Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2)

DNA sequencing and operon disruption experiments indicate that the genes glgBI and glgBII, which code for the two developmentally specific glycogen branching enzymes of Streptomyces coelicolor A3(2) each form part of larger duplicated operons consisting of at least four genes in the order pep1-treS-pep2-glgB. The sequences of the TreS proteins are 73% identical (93% similar) to that of an enzyme that converts maltose into trehalose in Pimelobacter, a distantly related actinomycete; and the Pep1 proteins show relatedness to the α-amylase superfamily. Disruptions of each operon have spatially specific effects on the nature of glycogen deposits, as assessed by electron microscopy. Upstream of the glgBI operon, and diverging from it, is a gene (glgP) that encodes a protein resembling glycogen phosphorylase from Thermatoga maritima and a homologue in Mycobacterium tuberculosis. These three proteins form a distinctive subgroup compared with glycogen phosphorylases from most other bacteria, which more closely resemble the enzymes from eukaryotes. Diverging from the glgBII operon, and separated from the pep1 gene by two very small ORFs, is a gene (glgX) encoding a probable glycogen debranching enzyme. It is suggested that most of these gene products participate in the developmentally modulated interconversion of immobile, inert glycogen reservoirs, and diffusible forms of carbon, both metabolically active (e.g. glucose-1-phosphate generated by glycogen phosphorylase) and metabolically inert but physiologically significant (trehalose). Received: 12 November 1999 / Accepted: 31 January 2000     

Comparative Genomic and Phylogenetic Analyses of Gammaproteobacterial glg Genes Traced the Origin of the Escherichia coli Glycogen glgBXCAP Operon to the Last Common Ancestor of the Sister Orders Enterobacteriales and Pasteurellales

Production of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria.     

Cell Size,Macromolecular Composition,and O2 Consumption During Agitated Cultivation of Naegleria gruberi*

SYNOPSIS. Cell size, macromolecular composition, carbohydrate utilization patterns, and O₂ concentrations were measured throughout the growth stages of Naegleria gruberi in agitated cultures in a complex medium. Biphasic logarithmic growth occurred during the initial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 × 10* amebaeJml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increased 31% to 35% per ameba at the end of each phase of log growth. DNA increased ～ 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O₂ tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation

The disaccharide trehalose is widely distributed in nature and can serve as a carbon reservoir, a signaling molecule for controlling glucose metabolism and a stress protectant. We demonstrated that in Escherichia coli ΔotsA cells, which are unable to synthesize trehalose, the aggregation of endogenous proteins during the stationary phase was increased in comparison to wild‐type cells. The lack of trehalose synthesis boosted Nε‐lysine acetylation of proteins, which in turn enhanced their hydrophobicity and aggregation. This increased Nε‐lysine acetylation could result from carbon overflow and the accumulation of acetyl phosphate caused by the ΔotsA mutation. These findings provide a better understanding of the previously reported protective functions of trehalose in protein stabilization and the prevention of protein aggregation. Our results indicate that trehalose may participate in proteostasis not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation. We propose that trehalose protects E. coli against carbon stress – the synthesis and storage of trehalose can prevent carbon overflow, which otherwise is manifested by protein acetylation and aggregation.     

Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa

Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA′ that contains two genes for agmatine deiminases (agu2A and agu2A′). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A′ contains a twin‐arginine translocation signal at its N‐terminus and site‐directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA′ promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA′ provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA′, specifically its secreted product Agu2A′, reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA′ operon in the biofilm development of P. aeruginosa.     

Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis.

Summary The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an M_r of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - ^H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53° C.     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Essential role of the plasmid hik31 operon in regulating central metabolism in the dark in Synechocystis sp. PCC 6803

The plasmid hik31 operon (P3, slr6039‐slr6041) is located on the pSYSX plasmid in Synechocystis sp. PCC 6803. A P3 mutant (ΔP3) had a growth defect in the dark and a pigment defect that was worsened by the addition of glucose. The glucose defect was from incomplete metabolism of the substrate, was pH dependent, and completely overcome by the addition of bicarbonate. Addition of organic carbon and nitrogen sources partly alleviated the defects of the mutant in the dark. Electron micrographs of the mutant revealed larger cells with division defects, glycogen limitation, lack of carboxysomes, deteriorated thylakoids and accumulation of polyhydroxybutyrate and cyanophycin. A microarray experiment over two days of growth in light‐dark plus glucose revealed downregulation of several photosynthesis, amino acid biosynthesis, energy metabolism genes; and an upregulation of cell envelope and transport and binding genes in the mutant. ΔP3 had an imbalance in carbon and nitrogen levels and many sugar catabolic and cell division genes were negatively affected after the first dark period. The mutant suffered from oxidative and osmotic stress, macronutrient limitation, and an energy deficit. Therefore, the P3 operon is an important regulator of central metabolism and cell division in the dark.     

Phenotypic characterization of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under osmotic stress conditions using elementary mode analysis

Corynebacterium glutamicum, a soil bacterium, is used to produce amino acids such as lysine and glutamate. C. glutamicum is often exposed to osmolality changes in its medium, and the bacterium has therefore evolved several adaptive response mechanisms to overcome them. In this study we quantify the metabolic response of C. glutamicum under osmotic stress using elementary mode analysis (EMA). Further, we obtain the optimal phenotypic space for the synthesis of lysine and formation of biomass. The analysis demonstrated that with increasing osmotic stress, the flux towards trehalose formation and energy-generating pathways increased, while the flux of anabolic reactions diminished. Nodal analysis indicated that glucose-6-phosphate, phosphoenol pyruvate, and pyruvate nodes were capable of adapting to osmotic stress, whereas the oxaloacetic acid node was relatively unresponsive. Fewer elementary modes were active under stress indicating the rigid behavior of the metabolism in response to high osmolality. Optimal phenotypic space analysis revealed that under normal conditions the organism optimized growth during the initial log phase and lysine and trehalose formation during the stationary phase. However, under osmotic stress, the analysis demonstrated that the organism operates under suboptimal conditions for growth, and lysine and trehalose formation.     

Dr Han L. Golterman,a Limnologist: a tribute on the occasion of his 65th birthday and retirement

The green algae D. tertiolecta, the flagellate I. galbana and the diatom C. gracilis were grown in batch cultures. The organisms were analysed for lipid class composition at the logarithmic and stationary growth phases using the Chromarod-Iatroscan thin layer chromatography with flame ionization detection (TLC-FID) system.There were major differences in lipid class production among the organisms investigated, but few differences in lipid class distribution between log phase and stationary phase cultures of D. tertiolecta and I. galbana. C. gracilis displayed the general trend exhibited in diatom metabolism, which can be characterized by an increase in triacylglycerol synthesis in situations of stress.     

Studies on cellulase production by a Bacillus subtilis

Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 °C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 °C, and the enzyme was highly stable up to 60 °C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.     

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber-mutated in E. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme II^Tre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.     

Biosynthesis of Glycogen and Starch in Cryptococcus laurentii

Cells of Cryptococcus laurentii, when grown in liquid culture on 2% glucose close to neutral pH, showed glycogen granules throughout the cytoplasm. Glycogen levels of C. laurentii cells reached maximal levels just before onset of stationary phase. Concomitantly, a sharp rise in total and specific activity of glycogen synthetase was observed. Conversely, glycogen phosphorylase reached its highest specific activity approximately 3 hr after the glycogen peaked and remained high until most of the endogenous glycogen was utilized. Uridine diphosphoglucose pyrophosphorylase activity was always an order of magnitude higher than glycogen synthetase during log phase, but fell off rapidly after the cells reached stationary growth. Kinetic properties of the glycogen synthetase showed that the enzyme is always activated by glucose-6-phosphate, although the degree of activation by glucose-6-phosphate was found to be somewhat variable. The accelerated uptake of glucose commencing with the onset of stationary phase is explained by the rapid formation of extracellular acidic polysaccharide, which continues as long as there is glucose in the medium. In cells grown at pH 3.4, where no detectable extracellular acidic polysaccharide was formed, glucose uptake drastically declined when the cells reached stationary phase. These cells also contained glycogen-like granules in the cytoplasm. The evidence presented indicates that these granules are in fact glycogen, and that its structure does not resemble that of the starch excreted by cells grown at acidic pH.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATα and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Glycogen metabolism of the anammox bacterium “Candidatus Brocadia sinica”

Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)⁻¹ while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.Subject terms: Applied microbiology, Water microbiology     

A Highlights from MBoC Selection: Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth

When conditions are unfavorable, virtually all living cells have the capability of entering a resting state termed quiescence or G0. Many aspects of the quiescence program as well as the mechanisms governing the entry and exit from quiescence remain poorly understood. Previous studies using the budding yeast Saccharomyces cerevisiae have shown that upon entry into stationary phase, a quiescent cell population emerges that is heavier in density than nonquiescent cells. Here, we show that total intracellular trehalose and glycogen content exhibits substantial correlation with the density of individual cells both in stationary phase batch cultures and during continuous growth. During prolonged quiescence, trehalose stores are often maintained in favor over glycogen, perhaps to fulfill its numerous stress-protectant functions. Immediately upon exit from quiescence, cells preferentially metabolize trehalose over other fuel sources. Moreover, cells lacking trehalose initiate growth more slowly and frequently exhibit poor survivability. Together, our results support the view that trehalose, which is more stable than other carbohydrates, provides an enduring source of energy that helps drive cell cycle progression upon return to growth.     

Intracellular accumulation of trehalose and glycogen in an extreme oligotroph,Rhodococcus erythropolis N9T-4

An extreme oligotroph, Rhodococcus erythropolis N9T-4, showed intracellular accumulation of trehalose and glycogen under oligotrophic conditions. No trehalose accumulation was observed in cells grown on the rich medium. Deletion of the polyphosphate kinase genes enhanced the trehalose accumulation and decreases the intracellular glycogen contents, suggesting an oligotrophic relationship between among the metabolic pathways of trehalose, glycogen, and inorganic polyphosphate biosyntheses.