipid A, a TLR4 ligand. Culture supernatants were analyzed by ELISA for secreted IL-6, MCP-1, TNF-α, and IL-8. HKCA augmented TLR ligand-induced proinflammatory cytokine production by J774.1 cells, mouse splenocytes, and THP-1 cells, but not A549 cells. However, IL-6, MCP-1, and TNF-α production induced by Pam3CSK4 or lipid A was not augmented when cells were pretreated with curdlan, a dectin-1 ligand, or mannan, a dectin-2 ligand. In contrast, pretreatment of cells with TLR ligands upregulated the production of IL-6 and TNF-α, but not MCP-1, induced by Pam3CSK4 or lipid A. The results suggest that C. albicans augments synthetic bacterial component-induced cytokine production by J774.1 cells via the TLR pathway, but not the dectin-1 or dectin-2 pathway.

Analysis of genetic variations of heat shock proteins Hsp70 and Hsp90 in Isaria farinosa strains from the Yunnan province of China Abstract In the present study, the cDNA sequences of Hsp70 and Hsp90 genes of Isaria farinosa (designated IFHSP70 and IFHSP90) were cloned and characterized using multiple techniques of molecular biology and bioinformatics. The genetic differentiation of the two genes was investigated among 10 geographically separated populations distributed in the Yunnan province. The complete sequence of the IFHSP70 cDNA had a length of 2158 bp, and contained an open reading frame (ORF) of 1962 bp, encoding a 71-kDa polypeptide comprising of 653 amino acids. IFHSP90 cDNA had a length of 2144 bp, and contained an ORF of 2103 bp, encoding a polypeptide of 79.23 kDa, comprising of 700 amino acids. The deduced amino acid sequences of IFHSP70 and IFHSP90 shared high sequence identities with other fungi. Fundamental information pertaining to the protein families, signatures, and conserved motifs of Hsp70 and Hsp90 were also identified. Analysis of molecular variances (AMOVA) from the Hsp70 and Hsp90 genes showed that the genetic variation within-population (83.26%, 83.08%) was greater than among the populations (16.74%, 16.92%). The values of nucleotide diversity (Pi), haplotype diversity (Hd), coefficient of genetic differentiation (Fst), and gene flow (Nm) were calculated. For Hsp70, Pi = 0.0425, Hd = 0.888, Fst = 0.167, Nm = 1.24; For Hsp90, Pi = 0.0420, Hd = 0.894, Fst = 0.169, and Nm = 1.22. These data indicated that the genetic differentiation among 10 different geographical populations of I. farinosa was limited. This study describes, for the first time, cloning, characterization and identification of Isaria farinosa Hsp70 and Hsp90 genes, and provides a preliminary basis for exploring the genetic structure of the genus Isaria using the sequences of Hsp70 and Hsp90 as molecular markers.

Quantitative evaluation of biofilm extracellular DNA by fluorescence-based techniques Abstract The formation of a hardly removable biofilm in food processing and clinical settings calls for a deeper understanding of composition of the matrix that protects the biofilm cells, as the crucial matrix component is extracellular DNA (eDNA), participating in adhesion, aggregation and penetration reduction, yet serving as a horizontal gene transfer reservoir. Therefore, we evaluated eDNA release from the biofilm of two pathogens, Listeria monocytogenes and Staphylococcus aureus, with respect to their origin under different culturing condition. Primarily, the biofilms were observed by confocal laser scanning microscopy (CLSM) under conditions mimicking the food processing environment and human body. The eDNA was quantitatively characterised based on its area by IMARIS. Next, the eDNA content and biofilm formation were quantified by spectrophotometry. Data from both sets of experiments were statistically evaluated. The eDNA release varied between the microorganism, culturing conditions and the origin of strains. Independent of the method used, the clinical strains of S. aureus released more eDNA than the food related strains at 37 °C. eDNA content can be crucial discriminating matrix component between food related and clinical strains. Deeper understanding of the eDNA role in such a phenomenon could facilitate the design of effective strategy for biofilm disruption.

Characterization of the bacteriocin produced by Enterococcus italicus ONU547 isolated from Thai fermented cabbage Abstract Seventy-eight isolates of lactic acid bacteria from Ukraine and Thailand were screened for bacteriocinogenic activity against indicator strain Lactobacillus sakei subsp. sakei JCM 1157. One isolate showed an antagonistic activity of cell-free supernatant eliminated after the treatment with Proteinase K. Based on 16S rRNA gene sequence, this isolate was identified as Enterococcus italicus. Bacteriocin produced by this strain showed antimicrobial activity against L. sakei subsp. sakei JCM 1157, Brochothrix thermosphacta DSMZ 20171, and Listeria ivanovii subsp. ivanovii DSMZ 20750 in agar well diffusion assay. This bacteriocin was cationic and hydrophobic. The partially purified bacteriocin was thermostable, while heating of cell-free supernatant increased its activity more than twofold. Molecular mass of the partially purified bacteriocin as determined by SDS-PAGE differed from enterocin A and B previously known for E. italicus. Concentrated bacteriocin decreased the level of biofilm formation in L. sakei subsp. sakei JCM 1157 and Pseudomonas aeruginosa PAO1 in 52.5 and 48.0%, respectively (p < 0.05). We suggest that the studied bacteriocin could be a perspective antibiofilm agent in food conservation and medicine.

Human gut microbes are susceptible to antimicrobial food additives in vitro Abstract The aim of this work was to test the hypothesis that antimicrobial food additives may alter the composition of human gut microbiota by selectively suppressing the growth of susceptible gut microbes. To explore the influence of antimicrobial food additives on the composition of the human gut microbiota, we examined the susceptibility of both aerobic and anaerobic gut bacteria to sodium benzoate, sodium nitrite, and potassium sorbate, and their combinations, using a broth microdilution method. The tested bacteria exhibited a wide range of susceptibilities to food additives. For example, the most susceptible strain, Bacteroides coprocola, was almost 580 times more susceptible to sodium nitrite than the most resistant strain, Enterococcus faecalis. However, most importantly, we found that gut microbes with known anti-inflammatory properties, such as Clostridium tyrobutyricum or Lactobacillus paracasei, were significantly more susceptible to additives than microbes with known proinflammatory or colitogenic properties, such as Bacteroides thetaiotaomicron or Enterococcus faecalis. Our data show that some human gut microbes are highly susceptible to antimicrobial food additives. We speculate that permanent exposure of human gut microbiota to even low levels of additives may modify the composition and function of gut microbiota and thus influence the host’s immune system. Whether the effect of additive-modified gut microbiota on the human immune system could explain, at least in part, the increasing incidence of allergies and autoimmune diseases remains to be shown.

Stb5p is involved in Kluyveromyces lactis response to 4-nitroquinoline-N-oxide stress Abstract In yeast, the STB5 gene encodes a transcriptional factor belonging to binuclear cluster class (Zn2Cys6) of transcriptional regulators specific to ascomycetes. In this study, we prepared the Kluyveromyces lactis stb5Δ strain and assessed its responses to different stresses. We showed that KlSTB5 gene is able to complement the deficiencies of Saccharomyces cerevisiae stb5Δ mutant. The results of phenotypic analysis suggested that KlSTB5 gene deletion did not sensitize K. lactis cells to oxidative stress inducing compounds but led to Klstb5Δ resistance to 4-nitroquinoline-N-oxide and hygromycin B. Expression analysis indicated that the loss of KlSTB5 gene function induced the transcription of drug efflux pump encoding genes that might contribute to increased 4-nitroquinoline-N-oxide and hygromycin B tolerance. Our results show that KlStb5p functions as negative regulator of some ABC transporter genes in K. lactis.

Polyphenols as resistance modulators in Arcobacter butzleri Abstract Arcobacter butzleri is an emerging human and animal pathogen for which an increased prevalence of resistance to antibiotics has been observed, and so alternative compounds to modulate resistance of A. butzleri are required. This work aims to study the potential use of several polyphenols as efflux pump inhibitors (EPIs) and to evaluate their interaction with antibiotics, in order to enhance antibiotic activity against A. butzleri. The minimum inhibitory concentration (MIC) of (−)-epicatechin, (+)-catechin, rutin, gallic acid, caffeic acid, chlorogenic acid, resveratrol, pterostilbene, and pinosylvin was determined, in absence and presence of four known EPIs. Subsequently, ethidium bromide accumulation in presence of subinhibitory concentrations of polyphenols was evaluated, and the synergistic potential of the compounds with antibiotics was assessed by checkerboard dilution test. Only stilbenes presented activity against A. butzleri, with MIC values ranging between 64 and 512 μg/mL. The MIC determination of the polyphenols in the presence of subinhibitory concentrations of known EPIs showed that efflux pumps play a role in the resistance to these compounds. Stilbenes also induced a higher intracellular accumulation of ethidium bromide, indicating that they may inhibit the activity of efflux pumps. Checkerboard assays showed that several combinations of polyphenol/antibiotic had an additive effect against A. butzleri. Overall, the results indicate that some polyphenols reduce A. butzleri resistance to antibiotics, suggesting the potential of stilbenes as EPIs. The potential of resveratrol and pinosylvin as resistance modulators was evidenced, insofar as these compounds can even revert antibiotic resistance. Therefore, the use of polyphenols as resistance modulators could be an alternative to overcome the decreasing susceptibility of A. butzleri to antibiotics.