Assessment of the effectiveness of an introductory general chemistry course in dentistry students enrolled in a biochemistry course.

As a strategy to carry out a better achievement in the Biochemistry course, undergraduate dentistry education manage a traditional course on the basic concepts of general chemistry necessary in the understanding of Biochemistry. In order to evaluate the effectiveness of learning outcome, we aimed to develop an evaluation tool that was applied to first-year dental students before and after receiving the general chemistry classes. Randomized trial consisted of 50 items distributed in 10 categories. The evaluation was applied to the students who took the Oral Biology course in the periods comprising 2020, 2021, and 2022 to a population of 109 students. Our results showed that after receiving the course the improvement rate was 20.71% with significant differences in each category. In conclusion, the introductory course allows students coming from different school systems to attend Biochemistry with similar knowledge of general chemistry.

Practical genetic control strategies for industrial bioprocesses.

Optimization of metabolism to maximize production of bio-based chemicals must consistently balance cellular resources for biocatalyst growth and desired compound synthesis. This mini-review discusses synthetic biology strategies for dynamically controlling expression of genes to enable dual-phase fermentations in which growth and production are separated into dedicated phases. Emphasis is placed on practical examples which can be reliably scaled to commercial production with the current state of technology. Recent case studies are presented, and recommendations are provided for environmental signals and genetic control circuits.

Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels of iron in excess of what is required for planktonic growth have been shown to promote biofilm formation, and therapies that interfere with ferric iron [Fe(III)] uptake combined with antibiotics may help treat P. aeruginosa infections. However, use of these therapies presumes that iron is in the Fe(III) state in the context of infection. Here we report the ability of phenazine-1-carboxylic acid (PCA), a common phenazine made by all phenazine-producing pseudomonads, to help P. aeruginosa alleviate Fe(III) limitation by reducing Fe(III) to ferrous iron [Fe(II)]. In the presence of PCA, a P. aeruginosa mutant lacking the ability to produce the siderophores pyoverdine and pyochelin can still develop into a biofilm. As has been previously reported (P. K. Singh, M. R. Parsek, E. P. Greenberg, and M. J. Welsh, Nature 417:552-555, 2002), biofilm formation by the wild type is blocked by subinhibitory concentrations of the Fe(III)-binding innate-immunity protein conalbumin, but here we show that this blockage can be rescued by PCA. FeoB, an Fe(II) uptake protein, is required for PCA to enable this rescue. Unlike PCA, the phenazine pyocyanin (PYO) can facilitate biofilm formation via an iron-independent pathway. While siderophore-mediated Fe(III) uptake is undoubtedly important at early stages of infection, these results suggest that at later stages of infection, PCA present in infected tissues may shift the redox equilibrium between Fe(III) and Fe(II), thereby making iron more bioavailable.

Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales.

Some pseudomonads produce phenazines, a group of small, redox-active compounds with diverse physiological functions. In this study, we compared the phenotypes of Pseudomonas aeruginosa strain PA14 and a mutant unable to synthesize phenazines in flow cell and colony biofilms quantitatively. Although phenazine production does not impact the ability of PA14 to attach to surfaces, as has been shown for Pseudomonas chlororaphis(Maddula et al., 2006; 2008), it influences swarming motility and the surface-to-volume ratio of mature biofilms. These results indicate that phenazines affect biofilm development across a large range of scales, but in unique ways for different Pseudomonas species.

Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo.

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

Glutamine phosphoribosylpyrophosphate amidotransferase-independent phosphoribosyl amine synthesis from ribose 5-phosphate and glutamine or asparagine.

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic l-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed.

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius.

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.

Glucose kinase alone cannot be responsible for carbon source regulation in Streptomyces peucetius var. caesius.

Using an antibiotic enrichment procedure, eight mutants of Streptomyces peucetius var. caesius were isolated for their sensitivity to the glucose analogue 2-deoxyglucose (DOG), from a DOG-resistant strain (Dog(R)). These mutants (Dog(S)) and their parent strain were examined for growth sensitivity to DOG, glucose kinase (Glk) activity, glucose uptake, and sensitivity to repression by glucose and other catabolites derived from it. No correlation was found between Glk levels or glucose uptake and carbon catabolite repression (CCR) in these strains. However, the ratio of glucose uptake to Glk activity, and thus the flux through glycolysis, seemed responsible for this effect. Among several products of glucose catabolism tested, fructose-1,6-bis-phosphate and phosphoenolpyruvate showed significant repression of anthracycline formation. These compounds also reduced anthracycline formation in a Dog(R) mutant insensitive to glucose repression. Our data suggest that Glk alone is not sufficient to elicit CCR in this microorganism, and gives the first physiological evidence supporting the hypothesis that some products of glucose catabolism are involved in CCR in Streptomyces.