M9 Minimal Medium Research Articles

Glyphosate-, carbofuran-, and chlorpyrifos-tolerant Priestia aryabhattai, isolated from agricultural soils

Objective: To isolate and carry out a molecular characterization of microorganisms potentially tolerant to high concentrations of different pesticides (glyphosate, carbofuran, and chlorpyrifos). Design/Methodology/Approach: Based on the isolate project SIP20170193, only the strain GVE 5 was chosen for the experiment, as a result of its morphological and growth characteristics. The tolerance capacity (TC) of GVE 5 to three different pesticides (glyphosate, carbofuran, and chlorpyrifos) was evaluated in two different media (LB and M9). The only carbon source was 200 mg/L of each pesticide. GVE 5 was identified through the Polymerase Chain Reaction (PCR) molecular techniques and amplified by the 16S rRNA marker. Results: Based on the TC analysis, the GVE 5 strain of Priestia aryabhattai recorded a growth with 200 mg L-1 of glyphosate, carbofuran, and chlorpyrifos in LB medium and M9 minimal medium. Study Limitations/Implications: There were no limitations or implications for this study. Findings/Conclusions: Priestia aryabhattai is tolerant to 200 mg L-1 of glyphosate, carbofuran, and chlorpyrifos. These results open new lines of research regarding the bioremediation of soils polluted by these agrochemicals. Priestia aryabhattai should be subjected to further evaluations as a plant growth promoter.

Effect of H2S and cysteine homeostasis disturbance on ciprofloxacin sensitivity of Escherichia coli in cystine-free and cystine-fed minimal medium.

Endogenous H2S has been proposed to be a universal defense mechanism against different antibiotics. Here, we studied the role of H2S transiently generated during ciprofloxacin (CF) treatment in M9 minimal medium with sulfate or produced by E. coli when fed with cystine. The cysM and mstA mutants did not produce H2S, while gshA generated more H2S in response to ciprofloxacin in cystine-free media. All mutants showed a reduced ability to maintain cysteine homeostasis under these conditions. We found no relationship between H2S generation, cysteine concentration and sensitivity to ciprofloxacin. Excess cysteine, which occurred during E. coli growth in cystine-fed media, triggered continuous H2S production, accelerated glutathione synthesis and cysteine export. This was accompanied by a twofold increase in ciprofloxacin tolerance in all strains except gshA, whose sensitivity increased 5-8-fold at high CF doses, indicating the importance of GSH in restoring the intracellular redox situation during growth in cystine-fed media.

Escherichia coli alcohol dehydrogenase YahK is a protein that binds both iron and zinc.

Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK's catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.

Analysis of the utility of a rapid vesicle isolation method for clinical strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a pathogen capable of causing diseases ranging from mild to life-threatening, has a large arsenal of virulence factors. Notably, extracellular vesicles have emerged as significant players in the pathogenesis of this organism. However, the full range of their functions is still being studied, and difficulties related to vesicle purification (long protocols, low yields, and specialized instruments) have become a major obstacle for their characterization. In this context, the utility of rapid new methods of vesicle isolation from clinical strains is still unknown. Here, we analyze the utility of the ExoBacteria OMV isolation kit for a collection of clinical strains of P. aeruginosa. We first phenotypically characterized 15 P. aeruginosa strains to ensure that our samples were heterogeneous. We then determined the best conditions for purifying vesicles from P. aeruginosa PAO1 reference strain by the rapid method and used them to isolate vesicles from clinical strains. Our results indicated that M9 minimal medium is the best for obtaining high purity with the rapid isolation kit. Although we were able to isolate vesicles from at least four strains, the low yield and the large number of strains with unpurifiable vesicles showed that the kit was not practical or convenient for clinical strains. Our findings suggest that although fast procedures for vesicle purification can be of great utility for Escherichia coli, the more complex phenotypes of clinical isolates of P. aeruginosa are a challenge for these protocols and new alternatives/optimizations need to be developed.IMPORTANCEPseudomonas aeruginosa is recognized as an opportunistic pathogen in humans and animals. It can effectively colonize various environments thanks to a large set of virulence factors that include extracellular vesicles. Different methods were recently developed to reduce the time and effort associated with vesicle purification. However, the utility of rapid vesicle isolation methods for clinical strains of P. aeruginosa (which are recognized as being highly diverse) is not yet known. In this context, we analyzed the utility of the ExoBacteria OMV Isolation kit for vesicle purification in P. aeruginosa clinical strains. Our findings showed that the kit does not seem to be convenient for research on clinical strains due to low vesicle recovery. Our results underscore the importance of developing new rapid vesicle purification protocols/techniques for specific clinical phenotypes.

CRP improves the survival and competitive fitness of Salmonella Typhimurium under starvation by controlling the cellular maintenance rate.

Catabolite repression is a mechanism of selectively utilizing preferred nutrient sources by redirecting the metabolic pathways. Therefore, it prevents non-essential energy expenditure by repressing the genes and proteins involved in the metabolism of other less favored nutrient sources. Catabolite repressor protein (CRP) is a chief mediator of catabolite repression in microorganisms. In this context, we investigated the role of CRP in starvation tolerance, at both cell physiology and molecular level, by comparing the growth, survival, competitive fitness, maintenance rate, and gene and protein expression of wild type (WT) and ∆crp of Salmonella Typhimurium, under nutrient-rich and minimal medium condition. The ∆crp shows slow growth upon the arrival of nutrient-limiting conditions, poor survival under prolong-starvation, and inability to compete with its counterpart WT strain in nutrient-rich [Luria broth (LB)] and glucose-supplemented M9 minimal medium. Surprisingly, we observed that the survival and competitive fitness of ∆crp are influenced by the composition of the growth medium. Consequently, compared to the glucose-supplemented M9 medium, ∆crp shows faster death and a higher maintenance rate in the LB medium. The comparative gene and protein expression studies of WT and ∆crp in LB medium show that ∆crp has partial or complete loss of repression from CRP-controlled genes, resulting in a high abundance of hundreds of proteins in ∆crp compared to WT. Subsequently, the addition of metabolizable sugar or fresh nutrients to the competing culture showed extended survival of ∆crp. Therefore, our results suggest that CRP-mediated gene repression improves starvation tolerance and competitive fitness of Salmonella Typhimurium by adapting its cellular maintenance rate to environmental conditions.IMPORTANCESalmonella Typhimurium is a master at adapting to chronic starvation conditions. However, the molecular mechanisms to adapt to such conditions are still unknown. In this context, we have evaluated the role of catabolite repressor protein (CRP), a dual transcriptional regulator, in providing survival and competitive fitness under starvation conditions. Also, it showed an association between CRP and nutrient composition. We observed that Δcrp growing on alternate carbon sources has lower survival and competitive fitness than Δcrp growing on glucose as a carbon source. We observed that this is due to the loss of repression from the glucose and CRP-controlled genes, resulting in elevated cellular metabolism (a high maintenance rate) of the Δcrp during growth in a medium having a carbon source other than glucose (e.g., Luria broth medium).

Competitive fitness of asymptomatic bacteriuria E. coli strain 83972 against uropathogens in human urine.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. The main causative agent of UTI is uropathogenic Escherichia coli (UPEC). There is an immediate need for novel prophylactic and treatment strategies against UTI because of the increasing incidence of antimicrobial resistance among uropathogens. ABU 83972, an asymptomatic bacteriuria-causing E. coli strain, prevents UTI by suppressing the colonization of UPEC. However, the nature of competition and growth repression of UPEC by ABU 83972 is unclear and is the subject of our investigation. Here, we characterized the growth kinetics of ABU 83972 and uropathogens in human urine and laboratory media. Next, we performed a series of competitive co-culture experiments where ABU 83972 and uropathogens were inoculated at a 1:1 ratio in human urine and in various media, and their relative abundance was determined. In human urine, ABU 83972 outcompeted UPEC and additional uropathogens, reaching up to 90% of the total population after 24 hours of incubation. In contrast, UPEC outcompeted ABU 83972 in LB and M9 minimal media and exhibited superior colonization than ABU 83972 in the mouse urinary bladder. Since engineered living materials (ELMs) can be used to retain an organism of interest in a particular location, we developed ABU 83972-containing ELMs that effectively outcompeted UPEC in human urine. In summary, our work establishes that ABU 83972 outcompetes UPEC in a milieu- and cell-density-dependent manner, highlighting the importance of the metabolites and nutrients found in the human urine as determinants of the competitive fitness of ABU 83972.

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.

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.

Revisiting the hydrolysis of ampicillin catalyzed by Temoneira-1 β-lactamase, and the effect of Ni(II), Cd(II) and Hg(II).

β-Lactamases grant resistance to bacteria against β-lactam antibiotics. The active center of TEM-1 β-lactamase accommodates a Ser-Xaa-Xaa-Lys motif. TEM-1 β-lactamase is not a metalloenzyme but it possesses several putative metal ion binding sites. The sites composed of His residue pairs chelate borderline transition metal ions such as Ni(II). In addition, there are many sulfur-containing donor groups that can coordinate soft metal ions such as Hg(II). Cd(II) may bind to both types of the above listed donor groups. No significant change was observed in the circular dichroism spectra of TEM-1 β-lactamase on increasing the metal ion content of the samples, with the exception of Hg(II) inducing a small change in the secondary structure of the protein. A weak nonspecific binding of Hg(II) was proven by mass spectrometry and 119m Hg perturbed angular correlation spectroscopy. The hydrolytic process of ampicillin catalyzed by TEM-1 β-lactamase was described by the kinetic analysis of the set of full catalytic progress curves, where the slow, yet observable conversion of the primary reaction product into a second one, identified as ampilloic acid by mass spectrometry, needed also to be considered in the applied model. Ni(II) and Cd(II) slightly promoted the catalytic activity of the enzyme while Hg(II) exerted a noticeable inhibitory effect. Hg(II) and Ni(II), applied at 10 μM concentration, inhibited the growth of E. coli BL21(DE3) in M9 minimal medium in the absence of ampicillin, but addition of the antibiotic could neutralize this toxic effect by complexing the metal ions.

Trace impurities in sodium phosphate influences the physiological activity of Escherichia coli in M9 minimal medium

In the field of applied microbiology, reproducibility and experimental variability are important factors that influence both basic research as well as process development for industrial applications. Experimental reproducibility and accuracy depend not only on culture conditions such as temperature and aeration but also on raw materials and procedures used for media preparation. The M9 minimal medium is one of the most common synthetic media for culturing Escherichia coli and other bacteria. This synthetic medium can be used to observe and evaluate the physiological activity of microbes under minimal nutritional requirements and determine the limiting factor for the desired phenotype. Although one of the advantages using the M9 medium is that its composition can be modulated, it is difficult to control presence of trace components and impurities from the reagents for preparing this medium. Herein, we showed that trace ingredients present in the reagents used for M9 media preparation affect the bacterial physiological activities (e.g., cell growth, substrate consumption, and byproduct formation). Additionally, we systematically identified the trace ingredient that influenced phenotypic differences. Our results showed that the selection of reagents and accuracy during reagent preparation is important for experimental reproducibility in the field of bio-engineering and systems biology focused on the systematic and continuous development of biomolecular systems (e.g., biorefinery, metabolic engineering, and synthetic biology).

SAT371 Gavaging Adult Female Rats with Rothia sp. Leads to Profound Disruption of the Estrous Cycles and Metabolic Consequences

Abstract Disclosure: R. Santos: None. G. Parodi: None. G. Leite: None. W. Morales: None. S.R. Weitsman: None. G. Barlow: None. I. Rivera: None. M. Sanchez: None. D. Flor: None. L. Choi: None. S.L. Gonzales: None. F. Faria: None. M. Villanueva-Millan: None. M. Pimentel: None. R. Mathur: None. Polycystic ovary syndrome (PCOS) is a major endocrine disorder affecting ∼12% of reproductive age women. It has diverse clinical manifestations, including hyperandrogenism, ovarian dysfunction and enlargement, infertility, insulin resistance, and others. The role of the gut microbiome in PCOS is poorly understood. We previously found higher testosterone (T) levels in PCOS stool samples vs. controls and identified bacterial candidates that correlated with testosterone levels. Here, we further investigate the role of one candidate microbe, Rothia sp., in the development of PCOS-like phenotypes. Methods: The presence of androgen biosynthesis associated genes in Rothia sp. was confirmed by RT-PCR. Rothia sp. was cultured in M9 minimal medium plus cholesterol. T production in the supernatant was measured by LC-MS/MS. Next, reproductive-age female rats were gavaged with 106 CFU/mL Rothia or with PBS (controls) daily for 2 weeks. H&E stained slides from daily vaginal lavages were used to assess estrous cycles. Serum T and progesterone (P) levels were measured by LC-MS/MS, and insulin levels by ELISA. Ovaries, uteri, and cardiac blood were harvested. Results: Genes encoding proteins associated with T biosynthesis (cytochrome P450, 3-beta hydroxysteroid dehydrogenase/isomerase family protein and NAD(P)H steroid dehydrogenase-like protein) were detected in Rothia sp. T production in Rothia sp. cultures was confirmed, with levels from 3.2±0.01 pg/mL after 48 hrs to 8.9±0.02 pg/mL after 6 days. T was not detected in negative controls (P<0.001). In gavaged rats, analysis of vaginal slides (controls n=10; Rothia n=10) revealed a disruption of estrous cycles in Rothia rats, with 68% of days in luteal phase (metestrus & diestrus) vs. 46% in controls (P=0.001). The predominance of luteal phase days in Rothia rats was driven by longer time in diestrus (1.96 vs 1.67 days in controls, P=0.04), and shorter time in estrus (1.24 vs 1.91 days in controls, P=0.001). Peak P and T levels occurred in diestrus in both groups. Rothia rats spent more days in diestrus, and the cumulative exposure times to P (P=0.046) and T (P=0.036) in the diestrus phase were significantly higher in Rothia rats vs controls. Final serum insulin levels were higher in Rothia rats (1.8±0.5 ng/dL) vs controls (1.0±0.1 ng/dL; P=0.07). Rothia rats also had heavier ovaries vs controls (0.128±0.007g vs 0.097±0.01g; P=0.07), and higher uterus weights (0.48±0.04g vs. 0.36±0.04g; P=0.04). Conclusion: Here we show that a specific Rothia sp. can produce testosterone in vitro. Gavaging female rats with this microbe results in PCOS-like phenotypes, including higher insulin levels, enlarged ovaries, significant disruption of reproductive cycles and longer exposure to peak progesterone and testosterone. This longer exposure to peak progesterone and testosterone may result in reproductive and metabolic sequelae that warrant further investigation. Presentation Date: Saturday, June 17, 2023

Microbial degradation of the benzimidazole fungicide carbendazim by Bacillus velezensis HY-3479.

Carbendazim (methyl benzimidazol-2-ylcarbamate: MBC) is a fungicide of the benzimidazole group that is widely used in the cultivation of pepper, ginseng, and many other crops. To remove the remnant carbendazim, many rhizobacteria are used as biodegradation agents. A bacterial strain of soil-isolated Bacillus velezensis HY-3479 was found to be capable of degrading MBC in M9 minimal medium supplemented with 250mg/L carbendazim. The strain had a significantly higher degradation efficiency compared to the control strain Bacillus subtilis KACC 15590 in high-performance liquid chromatography (HPLC) analysis, and HY-3479 had the best degradation efficiency of 76.99% at 48h. In gene expression analysis, upregulation of carbendazim-degrading genes (mheI, hdx) was observed in the strain. HY-3479 was able to use MBC as the sole source of carbon and nitrogen, but the addition of 12.5mM NH4NO3 significantly increased the degradation efficiency. HPLC analysis showed that the degradation efficiency increased to 87.19% when NH4NO3 was added. Relative gene expression of mheI and hdx also increased for samples with NH4NO3 supplementation. The enzyme activity of the carbendazim-degrading enzyme and the 2-aminobenzimidazole-degrading enzyme was found to be highly present in the HY-3479 strain. It is the first reported B. velezensis strain to biodegrade carbendazim (MBC). The biodegradation activity of strain HY-3479 may be developed as a useful means for bioremediation and used as a potential microbial agent in sustainable agriculture.

Production of Astaxanthin Using CBFD1/HFBD1 from Adonis aestivalis and the Isopentenol Utilization Pathway in Escherichia coli.

Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the β-carotene hydroxylase (CBFD1) and the β-carotene ketolase (HBFD1) from Adonis aestivalis, a flowering plant, to produce astaxanthin in E. coli equipped with the P. agglomerans β-carotene pathway and an over-expressed 4-methylerythritol-phosphate (MEP) pathway or the isopentenol utilization pathway (IUP). Introduction of the over-expressed MEP pathway and the IUP resulted in a 3.2-fold higher carotenoid content in LB media at 36 h post-induction compared to the strain containing only the endogenous MEP. However, in M9 minimal media, the IUP pathway dramatically outperformed the over-expressed MEP pathway with an 11-fold increase in total carotenoids produced. The final construct split the large operon into two smaller operons, both with a T7 promoter. This resulted in slightly lower productivity (70.0 ± 8.1 µg/g·h vs. 53.5 ± 3.8 µg/g·h) compared to the original constructs but resulted in the highest proportion of astaxanthin in the extracted carotenoids (73.5 ± 0.2%).

Biotransformation of Lignocellulosic Biomass Hydrolysate into Polyhydroxybutyrate Biopolymer via Ralstonia Eutropha

Currently, a rampant cultural shift is occurring in the modern world to progressively substitute fossil-derived plastics and shift to novel biomaterials that are benign to the environment owing to increased awareness of environmental sustainability along with the implementation of strict regulations worldwide. Polyhydroxyalkanoates (PHAs) are promising intracellular biodegradable polymers that have attracted considerable focus owing to their biocompatibility, biodegradability, non-toxicity and environment-friendly nature to function in diverse applications notably in the pharmaceutical, medical, textile, materials, fuel, agricultural industries. Nonetheless, despite its huge market potential, the commercial growth of PHA is achieved on a small extent only, since the cost-effectiveness of this product is highly debatable owing to the high production cost of processing the carbon substrate. The goal behind this research study is to explore the possibility of exploiting low-cost carbon substrates from low-value lignocellulosic materials that would have otherwise been discarded as waste and add stress to the landfill to manufacture biopolymer compounds that are used in everyday lives as well as to enhance the functionality and yields of glucose from PHA substrates that can undergo industrial upscaling. One of the major challenges of transforming lignocellulosic biomass into fermentable sugars is the recalcitrant nature of the fibre which renders it very resistant to the release of sugars for fermentation. Since lignocellulosic biomass has a specific attribute such as an extremely coordinated matrix which renders it very resistant to the release of sugars for fermentation owing to biological degradation, a pre-treatment phase is necessary prior to the hydrolysis stage for the transformation of the fermentable sugars. This study focuses on the biosynthesis of biopolymers from lignocellulosic biomass through sustainable approaches such as enzyme and microbial activities in order to examine its viability as a replacement for traditional polymers. Cupriavidus Necator H16 (Ralstonia Eutropha) having 8×108 CFU/ml viable colonies were cultured at 30 oC and was inoculated in submerged fermentation of M9 minimal salt medium using 1% reducing sugar from Furcraea Foetida as carbon source. Batch fermentation of PHB in submerged cultivation conducted for a residence time from 0 to 48h resulted in a dry cell weight from 0.32±0.05% to 1.62±0.05%. The nitrogen limiting phase was achieved after 48h and 17.05±0.35% of PHB was extracted from 3ml of the fermentation broth. The PHB yield was dramatically lower than reported optimal yields of 37.55 to 97.80% from works of literature. Nonetheless, Fourier-transform infrared spectroscopy (FTIR) spectroscopy revealed characteristics bands for carbonyl, methine and ester groups along with intermolecular hydrogen bonds in the biopolymer. Sudan Black B and FTIR spectrum demonstrate that PHB biosynthesis successfully bioaccumulates inside the cells of Ralstonia Eutropha using cellulose from Lignocellulosic biomass (LCB) as carbon source. Hence, the process needs to be optimized in terms of variables such as inoculum size, inoculum concentration, incubation time and salt medium conditions in order to maximise the production of PHB from Furcraea Foetida in Ralstonia Eutropha cultivation.

Flagella-related gene mutations in Vibrio cholerae during extended cultivation in nutrient-limited media impair cell motility and prolong culturability.

Understanding the survival strategies of pandemic cholera pathogens in aquatic environments is important for preventing their dissemination. Here, we report a phenomenon wherein long-term cultivation of Vibrio cholerae under nutrient-limited condition (M9 minimal medium supplemented with 0.2% glucose) causes mutations in flagella-related genes, thus inhibiting motility. Moreover, the motility defect reduced the transition of the viable but nonculturable state of the organisms. Maintenance of proliferative capability allows for the rapid population growth and prolonged survival in environments with nutrient sources. Diverse flagella-related genes, including flrA, flrC, flrD, flhA, flhF, fliD, fliF, fliG, fliH, fliI, fliP, fliQ, flgF, flgL, flgK, motX, and pomA, underwent mutations during the cultivation. However, only one gene (site) was mutated in a single V. cholerae. Longer cultivation for up to 300 days yielded further mutations in metabolism-related genes and the loss of virulence factors (e.g., CTX phage) and large DNA regions (~35 kb). These mutations were detected in most organisms of V. cholerae, accompanying the loss of genomic integrity. Motility-defective variants with mutations in the acetate kinase gene had become predominant in culturable cells after long-term cultivation in independently replicated experiments. These findings shed light on a survival mechanism in which V. cholerae adapts to an environmental niche by accumulating mutations. IMPORTANCE Vibrio cholerae undergoes a transition to a viable but non-culturable (VNC) state when subjected to various environmental stresses. We showed here that flagellar motility was involved in the development of the VNC state of V. cholerae. In this study, motility-defective isolates with mutations in various flagella-related genes, but not motile isolates, were predominantly obtained under the stress of long-term batch culture. Other genomic regions were highly conserved, suggesting that the mutations were selective. During the stationary phase of long-term culture, V. cholerae isolates with mutations in the acetate kinase and flagella-related genes were predominant. This study suggests that genes involved in specific functions in V. cholerae undergo mutations under certain environmental conditions.

Cytochrome c oxidase is one of the key enzymes providing the ability to produce phenazines in Pseudomonas chlororaphis subsp. aurantiaca.

Phenazines are heteroaromatic compounds consisting of a central pyrazine ring fused with two benzenes. Different functional groups attached to the dibenzopyrasin core cause differences in the chemical, physical, and biological properties of phenazines. Interest in these compounds has not diminished for decades. New biological activities and practical applications discovered in recent years force researchers to investigate all aspects of the synthesis, degradation, and mechanisms of action of phenazines. In this study, we have demonstrated the involvement of the coxA gene product (cytochrome c oxidase, su I) in the production of phenazines in P. chlororaphis subsp. aurantiaca. Overlap PCR was used to knock out the coxA gene and the resulting mutants were screened for their ability to grow on rich and minimal culture media and for phenazine production. The reintroduction of the full-length coxA gene into the B-162/coxA strains was used to further confirm the role of this gene product in the ability to produce phenazines. We were able to show that the product of the coxA gene is necessary for phenazine production in rich growth media. At the same time, the CoxA protein does not seem to have any effect on phenazine production in M9 minimal salt medium. We could show that knocking down even one subunit of the cytochrome c oxidase complex leads to a significant reduction (to trace concentrations) or complete suppression of phenazine antibiotic production on rich PCA medium in P. chlororaphis subsp. aurantiaca.

PilA Gene Contributes to Virulence, Motility, Biofilm Formation, and Interspecific Competition of Bacteria in Acidovorax citrulli.

Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-ΔpilA and Aac5-ΔpilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-ΔpilA also completely lost twitching motility, whereas Aac5-ΔpilA only partially lost motility. In King's B medium, there were no significant differences in biofilm formation between pslb65-ΔpilA and WT pslb65, but Aac5-ΔpilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-ΔpilA and Aac5-ΔpilA were significantly lower than in the WT strains; Aac5-ΔpilA was more strongly competitive than pslb65-ΔpilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-ΔpilA and Aac5-ΔpilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study.

Influence of Growth Medium Composition on Physiological Responses of Escherichia coli to the Action of Chloramphenicol and Ciprofloxacin.

The ability of hydrogen sulfide (H2S) to protect bacteria from bactericidal antibiotics has previously been described. The main source of H2S is the desulfurization of cysteine, which is either synthesized by cells from sulfate or transported from the medium, depending on its composition. Applying electrochemical sensors and a complex of biochemical and microbiological methods, changes in growth, respiration, membrane potential, SOS response, H2S production and bacterial survival under the action of bactericidal ciprofloxacin and bacteriostatic chloramphenicol in commonly used media were studied. Chloramphenicol caused a sharp inhibition of metabolism in all studied media. The physiological response of bacteria to ciprofloxacin strongly depended on its dose. In rich LB medium, cells retained metabolic activity at higher concentrations of ciprofloxacin than in minimal M9 medium. This decreased number of surviving cells (CFU) by 2-3 orders of magnitude in LB compared to M9 medium, and shifted optimal bactericidal concentration (OBC) from 0.3 µg/mL in M9 to 3 µg/mL in LB. Both drugs induced transient production of H2S in M9 medium. In media containing cystine, H2S was produced independently of antibiotics. Thus, medium composition significantly modifies physiological response of E. coli to bactericidal antibiotic, which should be taken into account when interpreting data and developing drugs.

Sarcosine sensitivity in Escherichia coli is mediated by activation of the glycine cleavage system.

Corynebacterium glutamicum AJ1511 and Escherichia coli BW25113 strains were compared in terms of resistance to sarcosine (N-methylglycine). The E. coli strain was more sensitive to sarcosine than C. glutamicum, especially when grown in minimal medium. Growth inhibition of the BW25113 strain in minimal M9 medium containing 0.5m sarcosine was overcome by the addition of glycine. Inactivation of the glycine cleavage (GCV) system (∆gcvP) as well as the removal of its activator (∆gcvA) in BW25113 cells increased the threshold for sarcosine inhibition up to 0.75m. Activation of the promoter of the E. coli gcvTHP operon by 0.1-0.4m sarcosine added to M9 medium was demonstrated in vivo using dasherGFP as the reporter. Sensitivity to sarcosine on glucose minimal medium is suggested to be a characteristic of Gram-negative bacteria with GcvA/GcvR regulation of the GCV system.

Omics data for sampling thermodynamically feasible kinetic models

Kinetic models are key to understanding and predicting the dynamic behaviour of metabolic systems. Traditional models require kinetic parameters which are not always available and are often estimated in vitro. Ensemble models overcome this challenge by sampling thermodynamically feasible models around a measured reference point. However, it is unclear if the convenient distributions used to generate the ensemble produce a natural distribution of model parameters and hence if the model predictions are reasonable.In this paper, we produced a detailed kinetic model for the central carbon metabolism of Escherichia coli. The model consists of 82 reactions (including 13 reactions with allosteric regulation) and 79 metabolites. To sample the model, we used metabolomic and fluxomic data from a single steady-state time point for E. coli K-12 MG1655 growing on glucose minimal M9 medium (average sampling time for 1000 models: 11.21 ± 0.14 min). Afterwards, in order to examine whether our sampled models are biologically sound, we calculated Km, Vmax and kcat for the reactions and compared them to previously published values. Finally, we used metabolic control analysis to identify enzymes with high control over the fluxes in the central carbon metabolism.Our analyses demonstrate that our platform samples thermodynamically feasible kinetic models, which are in agreement with previously published experimental results and can be used to investigate metabolic control patterns within cells. This renders it a valuable tool for the study of cellular metabolism and the design of metabolic pathways.