Growth In Standard Medium Research Articles

Unraveling the metabolism of Mycobacterium caprae using comparative genomics

In our laboratory, Mycobacterium caprae has poor growth in standard medium (SM) 7H9-OADC supplemented with pyruvate and Tween-80. Our objectives were to identify mutations affecting M. caprae metabolism and use this information to design a culture medium to improve its growth. We selected 77 M. caprae genomes and sequenced M. caprae NLA000201913 used in our experiments. Mutations present in >95% of the strains compared to Mycobacterium tuberculosis H37Rv were analyzed in silico for their deleterious effects on proteins of metabolic pathways. Apart from the known defect in the pyruvate kinase, M. caprae has important lesions in enzymes of the TCA cycle, methylmalonyl cycle, B12 metabolism, and electron-transport chain. We provide evidence of enzymatic redundancy elimination and epistatic mutations, and possible production of toxic metabolites hindering M. caprae growth in vitro. A newly designed SM supplemented with l-glutamate allowed faster growth and increased final microbial mass of M. caprae. However, possible accumulation of metabolic waste-products and/or nutritional limitations halted M. caprae growth prior to a M. tuberculosis-like stationary phase. Our findings suggest that M. caprae relies on GABA and/or glyoxylate shunts for in vitro growth in routine media. The newly developed medium will improve experiments with this bacterium by allowing faster growth in vitro.

Influence of selenium on the mycelia of the shaggy bracket fungus, Inonotus hispidus.

Selenium (Se) is a needed trace element for animals and humans. Many fungi have effective mechanisms to acquire, transform and accumulate Se in organic form. In this study, the effects of inorganic Se (sodium selenite) on the medicinal fungus Inonotus hispidus was investigated. Inonotus hispidus was capable of tolerating up to 3.85 mmol L-1 selenite, at which ~85% growth inhibition was seen, with 50% growth inhibition occurring at ~1 mmol L-1 selenite. Growth in 0.29 mmol L-1 Se resulted in I. hispidus mycelium with 115 times higher Se levels compared to growth in standard media, and an organic Se content of 86% to total Se content. The influence of Se accumulation on morphological features of I. hispidus were examined by microscopic and scanning electron microscopic observation. These data revealed significant shrinkage and deformations of I. hispidus hyphae with decreased branching and collapse of clamp connections under higher Se stress. However, conidial production in I. hispidus increased dramatically. The influence of Se on mycelial growth could be recovered by reinoculation in standard media. Se accumulation had only minimal impacts on the yield of the potential selenocompounds such as amino acids, proteins and polysaccharides. By contrast, Se-enriched I. hispidus mycelium was of higher quality due to reduction in crude fat and total ash contents. These data provide basic and applied information on the feasibility of producing selenized I. hispidus as an enriched and better quality product. © 2021 Society of Chemical Industry.

Molecular and Microscopic Methods of Quantifying Candida albicans Cell Wall PAMP Exposure.

The cell wall of Candida albicans is a multilayered structure consisting of polysaccharides and proteins. The inner cell wall layer is comprised of chitin and β1-3 and β1-6-glucan which contribute to the overall shape and structure of the cell, while the outer layer of highly glycosylated mannoproteins provides key functional traits such as cell adhesion required for virulence. However, the cell wall is not a static structure but is constantly being remodeled in response to the external environment. Given that all of the cell wall components act as pathogen-associated molecular patterns (PAMPs) that are recognized by a variety of receptors on the surface of innate immune cells, remodeling of the cell wall can have a dramatic impact on the host-pathogen interaction. For example, during growth in standard media, C. albicans shields its major cell wall PAMPs from the innate immune system, but during growth in acidic environments as encountered during colonization of the female reproductive tract, key PAMPs become exposed on the fungal cell surface initiating a strong pro-inflammatory innate immune response. The impact of environmental adaptation on fungal cell wall remodeling, and the subsequent impact this has on the host-pathogen interaction, has been the subject of much research. In this chapter, we outline techniques to assess cell wall components in both resting and environmentally adapted C. albicans cells.

Toward facilitating microalgae cope with effluent from anaerobic digestion of kitchen waste: the art of agricultural phytohormones

BackgroundAlthough numerous studies have used wastewater as substitutes to cultivate microalgae, most of them obtained weaker algal viability than standard media. Some studies demonstrated a promotion of phytohormones on algal growth in standard media. For exploiting a strategy to improve algal biomass accumulation in effluent from anaerobic digestion of kitchen waste (ADE-KW), the agricultural phytohormones gibberellin, indole-3-acetic acid, and brassinolide (GIB) were applied to Chlorella SDEC-11 and Scenedesmus SDEC-13 at different stages of algal growth. Previous studies have demonstrated a promotion of phytohormones on algal growth in standard media, but attempts have been scarce, focusing on wastewater cultivation system. In addition, the effects of wastewater on algal morphology and ultrastructure have not been revealed so far, much less on the mechanism of the role of phytohormones on algae.ResultsADE-KW disrupted the membranes of nuclear and chloroplast in ultrastructural cell of SDEC-11, and reduced the room between chloroplast and cell membrane and increased the starch size of SDEC-13. This reduced algal growth and biocompound accumulation, but SDEC-13 had greater adaptation to ADE-KW than SDEC-11. Moreover, inoculation with an algal seed pretreated with GIB aided the adaptability and viability of algae in ADE-KW, which for SDEC-13 was even promoted to the level in BG11. GIB mitigated the inhibition of ADE-KW on algal cell division and photosynthetic pigments and apparatus, and increased lipid droplets, which might result from the change in the synthesis and the fate of nicotinamide adenine dinucleotide phosphate. GIB addition significantly promoted lipid productivity of the two algal species, following 13 mg L−1 d−1 of SDEC-11 in B+ADE-KW and especially 13 mg L−1 d−1 of SDEC-13 achieved during the priming of algal seed with the hormones, which is 139% higher than 5 mg L−1 d−1 achieved in ADE-KW control.ConclusionsAgricultural phytohormones could be applied as a strategy for promoting biomass and biocompound accumulation of algae in ADE-KW, in which pretreatment of the algal inoculum with hormones is a unique way to help algae survive under stress. Considering our results and treatment technology for kitchen waste, a more feasible and economic plant can be built incorporating anaerobic digestion, algae cultivation with ADE-KW assisted with phytohormones, and biodiesel production.

High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.

Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.

The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila

The Gram-negative bacterium Legionella pneumophila elaborates the siderophore legiobactin. We previously showed that cytoplasmic LbtA helps mediate legiobactin synthesis, inner-membrane LbtB promotes export of legiobactin, and outer-membrane LbtU acts as the ferrisiderophore receptor. RT-PCR analyses now identified lbtC as an iron-repressed gene that is the final gene in an operon containing lbtA and lbtB. In silico analysis predicted that LbtC is an inner-membrane protein that belongs to the major facilitator superfamily (MFS). Although capable of normal growth in standard media, lbtC mutants were defective for growth on iron-depleted agar media. While producing normal levels of legiobactin, lbtC mutants were unable to utilize supplied legiobactin to stimulate growth on iron-depleted media and displayed an impaired ability to take up radiolabelled iron. All lbtC mutant phenotypes were complemented by reintroduction of an intact copy of lbtC. When a cloned copy of both lbtC and lbtU was introduced into a heterologous bacterium (Legionella longbeachae), the organism acquired the ability to utilize legiobactin to grow better on low-iron media. Together, these data indicate that LbtC is involved in the uptake of legiobactin, and based upon its predicted location is most likely the mediator of ferrilegiobactin transport across the inner membrane. The data are also a unique documentation of how an MFS protein can promote bacterial iron-siderophore import, standing in contrast to the vast majority of studies which have defined ABC-type permeases as the mediators of siderophore import across the Gram-negative inner membrane or the Gram-positive cytoplasmic membrane.

Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function

Knockout mutants of Plasmodium falciparum lacking pfpm1, pfpm2 and pfhap (triple-PM KO), and mutants lacking all four digestive vacuole (DV) plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap; quadruple-PM KO), were prepared by double cross-over integration effecting chromosomal deletions of up to 14.6 kb. The triple-PM KO was similar to the parental line (3D7) in growth rate, morphology and sensitivity to proteinase inhibitors. The quadruple-PM KO showed a significantly slower rate of growth in standard medium, which manifested as delayed schizont maturation accompanied by reduced formation of haemozoin. In amino acid-limited medium, the reduction in growth rate of the quadruple-PM KO was pronounced. The sensitivity of both the triple- and quadruple-PM KOs to six different HIV aspartic proteinase inhibitors was comparable to that of 3D7, thus establishing that the DV plasmepsins were not the primary targets of the antimalarial activity of these clinically important compounds. Electron microscopic analysis revealed the presence of multilamellar bodies resembling ceroid in the DV of the quadruple-PM KO, and intermediates of the autophagic pathway accumulated as determined by Western blot analysis. Thus, the DV plasmepsins, although not essential, contribute significantly to the fitness of the parasite and are required for efficient degradation of endosomal vesicles delivered to the DV.

The Legionella pneumophila tatB Gene Facilitates Secretion of Phospholipase C, Growth under Iron-Limiting Conditions, and Intracellular Infection

Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection.

The Yeast Plasma Membrane Protein Alr1 Controls Mg2+ Homeostasis and Is Subject to Mg2+-dependent Control of Its Synthesis and Degradation

The Saccharomyces cerevisiae ALR1 (YOL130w) gene product Alr1p is the first known candidate for a Mg(2+) transport system in eukaryotic cells and is distantly related to the bacterial CorA Mg(2+) transporter family. Here we provide the first experimental evidence for the location of Alr1p in the yeast plasma membrane and for the tight control of its expression and turnover by Mg(2+). Using well characterized npi1 and end3 mutants deficient in the endocytic pathway, we demonstrate that Alr1 protein turnover is dependent on ubiquitination and endocytosis. Furthermore, cells lacking the vacuolar protease Pep4p accumulated Alr1p in the vacuole. Mutants lacking Alr1p (Deltaalr1) showed a 60% reduction of total intracellular Mg(2+) compared with the wild type and failed to grow in standard media. When starved of Mg(2+), mutant and wild-type cells had similar low levels of intracellular Mg(2+); but upon addition of Mg(2+), wild-type cells replenished the intracellular Mg(2+) pool within a few hours, whereas Deltaalr1 mutant cells did not. Expression of the bacterial Mg(2+) transporter CorA in the yeast Deltaalr1 mutant partially restored growth in standard media. The results are discussed in terms of Alr1p being a plasma membrane transporter with high selectivity for Mg(2+).

Effect of Cerophyl growth medium on exocytosis in Tetrahymena thermophila.

Culturing the ciliate Tetrahymena thermophila in Cerophyl has provided an opportunity for studying the assembly and/or synthesis of the intramembrane particle array, the rosette, which marks the site of exocytosis in these cells. Cultures grown in this medium cease cell division after only 12h and enter 'stationary phase' earlier (12h of growth) relative to growth in standard medium (proteose peptone). In addition, the cell changes from the normally observed pear-shaped body to a thinner more ellipsoid form. Despite the initial similarities to starving cells, several differences are observed in the Cerophyl-grown cells. One is that cell size remains constant for at least 72h in contrast to starved cells. Secondly, in spite of this block in cell division, results from freeze-fracture replicas of the cell membrane of these cells show that they continue to assemble rosettes, the number of which increases approximately six times, from 0.34 rosette/microgram2 to 2.1 rosettes/microgram2. Addition of the protein synthesis inhibitor, cycloheximide (6h exposure), during growth in Cerophyl shows that 70% of rosettes can be assembled, despite the blockage of translation, by using pre-existing component(s) from a pool. The remaining 30% must involve de novo synthesis of one or more components; this percentage can be increased with longer exposure to the drug. Thirdly, an apparent increase in the number of mucocysts is observed by thin-section electron microscopy. At first (12-24h) only docked mucocysts seem to accumulate in the cell. However, by 36h a considerable increase seems to have taken place, particularly in the number of mucocysts located in the cytoplasm. In the cycloheximide-treated cells this increase in mucocysts begins to be blocked after 6h of exposure to the drug. These observations are in agreement with the results obtained from the freeze-fracture data on the concomitant increase in number of rosettes. This system therefore offers the first possibility of exploring the biosynthesis of these components.

The absence of calcium blocks impulse-evoked release of acetylcholine but not de novo formation of functional neuromuscular synaptic contacts in culture

We have examined the role of calcium-dependent, impulse-evoked release of acetylcholine (ACh) in the formation of functional neuromuscular contacts. Dissociated cell cultures prepared from Xenopus laevis embryos were maintained either in standard medium containing calcium or in calcium-free medium. Both the number of neuron-myocyte contacts and the number of terminations of neurites on myocytes were reduced in Ca-free medium. Intracellular recordings from neuron-myocyte pairs during perfusion with a standard saline revealed that functional synaptic contacts were formed in Ca-free medium, although with a reduced frequency (approximately 30% of controls). Postsynaptic potentials elicited by neuronal action potentials were smaller than those observed after growth in standard medium. The frequency of small, spontaneously occurring potentials was reduced by a factor of 10. Ca-free saline prevented the impulse-evoked release of transmitter from neurons in Ca-free and control cultures. Labeling with alpha-bungarotoxin and iontophoretic application of ACh revealed no ACh receptor clusters in the membrane of myocytes grown in Ca-free medium. Our results suggest that evoked, vesicular release is not required for the initial formation of neuromuscular contacts, although it may be involved in further maturation of synapses. Roles for spontaneous quantal or non-quantal release have not been excluded.

Characteristics of established myeloma and lymphoblastoid cell lines derived from an E myeloma patient: a comparative study.

AbstractThe in vitro characteristics of two types of cell lines established from bone marrow and peripheral blood of an E myeloma patient have been compared. Both types, one with a lymphoblast morphology and the other with plasma cell/plasmablast morphology, secreted monoclonal immunoglobulins in vitro. The former produced IgGK while the latter synthesized the IgEL myeloma protein, which in a previous report was shown to be identical with the myeloma protein in vivo.The myeloma cells were difficult to establish and more stringent in their in vitro requirements than the lymphoblastoid cells. Myeloma cells could only grow in the presence of feeder cells or medium harvested from such cells, while lymphoblasts were capable of independent growth in standard media. The lymphoblastoid line was principally similar to those obtainable from normal lymph nodes (Nilsson et al., 1968; Nilsson, 1971a) and is therefore regarded as being of non‐neoplastic origin. It is thus possible to obtain permanent immunoglobulin‐secreting lines of non‐neoplastic origin as well as myeloma lines of neoplastic origin from patients with myelomatosis. The dynamic and static morphology of the former most closely correspond to the appearance described for lymphoblasts or “immunoblasts”,i.e. lymphocytes stimulated by phyto‐hemagglutinin, while myeloma cells resemble plasma cells of varying maturity. The reasons for the morphologic differences are unknown, but they were sufficiently distinctive to permit unequivocal distinction between the two types of lines.