Minimal Agar Research Articles

Synthesis and Antibacterial Properties of Novel Quaternary Ammonium Lignins.

The ongoing demand for effective antimicrobial materials persists, and lignin emerges as a promising natural antibacterial material with renewable properties. The adaptability of lignin to various chemical modifications offers avenues to enhance its antimicrobial activity. Here, we employed chloromethylation and subsequent functionalization with variable tertiary N-alkyl dimethyl amines to produce C6-C18 quaternary ammonium lignins (QALs) from hardwood (aspen), softwood (pine), and grass (barley straw). Successful synthesis of QALs was confirmed through NMR and FTIR analysis results along with an increase in the surface ζ-potential. Antibacterial activity of QALs against clinical strains of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus was assessed using minimal bactericidal concentration (MBC) assay and agar growth inhibition zone (ZOI) test. The antibacterial activity of QALs was found to be higher than that of the unmodified lignins. QALs with longer alkyl chains demonstrated an MBC of 0.012 mg/L against K. pneumoniae already after 1 h of exposure with similar effect size reached after 24 h for S. aureus. For all the lignins, an increase in alkyl chain length resulted in an increase in their bactericidal activity. MBC values of C14-C18 QALs were consistently lower than the MBC values of QALs with shorter alkyl chains. Besides the alkyl chain length, MBC values of barley and pine QALs were negatively correlated with the surface ζ-potential. While alkyl chain length was one of the key properties affecting the MBC values in a liquid-based test, the agar-based ZOI test demonstrated an antibacterial optimum of QALs at C12-C14, likely due to limited diffusion of QALs with longer alkyl chains in a semisolid medium.

Auxotrophy of <I>Yersinia pestis</I> Strains of Antiqua Biovar and Its Genetic Basis

The aim of the work was to study the growth dependence on amino acids in Yersinia pestis strains of different phylogenetic branches of the antiqua biovar and to determine the genetic basis of auxotrophy of these strains.Materials and methods. We used 38 strains of Y. pestis, the main subspecies of the antiqua biovar, isolated in various foci of the world in the period of 1928–2020. The nutritional requirements of the strains were determined by incubation on Difco minimal agar with different sets of amino acids. Phylogenetic analysis of the strains was carried out using the Wombac 2.0 and SeaView 5.0.5 programs. Comparative analysis of the nucleotide sequences of the genes was performed using the BLAST algorithm and the Mega 7.0 program.Results and discussion. The phylogenetic appurtenance of the investigated Y. pestis strains of antiqua biovar to the phylogenetic branches 0.ANT3, 0.ANT5, 1.ANT, 2.ANT3, 3.ANT2, 4.ANT has been determined. It is established that all studied strains have a common dependence of growth on three amino acids – phenylalanine, threonine, and methionine. In the majority of the strains of antiqua biovar of all phylogenetic branches, growth is also dependent on the presence of cysteine in the medium, except for a part of the strains of the phylogenetic branch 4.ANT. In 14 genes of sulfur and cysteine metabolism, 19 mutations have been identified. Each phylogenetic group of the antique biovar has a distinct profile of mutations in genes involved in cysteine biosynthesis. The leucine requirement of the strains belonging to phylogenetic branch 0.ANT5 has been established, possibly caused by a frame shift in the leuA gene. Strains of the 1.ANT branch isolated in the Democratic Republic of the Congo demonstrate an additional proline-dependent growth. The defined nutritional requirements in the strains and the genetic causes of auxotrophy complement the phenotypic and genetic characteristics of the phylogenetic branches of the antiqua biovar and can be used as genetic markers for the differentiation of these strains.

Microbes Isolated from Landfill Soil Utilize Polyethylene Terephthalate (PET) as Their Sole Source of Carbon: An Unexplored Possibility of Bioremediation in Bangladesh

Plastic products are so extensively used that they continue to strain the already overburdened waste management system and, inevitably, the global climate. Biodegradation is a sustainable remedy. Here, we report a few microorganisms isolated from landfill soil near Dhaka that thrive especially on polyethylene terephthalate (PET) polymers. Soil samples were subjected to three enrichment cycles that contained no carbon except PET. Pure isolates were recovered and incubated on minimal agar containing PET as the sole carbon. A morphological examination was carried out. Potential PET-degrading enzyme sequences from the isolates and other microalgae were analyzed for homology using BLASTP and TBLASTN, and multiple sequence alignment (MSA) was performed to assess conserved domains. Six isolates were obtained. Two isolates grew around the PET film but did not grow sufficiently in other areas of the minimal agar. Two other isolates with greenish pigmentation flourished around the PET film as well as on other areas of the agar. One of the green cells resembled Aphanocapsa, with irregular shapes and occasionally brown dense bodies, while the others looked round like Microcystis. Homology analysis revealed the hypothetical PETases in green cells contained the highly conserved catalytic triad (Ser-His-Asp) at the active site, as always found in alpha-beta hydrolase fold containing enzymes. Microbes isolated from two landfill sites in the vicinity of Dhaka have been adapted to utilize PET as a carbon source. In the future, sequencing and further characterization would be necessary to validate the findings. Microalgal systems demand increased focus, given their potential to offer valuable resources for bioremediation.

Isolation of High Density Polyethelene Degradating Bacteria from Soil

Plastics are the most commonly used polymers for routine applications. These plastics are less biodegradable in the environment. So the plastics accumulated drastically in the environment and had bad effects on it. High-density polyethylene (HDPE) is a major cause of persistent and long-term environmental pollution. The main objective of the present study is the isolation and screening of bacterial species with the capability to degrade high-density polyethylene (HDPE). The two bacterial isolates were identified from soil collected from the dumping yard of village Atake (Tal-Karad, Dist-Satara). These two bacterial isolates were obtained on minimal agar medium incorporated with HDPE. These isolates are further tested for degradation of the plastic. The isolate 1 (Bacillus cereus) could degrade the plastic to the extent of 16% in terms of % weight loss, while the isolate 2 (Pseudomonas halobacterium) could degrade the plastic to the extent of 10% in terms of % weight loss, thus the isolate 1, which was identified as Bacillus cereus was found to be the best among the two isolates.

Screening and Functional Analyses of Novel Cecropins from Insect Transcriptome.

Antibiotic resistance is a significant and growing threat to global public health. However, antimicrobial peptides (AMPs) have shown promise as they exhibit a broad spectrum of antibacterial activities with low potential for resistance development. Insects, which inhabit a wide range of environments and are incredibly diverse, remain largely unexplored as a source of novel AMPs. To address this, we conducted a screening of the representative transcriptomes from the 1000 Insect Transcriptome Evolution (1KITE) dataset, focusing on the homologous reference genes of Cecropins, the first identified AMPs in insects known for its high efficiency. Our analysis identified 108 Cecropin genes from 105 insect transcriptomes, covering all major hexapod lineages. We validated the gene sequences and synthesized mature peptides for three identified Cecropin genes. Through minimal inhibition concentration and agar diffusion assays, we confirmed that these peptides exhibited antimicrobial activities against Gram-negative bacteria. Similar to the known Cecropin, the three Cecropins adopted an alpha-helical conformation in membrane-like environments, efficiently disrupting bacterial membranes through permeabilization. Importantly, none of the three Cecropins demonstrated cytotoxicity in erythrocyte hemolysis tests, suggesting their safety in real-world applications. Overall, this newly developed methodology provides a high-throughput bioinformatic pipeline for the discovery of AMP, taking advantage of the expanding genomic resources available for diverse organisms.

Mutagenicity assessment of high-power 1.6-THz pulse laser radiation.

The effect of terahertz (THz) radiation has been studied in medicine. However, there is a lack of scientific information regarding its possible mutagenicity. Therefore, the present study aimed to assess the mutagenicity of 1.6 THz laser irradiation. The Ames test was conducted using five bacterial tester strains. The bacteria were subjected to (i) 1.6 THz laser irradiation at 3.8 mW/cm2 for 60 min using a tabletop THz pulse laser system, (ii) ultraviolet irradiation, (iii) treatment with positive control chemicals (positive control) or (iv) treatment with the solvent used in the positive control (negative control). After treatment, the bacterial suspensions were cultured on minimal glucose agar to determine the number of revertant colonies. In addition, the comet assay was performed using fibroblasts (V79) to assess possible DNA damage caused by the THz laser irradiation. The Ames test demonstrated that the THz laser irradiation did not increase the number of revertant colonies compared to that in the negative control group, whereas the ultraviolet irradiation and positive control treatment increased the number of revertant colonies. Thus, 1.6 THz laser irradiation is unlikely to be mutagenic. The comet assay additionally suggests that the THz laser irradiation unlikely induce cellular DNA damage.

Biodegradation of Petroleum Sludge by Methylobacterium sp. Strain ZASH.

A bacterium was isolated from sludge-contaminated soil in a petroleum refinery and tested for its ability to degrade aliphatic hydrocarbon compounds present in petroleum sludge. The isolate was grown on minimal salt media agar supplemented with 1% (w/v) petroleum sludge. The isolate was tentatively identified as Methylobacterium s p. s t rain ZASH based on the partial 16s rDNA molecular phylogeny. The bacterium grew optimally between the temperatures of 30°C and 35°C, pH 7 and 7.5, 0.5% and 1.5% (v/v) Tween 80 as the surfactant, and between 1% and 2% (w/v) peptone as the nitrogen source. The constants derived from the Haldane equation were μmax = 0.039 hr-1, Ks = 0.385% (w/v) total petroleum hydrocarbons (TPH) or 3,850 mg/L TPH, and Ki =1.12% (w/v) TPH or 11,200 mg/L. The maximum biodegradation rate exhibited by this strain was 19 mg/L/hr at an initial TPH concentration of 10,000 mg/L. Gas chromatography analysis revealed that after 15 days the strain was able to degrade all aliphatic n-alkanes investigated with different efficiencies. Shorter n-alkanes were generally degraded more rapidly than longer n-alkanes with 90% removal for C-12 compared to only 30% removal for C-36. The addition of sawdust did not improve bacterial degradation of petroleum hydrocarbons, but it assisted in the removal of remaining undegraded hydrocarbons through adsorption.

Periodic Appraisal of Cholesterol Degradation by Two Bacteria Isolates from 'Iru' (Fermented Parkia Biglobosa)

This study was carried out to evaluate the incubation period on cholesterol degrading ability of bacteria isolated from 'iru' (fermented Parkia biglobosa seeds). The total bacterial count after culturing 'iru' (Parkia biglobosa) on nutrient agar were 7.30, 8.13, 7.28 and 8.11 respectively. The isolates were identified as Bacillus cereus and Bacillus subtilis. The isolates were screened on a salt agar of 1% cholesterol. Some of the isolates utilized cholesterol and grew well. IRUA and IRUC had the highest growth on the minimal salt cholesterol agar, followed by IRUB. The growth of 2IRUB was very low while IRUD, 2IRUA, 2IRUC, 2IRUD had no growth on the nutrient agar. Bacillus subtilis and Bacillus cereus degraded cholesterol maximally as 59.57mmol/L and 43.51mmol/L respectively. The cholesterol concentration degraded greatly with incubation period showing that there were high activities of the microorganisms and maximum cholesterol degradation was recorded on the 7th day. This study showed that the cholesterol degrading bacteria from Parkia biglobosa are endowed with the capability to degrade cholesterol and are good sources of cholesterol oxidase exploited for potential biomedical and industrial applications.

Biotransformation of Chitin to Chitosan using Bacillus cereus JCM44 isolated from Kanyakumari, India and its Characterization

Bacillus cereus JCM44 was isolated from the soil samples collected from the Kanyakumari district of Tamil Nadu, using a minimal agar medium. It was characterized by its ability to biotransform chitin into chitosan at 30°C for 48 h. The Chitosan (CS) was recovered by using 0.1N Sodium hydroxide and 2% acetic acid. It was confirmed by iodine solution with dark purple colouration and qualitatively identified by acetone and ethanol. CS formation has been identified from X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR). Scanning Electron Microscopy (SEM) analysis elucidated the formation of an average size of 349 nm to 1µm with the morphology of a non-porous, smooth membrane with irregular shape and crystallite. The Zeta potential study of the bacterially synthesized chitosan showed their excellent stability with a surface charge of -2.28 mV. Bacterially Synthesized Chitosan (BSC), with a polydispersity index (PDI) value of 0.626 indicates the aggregation of particles. The peaks of higher wavenumber confirm the formation of blend in Bacterially Synthesized Chitosan (BCS) at OH and NH was denoted. The XRD analysis of the BSC detected the crystallinity of the samples when compared to the commercial chitosan.

P330 Role of a D-amino acid oxidase (DAO) in an efficient human commensal Candida albicans

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Candida albicans is an opportunistic fungal pathogen residing as a harmless commensal component of the human microbiota. In the gut, C. albicans efficiently colonizes and successfully competes to limiting nutrient resources and overcomes the detrimental effect of an array of secondary metabolites released by the host and the dominant bacterial microflora. Among the metabolites, several free D-amino acids are released into the gut lumen, especially as a result of the degradation of bacterial cell walls. We sought to identify the genes that might confer advantages to C. albicans in metabolizing D-amino acids in diverse morphotypes, which might confer a competitive advantage.MethodsAll C. albicans genetically modified strains were constructed using a standard lithium acetate transformation method. The D-amino acid utilization and growth of C. albicans strains were assessed by spot dilution assays on minimal media agar plates at 30°C. Morphological switching between the yeast and hyphal forms was assessed in liquid media using hyphal-inducing conditions in the presence or absence of D-amino acids. Biofilm assays were performed using YNB-galactose media (with or without D-amino acids) on serum-treated 6-well plates at 30°C.Results C. albicans genome harbors three ORFs putatively encoding D-amino acid oxidase genes- DAO1orf19.3065, DAO2orf3365, and IFG3orf19.944. D-amino oxidase genes are lost from the genomes of several fungal species belonging to the Saccharomycetaceae family, including the free-living Saccharomyces cerevisiae. We found that C. albicans utilizes and grows on D-leucine, an amino acid known to be released by the gut bacteria into the intestinal lumen. This utilization is dependent on the growth morphotypes since the cells growing in biofilm fail to metabolize D-leucine. However, deletion of IFG3 but neither DAO1 nor DAO2 completely abrogates D-leucine utilization capabilities of C. albicans. The null ifg3 strain is moderately growth-sensitive in presence of D-alanine, an amino acid abundantly present and synthesized in the gut by bacteria for incorporation into peptidoglycan in their cell walls. By comparing Ifg3 protein levels in a leucine auxotrophic strain in different growth modes, we found that the expression of Ifg3 protein increases several folds in the biofilm growth mode in presence of D-leucine, but cells fail to grow optimally and make three-dimensional biofilm. Furthermore, ectopic expression of CaIFG3 in S. cerevisiae, which lost all D-amino oxidase genes, helps the organism to utilize D-leucine as a nutrient resource as well as overcome its inhibitory effect on growth.ConclusionOur results indicate that Ifg3 provides C. albicans the ability to metabolize D-leucine and helps it to overcome the growth inhibitory effect of D-alanine. Based on these primary observations we speculate a crucial role of Ifg3 in providing a competitive advantage against the resident microflora in the gut where continuous turnover of D-amino acids is crucial in maintaining host-microbe symbiotic interactions. Further in vivo commensalism experiments in mice will provide important clues on the role of this enzyme in C. albicans’s ability in gut colonization.

S6.3c Adaptive dynamics in experimental populations of Aspergillus nidulans

S6.3 Fungal adaptation and evolution, September 22, 2022, 4:45 PM - 6:15 PM A total of 12 replicate populations initiated with a laboratory strain of the ascomycete fungus Aspergillus nidulans evolved on synthetic minimal glucose agar medium for 1 year, using weekly transfers of 1% of the produced asexual spores to fresh medium. This Aspergillus short-term evolution experiment (ASEX) was designed to understand how filamentous fungi adapt to growth on limited carbon in a spatially structured environment. We observed no systematic improvement in the fitness components tested and neither in the competitive fitness relative to the ancestor. Instead, we observed the repeated evolution of at least two morphotypes, with a fluffy-like (FL) or an ancestor-like (AL) colony morphology, leading to non-transitive fitness interactions among isolates in two selected populations. The genomic analyses of clones from all 12 populations at an early (week 10) and the final time point (week 52), show a clear role of natural selection during ASEX. We also observed a shared genetic basis and different timing of adaptation of AL and FL types. In addition, in most populations, both morphotypes do not form monophyletic groups, but they frequently disappear and re-evolve from ancestral forms of both types. Reduction in asexual spore yield, the most evident parallel phenotypic change found in all our evolved populations, is not due to the direct selection of genes involved in asexual reproduction. Instead, we argue that reduced spore yield is a pleiotropic effect of adaptive changes in metabolism.

Copper Tolerance in Xanthomonas arboricola pv. pruni in South Carolina Peach Orchards.

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni, causes yield loss every year in southeastern U.S. peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in X. arboricola pv. pruni in the United States, adaptation of populations from frequent use is a concern. We collected X. arboricola pv. pruni from shoot cankers, leaves, and fruit of cultivar O'Henry over 2 years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. X. arboricola pv. pruni was isolated from four canker types (bud canker, tip canker, nonconcentric canker, and concentric canker) in early spring (bud break), as well as from leaf and fruit tissues later in the season at the phenological stages of pit hardening and final swell. X. arboricola pv. pruni was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. X. arboricola pv. pruni isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 μg/ml or 51 μg/ml of Cu2+. Two phenotypes of copper tolerance in X. arboricola pv. pruni were discovered: low copper tolerance (LCT; growth up to 38 μg/ml Cu2+) and high copper tolerance (HCT; growth up to 51 μg/ml Cu2+). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at the stages of delayed dormancy (high rate; 2,397 μg/ml Cu2+), shuck split (medium rate; 599 μg/ml Cu2+), and during summer cover sprays (low rate; 120 μg/ml Cu2+), and subsequently inoculated with sensitive, LCT, and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of X. arboricola pv. pruni tolerance to copper in commercial peach orchards in the southeastern United States, and suggests its contribution to bacterial spot development under current management practices.

Agar plate assay for rapid screening of aryl-alcohol oxidase mutant libraries in Pichia pastoris

Directed evolution is a powerful tool for developing biocatalysts with tailor-made properties for biocatalytic applications. High-throughput activity screening of large mutant libraries generated by e.g. means of directed evolution is usually performed in 96-well microtiter plates requiring, however, time-consuming and laborious enzyme expression, cell harvesting and activity measurements. In addition, automated liquid handling systems are needed to cope with the high number of colonies to be screened. Herein, we developed an agar plate-based assay for simple and fast screening of H2O2-producing aryl-alcohol oxidase (AAO) mutant libraries in Pichia pastoris. Buffered minimal methanol agar plates were supplemented with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP) and the target substrate. AAO activity is visualized by formation of green zones around AAO-secreting P. pastoris colonies due to ABTS oxidation by HRP which is fueled with H2O2 by AAO in course of substrate oxidation. Colonies were screened within 24 h for AAO activity using different AAO substrates like veratryl alcohol, p-anisyl alcohol or trans,trans-2,4-hexadien-1-ol and even low AAO activity towards 5-hydroxymethylfurfural could be detected within 48 h. The developed agar plate-based assay can be extended to other substrates and might also be applied for fast and substrate-specific screening of other H2O2-producing oxidases in P. pastoris.

A new release nanosystem mucoadhesive gel of Brazilian Red Propolis-containing chitosan: in vitro Citoxicity and Antimicrobial test. In vivo ligature induced periodontitis model- propolis treatment in rats

Red Propolis has been shown to be a potent antimicrobial, antinflammatory, antitumoral and antioxidant, however, is less studied against microorganisms and periodontal disease. This study aimed to investigate the antimicrobial, citoxicity properties and control of a induced periodontal pocket in rats of a red propolis mucoadhesive gel -containing chitosan. For this, we used two gels with concentrations of 5% (RPG5%) and 10% (RPG10%) of propolis and were compared with chitosan gel base 5% (CHG), propolis extract 5% (FRP5%) and chlorhexidine 0.12% (CHX0,12%). The products were tested against S. mutans, S. salivarius, S. sanguinis, L. casei, A. actinomycetemcomitans, E. faecalis, P. gingivalis, F. nucleatum, and C. albicans ATCC standards. The Minimum Inhibitory Concentration, Concentration Minimal Bactericidal and agar diffusion tests were performed according to CLSI standards. Ligature induced periodontitis model were used and the gel product were applied in test and control rats group. The results showed that all microorganisms were sensitive to the gel RPG5% and RPG10%. There was a reduction in insertion loss and alveolar resorption in the animals treated with propolis gel. Chitosan appears to have not affected the antimicrobial activity of propolis and there seems to have been a synergism between the products. On the other hand, new tests and clinical trials studies should be performed to confirm these parameters.

Floral origin, Antioxidant and Antimicrobial Activity of Some Floral Honey

The study was planned to assess botanic origin, antioxidant power, antimicrobial activity and physicochemical properties of four honey samples were collected in Turkey. The melissopalynologic results revealed that sample 1 and 2 are polyfloral, and others are unifloral. Total content of phenolic (116.47-1711.13 mg GAE/100 g) and flavonoids (1.46 - 27.25 mg CAE/100 g) were analysed, and the antioxidant activities measured by ferrous ion chelating activity (FICA) (71.32-93.35%), hydrogen peroxide scavenging activity (HPSA) (SC50: 217.8-1238.39 µg/mL), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging (SC50: 20.15-27.14 µg/mL), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (SC50: 22.93-308.17 µg/mL) and ferric reducing antioxidant power (FRAP) (71.03-99.54 %) differed notably. For comparison of the samples, butylated hydroxy anisole (BHA), rutin (RUT) and trolox (TRO) were used as standard antioxidant compounds. According to these results, samples showed more efficient antioxidant activity than TRO except for ABTS assay. Also, Sample 1 and 4 have higher FICA activity and Sample 2 and 3 demonstrated more FRAP activity than standards. The minimal inhibitory concentrations (MICs) and agar diffusion assays of honey samples against Staphylococcus aureus and Escherichia coli showed that these samples was the most effective in inhibition of their growth.

Isolation and Screening of Zinc and Potassium Solubilizing Microorganisms from Different Rhizospheric Soil

Zinc is considered as a crucial trace micronutrient for proper growth, reproduction and driving of many metabolic reactions in all crops. Potassium is classified as a macronutrient associated with movement of water and nutrients as well as enzyme activation. The present study was aimed for isolation and screening of zinc and potassium solubilizing microorganisms from different rhizospheric soil of rice. Eight different isolates- ZSB1, ZSB2, ZSB3, ZSB4, KSB1, KSB2, KSB3 and KSB4 were obtained and were screened for their Zinc and Potassium solubilizing activities on TRIS minimal agar medium and Aleksandrov’s medium, respectively. Upon characterization, ZSB1, ZSB4 and KSB3 were observed as motile Gram-negative organisms, ZSB2, KSB2 and KSB4 as motile Gram-positive organisms while ZSB3 and KSB1 had mycelial growth with some conidial spore. All isolates gave positive results for starch and lipid hydrolysis. In gelatin hydrolysis, only ZSB3, ZSB4, KSB1 and KSB2 gave positive results. The main purpose to obtain these zinc and potassium solubilizing microorganisms was for their use in plant growth promotion as biofertilizers to increase soil fertility and crop yield.

The Transcription-Repair Coupling Factor Mfd Prevents and Promotes Mutagenesis in a Context-Dependent Manner.

The mfd (mutation frequency decline) gene was identified by screening an auxotrophic Escherichia coli strain exposed to UV and held in a minimal medium before plating onto rich or minimal agar plates. It was found that, under these conditions, holding cells in minimal (nongrowth) conditions resulted in mutations that enabled cells to grow on minimal media. Using this observation as a starting point, a mutant was isolated that failed to mutate to auxotrophy under the prescribed conditions, and the gene responsible for this phenomenon (mutation frequency decline) was named mfd. Later work revealed that mfd encoded a translocase that recognizes a stalled RNA polymerase (RNAP) at damage sites and binds to the stalled RNAP, recruits the nucleotide excision repair damage recognition complex UvrA2UvrB to the site, and facilitates damage recognition and repair while dissociating the stalled RNAP from the DNA along with the truncated RNA. Recent single-molecule and genome-wide repair studies have revealed time-resolved features and structural aspects of this transcription-coupled repair (TCR) phenomenon. Interestingly, recent work has shown that in certain bacterial species, mfd also plays roles in recombination, bacterial virulence, and the development of drug resistance.

L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli.

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

In vitro inactivation effect of blue light emitting diode (LED) on Shiga-toxin-producing Escherichia coli (STEC)

The antimicrobial effects of blue LED were investigated on seven Shiga-toxin-producing Escherichia coli strains (STEC 7 serotypes) using two LED formats at 405 nm (Prototype A, bulb array emitting 22.77 ± 1.054 mW/cm2; Prototype B, strip array emitting 4.25 ± 0.25 mW/cm2). For both prototypes, cytotoxic effects were detected with time-dependent light dosages and antimicrobial activity of 405 nm blue LED was most effective on minimal medium agar and Luria-Bertani (LB) agar, less effective on cooked meat agar, and least effective on sheep blood agar. Inactivation kinetics of individual STEC strains were determined within a dosage of 163.96 ± 7.55 J/cm2, 5–7 log10 cfu reduction was observed in 7 log10 cfu/ml LB suspension test among seven strains and no viable cells were detected in 3 log10 cfu/ml LB suspension test for all seven tested strains. Difference in light susceptibility among the tested strains was observed between the two prototypes with O111 strain being the most sensitive strain. Multiple factors (e.g. nutrient availability, treatment time, distance, and temperature) could be associated with the antimicrobial effect of 405 nm blue light and need to be well considered when using the technology under different food processing scenarios.

Production of Bioemulsifiers from Bacteria Grown on Hydrocarbons

This work aimed to identify biosurfactant-producing bacterial strains isolated from hydrocarbon-contaminatedsites and to evaluate their biosurfactant properties. The drop-collapse method and minimal agar added witha layer of diesel as carbon source were used for screening, positive strains were able to grow in a liquidmedium, and surface tension and emulsification index were determined in cell-free supernatant and cellsuspension. 250 bacterial isolate were tested, and 16 were positive for the drop-collapse and hydrocarbonlayer agar method. Most of the strains were Pseudomonas, except for three strains (Acinetobacter, Bacillus,and Rhodococcus). Surface tension was similar in cell-free and cell suspension measurements, with valuesin the range of 58 to 26 (mN/m), and all formed stable emulsions with engine oil (77-92% E24). Consideringthe variety of molecular structures among microbial biosurfactants, they have different chemical propertiesthat can be exploited commercially, for applications as diverse as bioremediation or degradable detergents.