Evaluation of the Effect of Lactobacillus acidophilus ATCC 4356 Bacteriocin against Staphylococcus aureus.

Background: Lactobacillus acidophilus is lactic acid bacteria that produce bacteriocins. Bacteriocins are antimicrobial peptides or proteins that exhibit activity against closely related bacteria. The aim of this study was to determine the effect of L. acidophilus ATCC 4356 bacteriocin against Staphylococcus aureus. Material and Methods. We used four different phenotypic methods for antimicrobial activities against two standard strains: methicillin-resistant S. aureus (MRSA) ATCC 33591 and methicillin-susceptible S. aureus (MSSA) ATCC 25923. The methods were (1) agar well diffusion, (2) overlay soft agar, (3) paper disk, and (4) modification of punch hole. The ammonium sulfate method was used to concentrate crude bacteriocin, and ultrafiltration and dialysis tubes were used to remove ammonium sulfate from the bacteriocins. Each method was repeated in triplicate. Result: L. acidophilus ATCC 4356 showed antimicrobial activity against both MRSA and MSSA standard strains only by the overlay soft agar method and not by the agar well diffusion, punch hole modification, and paper disk methods. No antimicrobial effects were observed in crude bacteriocins concentrated. Conclusion: The growth inhibition of S. aureus in overlay soft agar method may be due to the production of bacteriocin-like substances. The overlay soft agar method is a qualitative test, so there is a need for further study to optimize the conditions for the production of bacteriocin-like substances in the culture supernatant and precise comparison between the inhibitory activity and pheromone secretion of different strains.

The comparative plastisphere microbial community profile at Kung Wiman beach unveils potential plastic-specific degrading microorganisms.

Background: Plastic waste is a global environmental issue that impacts the well-being of humans, animals, plants, and microorganisms. Microplastic contamination has been previously reported at Kung Wiman Beach, located in Chanthaburi province along with the Eastern Gulf of Thailand. Our research aimed to study the microbial population of the sand and plastisphere and isolate microorganisms with potential plastic degradation activity. Methods: Plastic and sand samples were collected from Kung Wiman Beach for microbial isolation on agar plates. The plastic samples were identified by Fourier-transform infrared spectroscopy. Plastic degradation properties were evaluated by observing the halo zone on mineral salts medium (MSM) supplemented with emulsified plastics, including polystyrene (PS), polylactic acid (PLA), polyvinyl chloride (PVC), and bis (2-hydroxyethyl) terephthalate (BHET). Bacteria and fungi were identified by analyzing nucleotide sequence analysis of the 16S rRNA and internal transcribed spacer (ITS) regions, respectively. 16S and ITS microbiomes analysis was conducted on the total DNA extracted from each sample to assess the microbial communities. Results: Of 16 plastic samples, five were identified as polypropylene (PP), four as polystyrene (PS), four as polyethylene terephthalate (PET), two as high-density polyethylene (HDPE), and one sample remained unidentified. Only 27 bacterial and 38 fungal isolates were found to have the ability to degrade PLA or BHET on MSM agar. However, none showed degradation capabilities for PS or PVC on MSM agar. Notably, Planococcus sp. PP5 showed the highest hydrolysis capacity of 1.64 ± 0.12. The 16S rRNA analysis revealed 13 bacterial genera, with seven showing plastic degradation abilities: Salipiger, Planococcus, Psychrobacter, Shewanella, Jonesia, Bacillus, and Kocuria. This study reports, for the first time of the BHET-degrading properties of the genera Planococcus and Jonesia. Additionally, The ITS analysis identified nine fungal genera, five of which demonstrated plastic degradation abilities: Aspergillus, Penicillium, Peacilomyces, Absidia, and Cochliobolus. Microbial community composition analysis and linear discriminant analysis effect size revealed certain dominant microbial groups in the plastic and sand samples that were absent under culture-dependent conditions. Furthermore, 16S and ITS amplicon microbiome analysis revealed microbial groups were significantly different in the plastic and sand samples collected. Conclusions: We reported on the microbial communities found on the plastisphere at Kung Wiman Beach and isolated and identified microbes with the capacity to degrade PLA and BHET.

Performance evaluation of Trichoderma reseei in tolerance and biodegradation of diuron herbicide in agar plate, liquid culture and solid-state fermentation.

The present study evaluated the performance of the fungus Trichoderma reesei to tolerate and biodegrade the herbicide diuron in its agrochemical presentation in agar plates, liquid culture, and solid-state fermentation. The tolerance of T. reesei to diuron was characterized through a non-competitive inhibition model of the fungal radial growth on the PDA agar plate and growth in liquid culture with glucose and ammonium nitrate, showing a higher tolerance to diuron on the PDA agar plate (inhibition constant 98.63 mg L-1) than in liquid culture (inhibition constant 39.4 mg L-1). Diuron biodegradation by T. reesei was characterized through model inhibition by the substrate on agar plate and liquid culture. In liquid culture, the fungus biotransformed diuron into 3,4-dichloroaniline using the amide group from the diuron structure as a carbon and nitrogen source, yielding 0.154 mg of biomass per mg of diuron. A mixture of barley straw and agrolite was used as the support and substrate for solid-state fermentation. The diuron removal percentage in solid-state fermentation was fitted by non-multiple linear regression to a parabolic surface response model and reached the higher removal (97.26%) with a specific aeration rate of 1.0 vkgm and inoculum of 2.6 × 108 spores g-1. The diuron removal in solid-state fermentation by sorption on barley straw and agrolite was discarded compared to the removal magnitude of the biosorption and biodegradation mechanisms of Trichoderma reesei. The findings in this work about the tolerance and capability of Trichoderma reesei to remove diuron in liquid and solid culture media demonstrate the potential of the fungus to be implemented in bioremediation technologies of herbicide-polluted sites.

LC-HRMS Profiling and Cytotoxic Potential of Actinomycetes Associated with the Red Sea Soft Coral Sarcophyton glaucum: In vitro and In silico Studies.

In the current study, the actinomycetes associated with the red sea-derived soft coral Sarcophyton glaucum were investigated in terms of biological and chemical diversity. Four different media, M1, ISP2, Marine Agar (MA), and Actinomycete isolation agar (AIA) were used for the isolation of three strains of actinomycetes that were identified as Streptomyces sp. UR 25, Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. LC-HRMS analysis was used to investigate the chemical diversity of the isolated actinobacteria. The LC-HRMS data were statistically processed using MetaboAnalyst 5.0 viz to differentiate the extract groups and determine the optimal growth culturing conditions. Multivariate data statistical analysis revealed that the Micromonospora sp. extract cultured on (MA) medium is the most distinctive extract in terms of chemical composition. While, the Streptomyces sp. UR 25 extracts are differ significantly from Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. Biological investigation using in vitro cytotoxic assay for actinobacteria extracts revealed the prominent potentiality of the Streptomyces sp. UR 25 cultured on oligotrophic medium against human hepatoma (HepG2), human breast adenocarcinoma (MCF-7) and human colon adenocarcinoma (CACO2) cell lines (IC50 =3.3, 4.2 and 6.8 µg/mL, respectively). SwissTarget Prediction speculated that among the identified compounds, 16-deethyl, indanomycin (8) could have reasonable affinity on HDM2 active site. In this respect, molecular docking study was performed for compound (8) to reveal a substantial affinity on HDM2 active site. In addition, molecular dynamics simulations were carried out at 200 ns for the most active compound (8) compared to the co-crystallized inhibitor DIZ giving deeper information regarding their thermodynamic and dynamic properties as well.

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormone signaling intersect to promote shallow lateral root angles.

Root system architecture (RSA) influences the acquisition of heterogeneously dispersed soil nutrients. Cytokinin and C-TERMINALLY ENCODED PEPTIDE (CEP) hormones affect RSA, in part by controlling the angle of lateral root (LR) growth. Both hormone pathways converge on CEP DOWNSTREAM 1 (CEPD1) and CEPD2 to control primary root growth; however, a role for CEPDs in controlling the growth angle of LRs is unknown. Using phenotyping combined with genetic and grafting approaches, we show that CEP hormone-mediated shallower LR growth requires cytokinin biosynthesis and perception in roots via ARABIDOPSIS HISTIDINE KINASE 2 (AHK2) and AHK3. Consistently, cytokinin biosynthesis and ahk2,3 mutants phenocopied the steeper root phenotype of cep receptor 1 (cepr1) mutants on agar plates, and CEPR1 was required for trans-Zeatin (tZ)-type cytokinin-mediated shallower LR growth. In addition, the cepd1,2 mutant was less sensitive to CEP and tZ, and showed basally steeper LRs on agar plates. Cytokinin and CEP pathway mutants were grown in rhizoboxes to define the role of these pathways in controlling RSA. Only cytokinin receptor mutants and cepd1,2 partially phenocopied the steeper-rooted phenotype of cepr1 mutants. These results show that CEP and cytokinin signaling intersect to promote shallower LR growth, but additional components contribute to the cepr1 phenotype in soil.

Agar plate-based activity assay for easy and fast screening of recombinant Pichia pastoris expressing unspecific peroxygenases.

Unspecific peroxygenases (UPOs) are promising biocatalysts that catalyze oxyfunctionalization reactions without the need for costly cofactors. Pichia pastoris (reclassified as Komagataella phaffii) is considered an attractive host for heterologous expression of UPOs. However, integration of UPO-expression cassettes into the genome via a single cross-over yields recombinant Pichia transformants with different UPO gene copy numbers resulting in different expression levels. Selection of the most productive Pichia transformants by a commonly used screening in liquid medium in 96-well plates is laborious and lasts up to 5 days. In this work, we developed a simple two-step agar plate-based assay to screen P. pastoris transformants for UPO activity with less effort, within shorter time, and without automated screening devices. After cell growth and protein expression on agar plates supplemented with methanol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), an additional top agar layer supplemented with ABTS and peroxide is added. UPO activity is visualized within 15 min by formation of green zones around UPO-secreting P. pastoris transformants. The assay was validated with two UPOs, AbrUPO from Aspergillus brasiliensis and evolved PaDa-I from Agrocybe aegerita. The assay results were confirmed in a quantitative 96-deep well plate screening in liquid medium.

Pathological granuloma fibrosis induced by agar-embedded Mycobacterium abscessus in C57BL/6JNarl mice.

Introduction: Pulmonary granuloma diseases caused by Mycobacterium abscessus (M. abscessus) have increased in past decades, and drug-resistance in this pathogen is a growing public health concern. Therefore, an animal model of chronic granuloma disease is urgently needed. Methods: In this study, M. abscessus embedded within agar beads (agar-AB) was used to develop such a model in C57BL/6JNarl mice. Results: Chronic infection was sustained for at least 3 months after agar-AB infection, visible granulomas spread in the lungs, and giant cells and foamy cells appeared in the granulomas. More importantly, pulmonary fibrosis progressed for 3 months, and collagen fibers were detected by Masson trichrome staining. Further, inducible nitric oxide synthase (iNOS) was highly expressed within the alveolar space, and the fibrosis-mediator transforming growth factor beta (TGF-ß) began to be expressed at 1 month. Hypoxia-inducible factor (HIF-1α) expression also increased, which aided in normalizing oxygen partial pressure. Discussion: Although the transient fibrosis persisted for only 3 months, and the pulmonary structure resolved when the pathogen was cleard, this pulmonary fibrosis model for M. abscessus infection will provide a novel test platform for development of new drugs, regimens, and therapies.

[Inhibition of GMA-induced ubiquitination process in 16HBE malignantly transformed cells on protein CD44 and MMP14].

OBJECTIVE: To observe the effect of the ubiquitination process on the expression of CD44 antigen(CD44) and matrix metalloproteinase-14(MMP14) in human bronchial epithelial(16HBE) malignantly transformed cells induced by glycidyl methacrylate(GMA). METHODS: Successfully resuscitated 16HBE cells were cultured using a final concentration of 8 µg/mL GMA as the treatment group and 1 µg/mL dimethyl sulfoxide as the solvent control group, each time stained for 72 h, and then stained again after an interval of 24 h. After repeating the staining three times, the cells were cultured in passages respectively. The 40th generation(P40) GMA-treated group and the same-generation solvent control group were subjected to soft agar colony formation assay and concanavalin A(ConA) agglutination test to confirm that the 40th generation of GMA-induced malignant transformed 16HBE cells possessed malignant transformed cell characteristics.5, 10, 20, 40, 60 µmol/L anacardic acid were used to inhibit the ubiquitination process of GMA-induced malignant transformed 16HBE cells. The protein expression of CD44 and MMP14 were detected by western blotting, while the transcript levels of CD44, MMP14, and TFAP2A were assessed by real-time fluorescence quantitative PCR(qPCR). RESULTS: (1) In the soft agar colony formation assay, the number of clones formed by the cells in the solvent control group was 22, and the number of clones created by the malignantly transformed cells in the GMA-treated group was 208. In the ConA agglutination test, the cells in the solvent control group were uniformly dispersed in ConA solution, and no obvious agglutination occurred for 30 min, whereas the cells in the GMA-treated group were agglutinated in the 5th min, and the agglutinated cells were larger and more rapidly agglutinated. The agglomerates were more significant and faster, and the sensitivity of agglutination was increased. (2) After differential inhibition of GMA-induced ubiquitination in malignantly transformed 16HBE cells, the expression levels of CD44 and MMP14 were reduced in GMA-induced malignantly transformed 16HBE cells compared with the control group(P<0.05). The transcript levels of MMP14 and CD44 decreased with increasing inhibitor concentration(P<0.05), and the transcript levels of the upstream transcription factor TFAP2A were also simultaneously reduced(P<0.05). CONCLUSION: Inhibition of the cellular ubiquitination process mediates the down-regulation of protein expression and transcriptional expression of CD44 and MMP14 in GMA-induced malignantly transformed 16HBE cells.

Isolation, characterization and screening of actinomycetes associated with fijian ant-plant symbioses.

In the search for novel therapeutics to combat the ongoing antimicrobial resistance crisis, scientists are turning to underexplored environments. Defensive mutualisms between hymenopteran insects and actinomycetes represent important reservoirs for bioactive compounds. In this study, we examined the association between actinomycetes and Squamellaria ant-plants spanning three different ant and plant species combinations (Squamellaria imberbis-Philidris nagasau, Squamellaria tenuiflora- Technomyrmex vitiensis, and Squamellaria tenuiflora-Tetramorium insolens). Eight Squamellaria plants were sampled including four containing T. vitiensis, three containing P. nagasau, and a single plant containing T. insolens. A total of 47 actinomycetes were obtained from the sampled material, with 5, 16, and 26 isolates originating from cuticle, tissue, and nest samples, respectively. Cross-streaking tests showed that 12 out of 47 isolates inhibited bacterial pathogens. The most frequently inhibited pathogens in the cross-streaking tests were S. aureus and E. coli while S. enterica was the least inhibited. Among the three primary screening media used, ISP2 agar was the most suitable for secondary metabolism as more isolates exhibited antibacterial activity when grown on the medium. TFS2010 and TFS2003, which matched to Streptomyces gramineus (>99% similarity), were the most bioactive isolates in cross-streaking tests. TFS2010 displayed the strong antibacterial on Nutrient agar, Mueller Hinton agar, and ISP2 agar while TFS2003 only exhibited strong antibacterial activity on Nutrient agar. Furthermore, a difference in potency of extracts based on batch culture medium was noted in TFS2010. DNA was extracted from 19 isolates and followed by 16SrRNA gene sequencing. Analysis of sequence data revealed the presence of six genera, including Amycolatopsis, Asanoa, Jiangella, Nocardia, Nocardiopsis, and Streptomyces, with the latter being the most abundant taxon. Among these, three isolates (PNS3002, PNS3005, and TFS3001) are likely to represent new species while one (TFS2015) is likely to be a member of a novel genus. Our work represents the first attempt to study actinomycetes from Squamellaria-ant mutualisms.

High-throughput bacterial co-encapsulation in microfluidic gel beads for discovery of antibiotic-producing strains.

Bacteria with antagonistic activity inhibit the growth of other bacteria through different mechanisms, including the production of antibiotics. As a result, these microorganisms are a prolific source of such compounds. However, searching for antibiotic-producing strains requires high-throughput techniques due to the vast diversity of microorganisms. Here, we screened and isolated bacteria with antagonistic activity against Escherichia coli expressing the green fluorescent protein (E. coli-GFP). We used microfluidics to co-encapsulate and co-culture single cells from different strains within picoliter gel beads and analyzed them using fluorescence-activated cell sorting (FACS). To test the methodology, we used three bacterial isolates obtained from Mexican maize, which exhibit high, moderate, or no antagonistic activity against E. coli-GFP, as determined previously using agar plate assays. Single cells from each strain were separately co-incubated into gel beads with E. coli-GFP. We monitored the development of the maize bacteria microcolonies and tracked the growth or inhibition of E. coli-GFP using bright-field and fluorescent microscopy. We correlated these images with distinctive light scatter and fluorescence signatures of each incubated bead type using FACS. This analysis enabled us to sort gel beads filled with an antagonistic strain, starting from a mixture of the three different types of maize bacteria and E. coli-GFP. Likewise, culturing the FACS-sorted beads on agar plates confirmed the isolation and recovery of the two antagonistic strains. In addition, enrichment assays demonstrated the methodology's effectiveness in isolating rare antibiotic-producer strains (0.01% abundance) present in a mixture of microorganisms. These results show that associating light side scatter and fluorescent flow cytometry signals with microscopy images provides valuable controls to establish successful high-throughput methods for sorting beads in which microbial interaction assays are performed.

A layered solid-state culture system for investigating the fungal growth and decay behaviour on the cellulosic substrate.

Mycelium-based materials have attracted growing interest, facilitating their development for various new applications. Finding for suitable fungal species and strains enables further technical breakthroughs in their development and quality control. Here, we developed a model solid-state culture system to screen fungal strains efficiently for mycelium-based materials production. A piece of silicone-coated paper set on the general agar plate allows for isolation of the mycelial mat from the substrate. The mycelial growth and density can be evaluated by weighing the mycelial mat. We used the paper substrate after fungal incubation to investigate the relationship between substrate degradation and the contact time with hyphae. It yielded further insights into the fungal decay. Ten basidiomycetes were assessed for their fungal growth and degradation behaviour of the substrate using this method. Pleurotus floridanus FBCC375 showed a dense and elastic mycelial mat and mild degradation of the substrate. A unique decay behaviour was found in Hypsizygus ulmarius FBCC573 and Trametes versicolor FBCC564. They indicated a positional imbalance in the decay activity within the colony. This simple method is helpful for screening fungal strains and facilitates the further development of mycelium-based materials.

Detection, production, modification, and application of arylsulfatases.

Arylsulfatase is a subset of sulfatase which catalyzes the hydrolysis of aryl sulfate ester. Arylsulfatase is widely distributed among microorganisms, mammals and green algae, but the arylsulfatase-encoding gene has not yet been found in the genomes of higher plants so far. Arylsulfatase plays an important role in the sulfur flows between nature and organisms. In this review, we present the maturation and catalytic mechanism of arylsulfatase, and the recent literature on the expression and production of arylsulfatase in wild-type and engineered microorganisms, as well as the modification of arylsulfatase by genetic engineering are summarized. We focus on arylsulfatases from microbial origin and give an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the researches about arylsulfatase application on the field of agar desulfation, soil sulfur cycle and soil evaluation are also discussed. Finally, the perspectives concerning the future research on arylsulfatase are prospected.

Screening and characterization of agarolytic bacteria from different sources.

According to the results of our investigation, distinct bacterial isolates capable of breaking down agar were found in various nonmarine environments. The deficiency of reducing sugar in the control media demonstrates that the agar in the experiment is broken down by the bacteria to produce various oligosaccharides because the viscosity of the medium containing the agar was found to have been extremely high before inoculation, reducing with incubation duration and attaining a maximum after 48 hours. These isolates were subsequently used in tests along with additional investigation since they could create reducing sugar. Interestingly, the deterioration of agar appears to be mainly caused by Gram-negative bacteria. In order to study the agarase properties, the relative quantity of the enzyme secreted by the bacteria that hydrolyze the agar was used. The detection of extracellular agarase surrounding the colonies and the absence of stained halos on iodine-treated agar plates show that the agarase diffusing from the bacteria impacted the characteristics of the gel. Inconclusion, these agarsase-producing bacteria can be exploited for industrial applications. Waste agar from the plant tissue culture business can be utilized for a range of applications and this degraded agar can be explored for reliable and ecologically safe alternatives.

Excessive ultraviolet C irradiation causes spore protein denaturation and prohibits the initiation of spore germination in Bacillus subtilis.

Ultraviolet (UV) -C is widely used to kill bacteria as it damages chromosomal DNA. We analyzed the denaturation of the protein function of Bacillus subtilis spores after UV-C irradiation. Almost all of the B. subtilis spores germinated in Luria-Bertani (LB) liquid medium, but the colony-forming unit (CFU) of the spores on LB agar plates decreased to approximately 1/103 by 100 mJ/cm2 of UV-C irradiation. Some of the spores germinated in LB liquid medium under phase-contrast microscopy, but almost no colonies formed on the LB agar plates after 1 J/cm2 of UV-C irradiation. The fluorescence of the green fluorescent protein (GFP) -fused spore proteins, YeeK-GFP, YeeK is a coat protein, decreased following UV-C irradiation of over 1 J/cm2, while that of SspA-GFP, SspA is a core protein, decreased following UV-C irradiation of over 2 J/ cm2, respectively. These results revealed that UV-C affected on coat proteins more than core proteins. We conclude that 25 to 100 mJ/cm2 of UV-C irradiation can cause DNA damage, and more than 1 J/cm2 of UV-C irradiation can cause the denaturation of spore proteins involved in germination. Our study would contribute to improve the technology to detect the bacterial spores, especially after UV sterilization.

Analysis of biofilm expansion rate of Bacillus subtilis (MTC871) on agar substrates with different stiffness.

The surface morphology of mature biofilms is heterogeneous and can be divided into concentric rings wrinkles (I), labyrinthine networks wrinkles (II), radial ridges wrinkles (III), and branches wrinkles (IV), according to surface wrinkle structure and distribution characteristics. Due to the wrinkle structures, channels are formed between the biofilm and substrate and transport nutrients, water, metabolic products, etc. We find that expansion rate variations of biofilms growing on substrates with high and low agar concentrations (1.5, 2.0, 2.5 wt.%) are not in the same phase. In the first 3 days' growth, the interaction stress between biofilm and each agar substrate increases, which makes the biofilm expansion rate decreases before wrinkle pattern IV (branches) comes up. After 3 days, in the later growth stage after wrinkle pattern IV appears, the biofilm has larger expansion rate growing on 2.0 wt.% agar concentration, which has the larger wrinkle distance in wrinkle pattern IV reducing energy consumption. Our study shows that the stiff substrate does not always inhibit the biofilm expansion, although it does in the earlier stage; after that, mature biofilms acquire larger expansion rate by adjusting the growth mode through the wrinkle evolution even in nutrient extremely depletion.

Exploring the use of Congo red agar in improving the serotyping of non-serotypeable Shigella with In-silico evidence.

OBJECTIVES: To assess if congo red could make non-serotypeable Shigella strains serotypeable and to employ molecular docking to determine the basis of the same phenomenon. METHODS: We used 42 bacterial strains of non-serotypeable Shigella collected from 2012 to 2019 for this study. Each bacterial strain was freshly inoculated onto congo red agar and incubated at 37° C for 18-24 h. Bacterial colonies obtained were re-subjected to biochemical tests followed by serotyping and serogrouping. In-silico studies to investigate the interaction between MxiC protein of T3SS and O-antigen LPS with congo red were performed. RESULTS: Of the total 42 non-serotypeable Shigella studied, (26/42)62% were capable of being serotyped following the use of congo red agar, 65% were Shigella flexneri, 19% were Shigella dysenteriae, while 2 strains (7%) each of Shigella boydii and Shigella sonnei were detected. We observed no change in their biochemical properties. The in-silico molecular docking studies revealed high binding affinity between congo red and the B-Chain of Mxi C. Out of the 5 chains of the O-Antigen, congo red showed robust binding with the B-chain with the involvement of a cluster of hydrophobic interactions between them. This may have a crucial role in the conversion of non-serotypeable strains to serotypeable strains on exposure to congo red as observed in our study. CONCLUSION: Congo red agar as a medium converts a sizeable percentage of non-serotypeable Shigella strains to serotypeable Shigella strains.

Toward standardized solid medium cultivations: Online microbial monitoring based on respiration activity.

Cultivating microorganisms on solid agar media is a fundamental technique in microbiology and other related disciplines. For the evaluation, most often, a subjective visual examination is performed. Crucial information, such as metabolic activity, is not assessed. Thus, time-resolved monitoring of the respiration activity in agar cultivations is presented to provide additional insightful data on the metabolism. A modified version of the Respiration Activity MOnitoring System (RAMOS) was used to determine area-specific oxygen and carbon dioxide transfer rates and the resulting respiratory quotients of agar cultivations. Therewith, information on growth, substrate consumption, and product formation was obtained. The validity of the presented method was tested for different prokaryotic and eukaryotic organisms on agar, such as Escherichia coli BL21, Pseudomonas putida KT2440, Streptomyces coelicolor A3(2), Saccharomyces cerevisiae WT, Pichia pastoris WT, and Trichoderma reesei RUT-C30. Furthermore, it is showcased that several potential applications, including the determination of colony forming units, antibiotic diffusion tests, quality control for spore production or for pre-cultures and media optimization, can be quantitatively evaluated by interpretation of the respiration activity.

Genome sequencing of Cladophialophora exuberans, a novel candidate for bioremediation of hydrocarbon and heavy metal polluted habitats.

Cladophialophora exuberans is a filamentous fungus related to black yeasts in the order Chaetothyriales. These melanized fungi are known for their 'dual ecology', often occurring in toxic environments and also being frequently involved in human infection. Particularly Cladophialophora exuberans, C. immunda, C. psammophila, and Exophiala mesophila have been described with a pronounced ability to degrade aromatic compounds and xenobiotic volatiles, such as benzene, toluene, ethyl-benzene, and xylene, and are candidates for bioremediation applications. The objective of the present study is the sequencing, assembly, and description of the whole genome of C. exuberans focusing on genes and pathways related to carbon and toxin management, assessing the tolerance and bioremediation of lead and copper, and verifying the presence of genes for metal homeostasis. Genomic evaluations were carried out through a comparison with sibling species including clinical and environmental strains. Tolerance of metals was evaluated via a microdilution method establishing minimum inhibitory (MIC) and fungicidal concentrations (MFC), and agar diffusion assays. Heavy metal bioremediation was evaluated via graphite furnace atomic absorption spectroscopy (GFAAS). The final assembly of C. exuberans comprised 661 contigs, with genome size of 38.10 Mb, coverage of 89.9X and a GC content of 50.8%. In addition, inhibition of growth was shown at concentrations of 1250 ppm for copper and at 625 ppm for lead, using the MIC method. In the agar tests, the strain grew at 2500 ppm of copper and lead. In GFAAS tests, uptake capacities were observed of 89.2% and 95.7% for copper and lead, respectively, after 21 experimental days. This study enabled the annotation of genes involved in heavy metal homeostasis and also contributed to a better understanding of the mechanisms used in tolerance of and adaptation to extreme conditions.

Bifunctional and monofunctional α-neoagarooligosaccharide hydrolases from Streptomyces coelicolor A3(2).

Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: • SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. • SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. • SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.

The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction.

In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method-the soft agar colony formation assay using polymerase chain reaction (PCR)-to evaluate tumorigenicity. MRC-5 cells, contaminated with HeLa cells, were cultured for up to 4 weeks in soft agar medium. Cell-proliferation-related mRNAs, Ki-67 and cyclin B, could be detected in 0.01% of HeLa cells after 5 days of culture, whereas cyclin-dependent kinase 1 (CDK1) could be detected after 2 weeks. On the other hand, CDK2, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance protein 7 (MCM7) were not useful to detect HeLa cells even after 4 weeks of culture. The cancer stem cell (CSC) markers, aldehyde dehydrogenase 1 (ALDH1) and CD133 in 0.01% of HeLa cells, could be detected 2 and 4 weeks after culture, respectively. However, another CSC marker CD44 was not useful because its expression was also detected in MRC-5 cells alone. This study suggests that the application of the PCR method to the soft agar colony formation assay could evaluate not only the tumorigenic potency in the short-term but also characterize the colonies, eventually improving the safety of CTPs.