Minimal Medium Research Articles

ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets.

Methods that measure the transcriptomic state of thousands of individual cells have transformed our understanding of cellular heterogeneity in eukaryotic cells since their introduction in the past decade. While simple and accessible protocols and commercial products are now available for the processing of mammalian cells, these existing technologies are incompatible with use in bacterial samples for several fundamental reasons including the absence of polyadenylation on bacterial messenger RNA, the instability of bacterial transcripts and the incompatibility of bacterial cell morphology with existing methodologies. Recently, we developed ProBac sequencing (ProBac-seq), a method that overcomes these technical difficulties and provides high-quality single-cell gene expression data from thousands of bacterial cells by using messenger RNA-specific probes. Here we provide details for designing large oligonucleotide probe sets for an organism of choice, amplifying probe sets to produce sufficient quantities for repeated experiments, adding unique molecular indexes and poly-A tails to produce finalized probes, in situ probe hybridization and single-cell encapsulation and library preparation. This protocol, from the probe amplification to the library preparation, requires ~7 d to complete. ProBac-seq offers several advantages over other methods by capturing only the desired target sequences and avoiding nondesired transcripts, such as highly abundant ribosomal RNA, thus enriching for signal that better informs on cellular state. The use of multiple probes per gene can detect meaningful single-cell signals from cells expressing transcripts to a lesser degree or those grown in minimal media and other environmentally relevant conditions in which cells are less active. ProBac-seq is also compatible with other organisms that can be profiled by in situ hybridization techniques.

Delayed Enhancement in Cardiac CT: A Potential Alternative to Cardiac MRI? Technical Updates and Clinical Considerations

Despite cardiac magnetic resonance (CMR) with late gadolinium enhancement (LGE) being the current gold standard for non-invasive myocardial characterization and fibrosis quantification, its accessibility is limited, particularly in acute settings and in certain patient populations with contraindications to magnetic resonance imaging. Late iodine enhancement (LIE) in computed tomography (CT) imaging has emerged as a potential alternative, capitalizing on the similarities in the contrast kinetics between gadolinium and iodinated contrast agents. Studies have investigated LIE-CT’s effectiveness in myocardial infarction (MI) detection, revealing promising outcomes alongside some disparities compared to LGE-CMR. LIE-CT also proves beneficial in diagnosing non-ischemic heart diseases such as myocarditis, hypertrophic cardiomyopathy, and sarcoidosis. While LIE-CT demonstrates good accuracy in detecting certain myocardial pathologies, including acute MI and chronic fibrotic changes, it has limitations, such as the inability to detect diffuse myocardial enhancement. Nonetheless, thanks to the availability of optimized protocols with minimal radiation doses and contrast medium administration, integrating LIE-CT into cardiac CT protocols could enhance its clinical utility, particularly in acute settings, providing valuable prognostic and management insights across a spectrum of cardiac ischemic and non-ischemic conditions.

Proteome partitioning constraints in long-term laboratory evolution

Adaptive laboratory evolution experiments provide a controlled context in which the dynamics of selection and adaptation can be followed in real-time at the single-nucleotide level. And yet this precision introduces hundreds of degrees-of-freedom as genetic changes accrue in parallel lineages over generations. On short timescales, physiological constraints have been leveraged to provide a coarse-grained view of bacterial gene expression characterized by a small set of phenomenological parameters. Here, we ask whether this same framework, operating at a level between genotype and fitness, informs physiological changes that occur on evolutionary timescales. Using a strain adapted to growth in glucose minimal medium, we find that the proteome is substantially remodeled over 40 000 generations. The most striking change is an apparent increase in enzyme efficiency, particularly in the enzymes of lower-glycolysis. We propose that deletion of metabolic flux-sensing regulation early in the adaptation results in increased enzyme saturation and can account for the observed proteome remodeling.

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Biodegradation of DEP, DIBP, and BBP by a psychrotolerant Sphingobium yanoikuyae strain P4: Degradation potentiality and mechanism study.

Phthalic acid esters (PAEs) are human made chemicals widely used as plasticizers to enhance the flexibility of plastic products. Due to the lack of chemical bonding between phthalates and plastics, these materials can easily enter the environment. Deleterious effects caused by this chemo-pollutant have drawn the attention of the scientific community to remediate them from different ecosystem. In this context, many bacterial strains have been reported across different habitats and Sphingobium yanoikuyae strain P4 is among the few psychrotolerant bacterial species reported to biodegrade simple and complex phthalates. In the present study, biodegradation of three structurally different PAEs viz., diethyl phthalate (DEP), di-isobutyl phthalate (DIBP), and butyl benzyl phthalate (BBP) have been investigated by the strain P4. Quantitative analyses through High-performance liquid chromatography (HPLC) revealed that the bacterium completely degraded 1g/L of DEP, DIBP, and BBP supplemented individually in minimal media pH 7.0 within 72, 54, and 120h of incubation, respectively, at 28°C and under shake culture condition (180rpm). In addition, the strain could grow in minimal media supplemented individually with up to 3g/L of DEP and 10.0g/L of DIBP and BBP at 28°C and pH 7.0. The strain also could grow in metabolites resulting from biodegradation of DEP, DIBP, and BBP, viz. n-butanol, isobutanol, butyric acid, ethanol, benzyl alcohol, benzoic acid, phthalic acid, and protocatechuic acid. Furthermore, phthalic acid and protocatechuic acid were also detected as degradation pathway metabolites of DEP and DIBP by HPLC, which gave an initial idea about the biodegradation pathway(s) of these phthalates.

Substrate Analogues Entering the Lipoic Acid Salvage Pathway via Lipoate-Protein Ligase 2 Interfere with Staphylococcus aureus Virulence.

Lipoic acid (LA) is an essential cofactor in prokaryotic and eukaryotic organisms, required for the function of several multienzyme complexes such as oxoacid dehydrogenases. Prokaryotes either synthesize LA or salvage it from the environment. The salvage pathway in Staphylococcus aureus includes two lipoate-protein ligases, LplA1 and LplA2, as well as the amidotransferase LipL. In this study, we intended to hijack the salvage pathway by LA analogues that are transferred via LplA2 and LipL to the E2 subunits of various dehydrogenases, thereby resulting in nonfunctional enzymes that eventually impair viability of the bacterium. Initially, a virtual screening campaign was carried out to identify potential LA analogues that bind to LplA2. Three selected compounds affected S. aureus USA300 growth in minimal medium at concentrations ranging from 2.5 to 10 μg/mL. Further analysis of the most potent compound (Lpl-004) revealed its transfer to E2 subunits of dehydrogenase complexes and a negative impact on its functionality. Growth impairment caused by Lpl-004 treatment was restored by adding products of the lipoate-dependent enzyme complexes. In addition, Caenorhabditis elegans infected with LpL-004-treated USA300 demonstrated a significantly expanded lifespan compared to worms infected with untreated bacteria. Our results provide evidence that LA analogues exploiting the LA salvage pathway represent an innovative strategy for the development of novel antimicrobial substances.

Biosynthesis of bioplastic polyhydroxybutyrate (PHB) from microbes isolated from paddy/sugarcane fields and fabrication of biodegradable thin film

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA) family microbial polyester. Three Gram-negative bacteria that produce PHB, Acinetobacter baumannii, Enterobacter hormaechei, and Pseudomonas xanthomarina were isolated and cultured in nitrogen-limited minimal salt media. The pH and culture media were further optimized with various glucose and peptone as carbon and nitrogen sources to improve PHB production. FTIR, UV–vis, NMR, TGA, and XRD were used to characterize the PHB produced. The optimal pH for PHB production by Acinetobacter baumannii, Enterobacter hormaechei, and Pseudomonas xanthomarina was discovered to be 6.5, 7.5, and 7.5, respectively. P. xanthomarina produced the highest PHB (7.5 gm/L) in the optimized culture media. Furthermore, PHB-starch thin films were prepared and characterized by XRD before their biodegradability was investigated. The degradation study reveals that 25% of PHB-starch blended thin film has been degraded within 30 days.

Impact of oil supply conditions on water lubrication assisted with minimal secondary lubricating medium

Abstract Previous research has revealed that introducing a secondary lubricating medium can temporarily enhance water-lubricated bearing performance, thereby reducing the risk of lubrication failure under challenging conditions. This study aims to identify the optimal injection point for the secondary lubricating medium and evaluates oil supply rate effects. Experiments are conducted using a block-on-ring test rig, complemented by CFD simulations to elucidate the underlying mechanisms. Three oil supply settings, upstream, inlet and outlet of the contact region, are compared. Results shows that upstream oil supply led to a lower critical speed at which the added oil loses any effect compared to the inlet oil supply and outlet oil supply. The effect of oil supply rates on friction reduction varies among the oil supply settings and ring rotational speeds. In conclusion, the most effective oil supply setting positions the injector at the outlet of the contact region, outperforming placement at the entrance of the contact region.

Biodegradation of polyurethanes by Staphylococcus warneri and by microbial co-culture

With the increasing production and use of polyurethanes (PUs), it is necessary to develop sustainable techniques for the remediation of plastic pollution. The use of microorganisms capable of biodegrading PUs may be an environmentally desirable solution for controlling these plastic contaminants. To contribute to the discovery of alternatives for the mitigation of plastics in the environment, this study aimed to explore the potential of StaphylococcuswarneriUFV_01.21, isolated from the gut of Galleria mellonellalarvae, for biodegradation of PU in pure culture and microbial co-culture with Serratia liquefaciensL135. S. warneri grew using Impranil® PU as the sole carbon source in pure culture and co-culture. With six days of incubation, the biodegradation of Impranil® in Luria Bertani broth was 96, 88 and 76%, while in minimal medium, it was 58, 54 and 42% for S. warneri, S. liquefaciens, and co-culture, respectively. In addition, S. warneri in pure culture or co-culture was able to biodegrade, adhere and form biofilms on the surfaces of Impranil® disks and poly[4,4′-methylenebis (phenyl isocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone] (PCLMDI) films. Scanning electron microscopy also revealed biodegradation by detecting the formation of cracks, furrows, pores, and roughness on the surfaces of inoculated PU, both with pure culture and microbial co-culture. This study is the first to demonstrate the potential of S. warneriin PU biodegradation.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

A screening method for plastic-degrading fungi

The growing amount of plastic waste requires new ways of disposal or recycling. Research into the biodegradation of recalcitrant plastic polymers is gathering pace. Despite some progress, these efforts have not yet led to technologically and economically viable applications. In this study, we show that respirometric screening of environmental fungal isolates in combination with scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy can be used to identify new strains with the potential for the degradation of plastic polymers. We screened 146 fungal strains, 71 isolated from car repair shops, an environment rich in long-chain hydrocarbons, and 75 isolated from hypersaline water capable of growing at high concentrations of NaCl. When grown in a minimal medium with no carbon source, some strains produced significantly more CO2 when a pure plastic polymer was added to the medium, some only at high salinity. A selection of these strains was shown by FTIR and Raman spectroscopy to alter the properties of plastic polymers: Cladosporium sp. EXF-13502 on polyamide, Rhodotorula dairenensis EXF-13500 on polypropylene, Rhodotorula sp. EXF-10630 on low-density polyethylene and Wickerhamomyces anomalus EXF-6848 on polyethylene terephthalate. Respirometry in combination with specific spectroscopic methods is an efficient method for screening microorganisms capable of at least partial plastic degradation and can be used to expand the repertoire of potential plastic degraders. This is of particular importance as our results also show that individual strains are only active against certain polymers and under certain conditions. Therefore, efficient biodegradation of plastics is likely to depend on a collection of specialized microorganisms rather than a single universal plastic degrader.

Some selected properties of the recombinant aminoacylase from Escherichia coli LGE 36

Background: Aminoacylase has seen extensive use in the synthesis of L-amino acids. L-amino acids are widely used in the food and medical industries, as well as in healthcare. The effect of metal ions and inhibitors on the enzyme activity of recombinant intracellular aminoacylase of Escherichia coli LGE 36 was studied. Acetyl-D,L-methionine and glycyl-L-methionine dipeptide were used as substrates. It has been shown that with the addition of Co2+ ions, the acylase activity of the recombinant enzyme towards N-acetyl-D,L-methionine increases 3 times. In the case of glycyl-L-methionine, the peptidase activity of the recombinant aminoacylase of Escherichia coli LGE 36 increases more than 40 times. A number of other metal ions studied do not stimulate the hydrolytic activity of the enzyme, moreover some of them even inhibit it. PCMB completely inactivates the recombinant aminoacylase, EDTA reduces enzyme activity by 85%. Objective: This study aims to investigate how metal ions and inhibitors affect the enzyme activity of recombinant intracellular aminoacylase Escherichia coli LGE 36. Methods: In this study, a recombinant strain-producer of aminoacylase derived from Escherichia coli LGE 36 was employed. Escherichia coli cells were cultured in M9 minimal medium supplemented at 37°C. To assess aminoacylase activity, assays were conducted at 37°C using a reaction mixture of 0.2 ml final volume comprising 100 mM Na, K-phosphate buffer at pH 7.0, 0.2 mM CoCI2, 40 mM N-acetyl-D, L-methionine, and the enzyme in the appropriate concentration. Acylase activity was quantified as the amount of enzyme catalyzing the formation of 1µmol of L-methionine within 1 minute at 37°C and pH 7.0. Results: Some characteristics of the recombinant intracellular aminoacylase from E. coli LGE 36 were determined. For investigating the impact of metallic ions and inhibitors, acetylmethionine and glycylmethionine were employed as the substrates. Conclusion: The present study and the choice of right strategies for functional food products allows optimizing the process for production of optically active amino acids using the recombinant enzyme to obtain food and feed additives. Keywords: ion, metals, inhibitors, acetylmethionine, glycylmethionine, recombinant aminoacylase from Escherichia coli, enzyme.

Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform.Key pointsA comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed.Experiments and simulations predict N limitation in minimal media under aerobiosis.In silico media design increased experimental biomass yield and PHB titer.

Deletion of yghZ in Escherichia coli promotes growth in presence of furfural with xylose as carbon source.

Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain-SSK201-was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96h and in presence of 1.0g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

Impact of Carao (Cassia grandis) on Lactobacillus plantarum Immunomodulatory and Probiotic Capacity

Lactobacillus plantarum has beneficial effects on the reduction of symptoms of poor lactose digestion and hypercholesterolemia, removal of the duration and severity of diarrheal processes, improvement of the intestinal permeability barrier, prevention of some types of cancer by adsorption or inactivation of genotoxic agents, increased resistance to intestinal and extraintestinal infections, attenuation of inflammatory bowel disease, and prevention of allergies (especially food). On the other hand, carao (Cassia grandis) has shown remarkable nutritious content with influential dietary applications. As a result, this investigation aimed to explore the effect of Cassia grandis pulp on viability of Lactobacillus plantarum under gastrointestinal conditions, immunomodulatory capacity, and probiotic potential. Adding carao to the medium under different experimental conditions, including rich and minimal culture media and a gastrointestinal digestion process of skimmed milk, did not substantially affect Lactobacillus plantarum’s growth but prolonged its viability. The administration of Lactobacillus plantarum with carao in mice did not induce a proinflammatory response at a systemic level. Still, it did cause an increase in the production of immunoregulatory cytokines. Also, the viability of TSB broth was improved by adding carao. Carao improved the growth of acid tolerance, bile tolerance, growth in TSB broth, and NaCl resistance. According to the results, carao may enhance the characteristics of L. plantarum when enriching fermented dairy products.

Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium.

Cysteine and its derivatives, including H2S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H2S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H2S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H2S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Adhesion and biofilm formation by two clinical isolates of Trichosporon Cutaneum in various environmental conditions.

Trichosporon spp. is an emerging opportunistic pathogen and a common cause of both superficial and invasive infections. Although Trichosporon asahii is the most frequently isolated species, Trichosporon cutaneum is also widely observed, as it is the predominant agent in cases of white Piedra and onychomycosis. Trichosporon spp. is a known to produce biofilms, which serve as one of its virulence mechanisms, however, there is limited data available on biofilms formed by T. cutaneum. Thus, the aim of this study was to assess the adhesion and biofilm formation of two clinical isolates of T. cutaneum under various environmental conditions (including temperature, nutrient availability, and carbon source), as well as their tolerance to fluconazole. Adhesion was tested on common abiotic substrates (such as silicone, glass, and stainless steel), revealing that T. cutaneum readily adhered to all surfaces tested. CV staining was applied for the evaluation of the environment influence on biofilm efficiency and it was proved that the nutrient availability has a major impact. Additionaly, fluorescent staining was employed to visualize the morphology of T. cutaneum biofilm and its survival in the presence of fluconazole. Hyphae production was shown to play a role in elevated biofilm production in minimal medium and increased tolerance to fluconazole.

Brilliant green decolorization by dye decolorizing peroxidase producing bacterium, Achromobacter insolitus isolated from Forest soils of Similipal Biosphere Reserve, Odisha

Dye-decolorizing peroxidases (DyPs) are recently identified superfamily of heme-containing peroxidases which has received much attention for its potential application in dye decolourization. In the current research, six bacterial colonies (SDB1-6) showing lignolytic activity were isolated from forest soils of SBR using a minimal salt medium supplemented with alkali lignin and screened for DyP activity using brilliant green dye supplemented luria bertani agar medium. The highest DyP activity showing bacterium was identified using biochemical and 16SrRNA sequencing and was identified as Achromobacter insolitus. A. insolitus was found to be an efficient strain showing the highest DyP activity of 2.7 U/mL/ml at un-optimized condition. The RSM optimization was conducted to increase the DyP enzyme activity. A. insolitus demonstrated DyP activity of 6.20 U/mL/min with an increase of 2.29 times of enzyme activity in optimal condition of pH 7.0, temperature 25 °C, H2O2 concentration 0.1 mM, and time 24 h. The bacterium was further screened for dye decolorization taking crystal violet, methyl orange, methylene blue, brilliant green and methyl red dyes supplemented in LB medium. The bacterium showed maximum brilliant green degradation ability was analyzed using UV-Vis and FTIR analyses to characterize the degraded product. The highest brilliant green degradation activity of the A. insolitus in the present study could be exploited for bioremediation of brilliant green contaminated industrial effluents in a ecofriendly and cost effective way.