The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

Bovine Colostrum Exosomes Are a Promising Natural Bacteriostatic Agent against Staphylococcus aureus.

Bioactive molecules and immune factors in the bovine colostrum (BC) are important elements of passive immunity that prevent bacterial infection. However, the mechanisms underlying the antimicrobial activity of BC are not fully understood. We assessed the antibacterial properties of BC-derived exosomes (BC-Exo) and found that they had bacteriostatic, anti-hemolytic, and biofilm-eradication effects on Staphylococcus aureus. Moreover, cell surface deformation and reduced ATP production were observed following BC-Exo treatment. The most reasonable explanation for this finding is that BC-Exo has a strong inhibitory effect on the oxidative phosphorylation pathway in S. aureus. We demonstrated, for the first time, that BC-Exo can exhibit clear antimicrobial activity against S. aureus. Our findings constitute an important basis for future antibiotic discovery.

Effect of Corynebacterium glutamicum on Livestock Material Burial Treatment.

In recent years, foot-and-mouth disease has occurred in all parts of the world. The animals with the disease are buried in the ground; therefore, their concentration could affect ground or groundwater. Moreover, the complete degradation of carcasses is not a certainty, and their disposal is important to prevent humans, livestock, and the environment from being affected with the disease. The treatment of Corynebacterium glutamicum is a feasible method to reduce the risk of carcass decomposition affecting humans or the environment. Therefore, this study aimed to investigate the effect of C. glutamicum on the soil environment with a carcass. The composition of amino acids in the soil treated with C. glutamicum was generally higher than those in the untreated soil. Moreover, the plant root in the soil samples treated with C. glutamicum had 84.0% amino acids relative to the standard value and was similar to that of the control. The results of this study suggest the possibility to reduce the toxicity of a grave land containing animals with this disease.

Enhanced Hydrogen Production by Co-cultures of Hydrogenase and Nitrogenase in Escherichia coli.

Rhodobacter sphaeroides is a bacterium that can produce hydrogen by interaction with hydrogenase and nitrogenase. We report a hydrogen production system using co-cultivation of hydrogenase in liquid medium and immobilized nitrogenase in Escherichia coli. The recombinant plasmid has been constructed to analyze the effect of hydrogen production on the expression of hupSL hydrogenase and nifHDK nitrogenase isolated from R. sphaeroides. All recombinant E. coli strains were cultured anaerobically, and cells for nitrogenase were immobilized in agar gel, whereas cells for hydrogenase were supplemented on the nitrogenase agar gel. The hupSL hydrogenase has been observed to enhance hydrogen production and hydrogenase activity under co-culture with nifHDK nitrogenase. The maximum hydrogen production has been obtained at an agar gel concentration and a cell concentration for co-culture of 2 % and 6.4 × 10(8) CFU. Thus, co-culture of hupSL hydrogenase and nifHDK nitrogenase provides a promising route for enhancing the hydrogen production and hydrogenase activity.

Improvement of bacterial hydrogen production by ATP in mixed organic compounds extracted from Rhodobacter sphaeroides aerobically cultured under dark conditions.

The aim of this study was to increase the hydrogen production of recombinant Escherichia coli harboring HupSL hydrogenase by supplementing physiologically activating compounds extracted from Rhodobacter sphaeroides cultured under anaerobic dark condition after treating them with dimethyl sulfoxide, and the 0.5% extracts contained 4×10(-8)M ATP, which was 100-fold higher than that in the extracts from E. coli. In addition, it was found that the hydrogen production from recombinant E. coli harboring HupSL hydrogenase isolated from R. sphaeroides was doubled under anaerobic conditions when it was supplemented by the extracts from R. sphaeroides cultured aerobically in dark conditions, and this also showed consistent pattern with the increased level of HupSL hydrogenase expression. Therefore, we conclude that the mixed organic compounds extracted from R. sphaeroides have an ATP which enhances the hydrogen production by increasing the amount of HupSL hydrogenase.

Evaluation of whole lysosomal enzymes directly immobilized on titanium (IV) oxide used in the development of antimicrobial agents.

Lysosomal enzymes isolated from egg white were directly immobilized on titanium (IV) oxide (TiO(2)) particles using shaking methods (150 rpm, room temperature, 10 min), and the immobilization efficiency, activity, and stability of lysosomal enzymes immobilized on TiO(2) were evaluated. Of the various mass ratios (w/w) of lysosomal enzymes to TiO(2) tested, we found that 100% immobilization efficiency was observed at a ratio of 1:20 (enzymes:TiO(2); w/w). Furthermore, the antimicrobial activities of the immobilized lysosomal enzymes were confirmed using viable cell counts against Escherichia coli. Our results showed that the antimicrobial activity of immobilized lysosomal enzymes is stable and can be maintained up to one month, but the antimicrobial activity of free enzymes without immobilization completely disappeared after five days in storage. In addition, enhanced immobilization efficiency was shown in TiO(2) pretreated with a divalent, positively charged ion, Ca(2+), and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized enzymes. However, K(+), a monovalent, positively charged ion, did not have any positive effect on immobilization or antimicrobial activity. Finally, we suggest that activity and stability of immobilized lysosomal enzymes can be maintained for a longer time than those properties of free lysosomal enzymes.

Interaction of transcriptional repressor ArgR with transcriptional regulator FarR at the argB promoter region in Corynebacterium glutamicum.

In Corynebacterium glutamicum, the ArgR protein, a transcriptional repressor, affects the expression level of the argB gene through binding to its promoter region. The argB promoter region (positions -77 to -25) has been found by in vitro electrophoretic mobility shift assay (EMSA) results and in silico analysis to be important for the DNA binding of ArgR. Proline supplementation prevented the DNA binding of ArgR to the argB promoter region and triggered an increase of the argB mRNA level. Additional mutational analyses of the argB promoter region found nucleotides critical for ArgR binding (G located at position -58, C at position -55, and A at position -41 of the argB promoter) in that region. Another transcriptional repressor, FarR, was also demonstrated to bind to the argB promoter region. This binding was delimited to positions -57 to -77 on the argB promoter. FarR has only one putative binding domain located at positions -57 to -77, but this region exactly overlapped with the binding region located from positions -55 to -77 for the binding of ArgR within the argB promoter; thus, if ArgR bound with the argB promoter first, the binding of FarR was not observed in this region. However, if FarR bound to the binding domain located at positions -57 to -77 first, ArgR could bind other binding sites located at positions -49 to -25 within the argB promoter. Finally, this study suggests that ArgR can affect FarR binding to the argB promoter region, as protein binding is dominated by the protein most able to do so.

Utilization of phenol and naphthalene affects synthesis of various amino acids in Corynebacterium glutamicum.

This article reports multiple metabolic pathways of amino acid production via phenol and naphthalene use by Corynebacterium glutamicum. Biodegradation of phenol and naphthalene by C. glutamicum occurred in a mineral salt medium containing 1% yeast extract without any additional carbon sources. Among the amino acids synthesized via the TCA-cycle, glutamate synthesis increased in C. glutamicum supplemented with 8.5 mM phenol or with 4.2 mM naphthalene. Aspartate synthesis significantly increased when cultured with 4.2 mM naphthalene, and increased synthesis of threonine and histidine was observed only with the addition of phenol. In addition, synthesis of valine and leucine decreased considerably under both conditions. Moreover, the bioconversion of glutamate from phenol and naphthalene is regulated by a transcriptional regulator, FarR, at the transcription level of the gltBD and gdh genes. In this study, we found that the utilization of phenol and naphthalene enhances biosynthesis of several amino acids and that this mechanism is controlled by a transcriptional regulator.

Enhancement of ornithine production in proline-supplemented Corynebacterium glutamicum by ornithine cyclodeaminase.

In this study, Corynebacterium glutamicum and its derived mutants were used to demonstrate the relationship between proline, glutamate and ornithine. The maximum ornithine production was shown in the culture medium (3295.0 mg/l) when the cells were cultured with 20 mM proline and was 15.5 times higher than in the presence of 1 mM proline. However, glutamate, which known as an intermediate in the process of converting proline to ornithine, did not have any positive effect on ornithine production. This suggests that the conversion of proline to ornithine through glutamate, is not possible in C. glutamicum. Comparative analysis between the wild-type strain, SJC8043 (argF-, argR-) and SJC8064 (argF-, argR- and ocd-), showed that C. glutamicum could regulate ornithine production by ornithine cyclodeaminase (Ocd) under proline-supplemented conditions. Therefore, proline directly caused an increase in the endogenous level of ornithine by Ocd, which would be a primary metabolite in the ornithine biosynthesis pathway.

Conversion of phenol to glutamate and proline in Corynebacterium glutamicum is regulated by transcriptional regulator ArgR.

This paper reports a novel integrated metabolic pathway of amino acid production through the utilization of phenol in Corynebacterium glutamicum. In the presence of 8.5 mM phenol, the level of glutamate and proline production increased up to 1.2- and 14.7-fold, respectively, compared to the control condition. In addition, their productivities increased 1.6- and 20-fold in the culture medium using phenol as the sole carbon source with 72 microM FeSO(4) as iron supplementation. Chromatin immunoprecipitation assay showed that the DNA-binding affinity of ArgR as a transcriptional repressor to the upstream of gltB and gdh gene was reduced significantly in the presence of 8.5 mM phenol. In addition, the DNA-binding affinity of ArgR to the upstream of gltB and gdh gene by iron supplementation was severely reduced, more than that under only 8.5 mM phenol. These results are consistent with those showing an increase in the mRNA levels of the two genes in the presence of 8.5 mM phenol and iron supplementation. Overall, iron enhances the biotransformation to glutamate and proline from phenol because of the regulated transcriptional levels. Furthermore, the biotransformation of phenol to glutamate and proline probably occurs through an interaction between ArgR and the gltB and gdh genes.

Proline reduces the binding of transcriptional regulator ArgR to upstream of argB in Corynebacterium glutamicum.

In this study, the ArgR-binding sites on the arg operon Corynbebacterium glutamicum were characterized by in vivo chromatin immunoprecipitation (ChIP). In addition, the ArgR-binding affinity in the presence of glutamate, proline, or arginine was examined to get further information on expression control. The ChIP assay showed that the ArgR protein binds specifically to the upstream regions of argC, argB, argF, and argG. Upon proline supplementation, ArgR-binding affinity was significantly reduced upstream of argB, resulting in increased ornithine production. In contrast, there was no change in the binding affinity of ArgR to the upstream regions of argC, argF, argG, or argB following the addition of glutamate and arginine. These results suggest that the upstream region of argB on the arg operon plays an important role in interacting with ArgR under proline-supplemented conditions and that proline causes an increase in the endogenous level of ornithine by reducing the binding affinity of ArgR to the upstream region of argB.

The effect of ArgR-DNA binding affinity on ornithine production in Corynebacterium glutamicum.

pEMBTL-SY1, which can over produce the ArgR protein in Corynebacterium glutamicum, was constructed. The DNA-binding affinity of ArgR was analyzed using a Chromatin Immunoprecipitation (ChIP) assay. The level of ArgR protein expression in the plasmid-carrying C. glutamicum (pEMBTL-SY1) was higher than that in the wild-type strain. On the other hand, there was no increase in the DNA-binding affinity of ArgR on the upstream of argB and the level of ornithine production. The DNA-binding affinity of ArgR on the arg operon and the level of ornithine production in the presence of three metabolites, ornithine, arginine, and proline, were examined as feedback controlling effectors in the arginine biosynthesis pathway in C. glutamicum. The ChIP assay showed that the supplemented metabolites altered the ArgR-binding affinity on the upstream of argB, which is consistent with the change in ornithine production. This suggests that the regulation of ornithine biosynthesis by the transcriptional regulator, ArgR, depends on the DNA-binding affinity of the arg operon, which is regulated by the feedback controlling effectors, rather than on the level of ArgR protein expression.

The ars genotype characterization of arsenic-resistant bacteria from arsenic-contaminated gold-silver mines in the Republic of Korea.

The isolates were identified on the basis of ars genotype characteristics as well as arsenic oxidation/reduction analysis based on the molecular detection characterization. Diversity, pH range (4.0 to 7.0), location, and ars features were assessed for four arsenic-contaminated pond sites and six arsenic tailings located in the Duck-um mine and Myoung-bong mine areas. The presence of ars genes in the genomes of each bacterial strain was evaluated using polymerase chain reaction. Batch experiment results showed that Pseudomonas putida strains OS-3 and -18 completely oxidized 1 mM of arsenite(III) to arsenate(V) within 35-40 h. In contrast, two arsenate-reducing bacteria isolated from mines, P. putida RS-4 and RS-5, were capable of growing aerobically in growth medium supplemented with up to 66.7 mM arsenate(V), which are significantly higher concentration than those tolerated by other arsenic-resistant bacteria. These results suggest that newly isolated indigenous arsenic-resistant bacteria may provide a better understanding of the molecular geomicrobiology and may be applied to the bioremediation of arsenic-contaminated mines in Korea. Ecologically, the redox potential plays an important role in arsenic toxicity and mobility in As-contaminated mine areas, as it facilitates the biogeochemical cycling activity of Pseudomonas sp. groups.