Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the forth reaction in the shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.     

Gene replacement and quantitative mass spectrometry approaches validate guanosine monophosphate synthetase as essential for Mycobacterium tuberculosis growth

Guanosine monophosphate synthetase (GMPS), encoded by guaA gene, is a key enzyme for guanine nucleotide biosynthesis in Mycobacterium tuberculosis. The guaA gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct guaA deletion in M. tuberculosis has been described so far. Here, we demonstrated that the guaA gene is essential for M. tuberculosis H37Rv growth. The lethal phenotype of guaA gene disruption was avoided by insertion of a copy of the ortholog gene from Mycobacterium smegmatis, indicating that this GMPS protein is functional in M. tuberculosis. Protein validation of the guaA essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from M. tuberculosis and M. smegmatis GMPS proteins. These results validate GMPS as a molecular target for drug design against M. tuberculosis, and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.     

Influence of Different Filling,Cooling, and Storage Conditions on the Growth of Alicyclobacillus acidoterrestris CRA7152 in Orange Juice

The prevention of spoilage by Alicyclobacillus acidoterrestris is a current challenge for fruit juice and beverage industries worldwide due to the bacterium''s acidothermophilic growth capability, heat resistance, and spoilage potential. This study examined the effect of storage temperature on A. acidoterrestris growth in hot-filled orange juice. The evolution of the A. acidoterrestris population was monitored under six different storage conditions after pasteurization (at 92°C for 10 s), maintenance at 85°C for 150 s, and cooling with water spray to 35°C in about 30 min and using two inoculum levels: <10¹ and 10¹ spores/ml. Final cooling and storage conditions were as follows: treatment 1, 30°C for the bottle cold point and storage at 35°C; treatment 2, 30°C for 48 h and storage at 35°C; treatment 3, 25°C for the bottle cold point and storage at 35°C; treatment 4, 25°C for 48 h and storage at 35°C; treatment 5, storage at 20°C (control); and treatment 6, filling and storage at 25°C. It was found that only in treatment 5 did the population remain inhibited during the 6 months of orange juice shelf life. By examining treatments 1 to 4, it was observed that A. acidoterrestris predicted growth parameters were significantly influenced (P < 0.05) either by inoculum level or cooling and storage conditions. The time required to reach a 10⁴ CFU/ml population of A. acidoterrestris was considered to be an adequate parameter to indicate orange juice spoilage by A. acidoterrestris. Therefore, hot-filled orange juice should be stored at or below 20°C to avoid spoilage by this microorganism. This procedure can be considered a safe and inexpensive alternative to other treatments proposed earlier.The first Alicyclobacillus sp. discovered was isolated in 1982 from spoiled apple juice aseptically packed in Germany and was considered at that time strictly limited to thermophilic and acidic environments (5). The spoilage of fruit juices by Alicyclobacillus is characterized by “off” flavors (medicinal or phenolic) due to guaiacol, 2,6-dibromophenol, and 2,6-dichlorophenol (20, 32, 25). As the spoilage does not show any evident signs like swelling of the container or any overt changes in the fruit juice (e.g., pH or turbidity), it is often not recognized until the packages are opened, the product is tasted, and consumer complaints are received by the manufacturer (6).Alicyclobacillus acidoterrestris, Alicyclobacillus cycloheptanicus and Alicyclobacillus acidocaldarius were the first three species described when the Alicyclobacillus genus was created in 1992 (31). Although there are currently more than 15 species described (27), only four Alicyclobacillus species (A. acidoterrestris, A. pomorum, A. herbarius and A. acidophilus) have shown the ability to produce off flavors in fruit juices or beverages (5, 13, 9, 1). Of these, A. acidoterrestris is considered the most important spoilage species within the Alicyclobacillus genus either by its frequency of occurrence or by its linkage to the spoilage problems of fruit juices and beverages. The broad temperature range for A. acidoterrestris growth (25 to 60°C) (33, 21, 20, 30, 11), its ability to grow under acidic environments (pH 2.5 to 6.0) (20, 30, 19, 22, 6), and its high heat resistance in orange juice (D at 95°C of 2.7 min) (8) together provide adequate conditions for both survival through pasteurization and growth during juice storage.A. acidoterrestris growth and the consequent orange juice spoilage can lead to enormous economic losses; therefore, this microorganism is currently considered a major challenge for the fruit juice industries. It is known to be difficult, if not impossible, to guarantee the absence of Alicyclobacillus spores on the surface of fruits used to make juices since the soil is the primary niche of Alicyclobacillus spp. (7). Subsequently, control measures such as avoiding fruit contact with soil and the use of sanitizers during the fruit washing step before crushing have been studied (16, 12). In addition, fruit juice producers need to better control the pasteurization conditions and to redesign their thermal processes for targeting Alicyclobacillus spp. (28, 24). However, the limited effectiveness of sanitizers against Alicyclobacillus spores and the sensory and nutritional problems that may arise from increased time and temperature regimes in pasteurization are recognized. Since the complete inactivation of Alicyclobacillus spores from raw materials may not be feasible and since juice spoilage by this microorganism depends on the germination of spores and outgrowth, studies reporting conditions that avoid spoilage by controlling Alicyclobacillus spore germination are necessary. The best option to manage the challenge that Alicyclobacillus presents to the fruit juice industries will be one that results in the fewest alterations in processing and storage conditions, preserves the nutritional and sensory aspects of the final products, does not impact production costs and commercial practices, and at the same time ensures the control of the microorganism.In the fruit juice industry, two main types of thermal processes are commonly applied: pasteurization followed by a hot-fill process or pasteurization followed by a cold-fill process (6). In the former, after the product is heated to >90 to 95°C, it is held hot for 15 to 20 s. As the temperature decreases to 82 to 84°C, the product is filled into the package. Next, the product is held for approximately 2 min before the packages are cooled to room temperature. Hot filling has been extensively used in the manufacturing processes of fruit-based drinks and beverages, but problems due to spoilage caused by Alicyclobacillus may arise during fruit juice shelf life. This is due to the extended time that the product is maintained at temperature conditions adequate for the germination and outgrowth of acidothermophilic spore-forming microbes. Despite several studies regarding the factors that affect A. acidoterrestris growth and heat resistance (12, 18, 2, 29), there is a lack of research on the effects of hot-filled fruit juice storage conditions on A. acidoterrestris growth during juice storage. Thus, this study aimed at estimating and comparing the growth parameters (maximum population ratio, κ; lag time, λ; and maximum growth rate, μ) of A. acidoterrestris survival in hot-filled orange juice that was cooled and stored under several conditions that simulate industrial and commercial practices. Primary growth parameters were estimated by using the Baranyi predictive model (3). Additionally, orange juice cooling and storage conditions that avoided germination, growth, and guaiacol production by A. acidoterrestris CRA 7152 were determined.     

The pleurogram,an under-investigated functional trait in seeds

BackgroundA structure called the pleurogram makes up a large part of the seed coat of some species in subfamilies Caesalpinioideae and Mimosoideae of Fabaceae, but little is known about its function. It has been hypothesized that this structure acts as a hygroscopic valve during the maturation drying of seeds. However, a new hypothesis has recently emerged that proposes a distinct function for the pleurogram.ScopeHere, we provide an overview of the structure and function of the pleurogram, which is diverse and complex. This large structure can be dislodged, thereby creating a pathway for water entry into water-impermeable seeds. However, the pleurogram is non-functional as a pathway of water into the seed of some species. Thus, the evolutionary history of species with a pleurogram may be related to a loss/gain in its function. A complete model for the function of the pleurogram is proposed.ConclusionsThe pleurogram may act on several stages of the seed, from maturation to germination. As a hygroscopic valve, it regulates dehydration of the seed during maturation. As a pathway for water entry into the seed, the pleurogram acts as a water gap in seeds with physical dormancy, thereby regulating dormancy break/germination. The occurrence of a pleurogram in several genera of legumes and Cucurbitaceae is confirmed. Single or multiple pleurograms can serve as (the) point(s) of water entry into seeds that do not otherwise have a hilar water gap.