Investigations of the influence of carbon starvation on the carbohydrate storage compounds (trehalose, glycogen), viability, adenylate pool, and adenylate energy charge in Cellulomonas sp. (DSM20108)

During conditions of energy and carbon excess Cellulomonas sp. accumulates intracellularly two different carbohydrate storage products in different relative concentrations: trehalose and glycogen. During carbon starvation these compounds are degraded at different rates and are therefore characterized metabolically by different half-life periods (glycogen 1.6 h, trehalose 34 h). Other parameters which bear some relation to viability during conditions of stress are compared with these half-life periods. The half-life period of the adenylate energy charge EC_A (52 h) is similar to the trehalose half-life period, and it is concluded that it is trehalose which is essential for long-term survival while glycogen is used in the very early stages of carbon starvation to produce energy for metabolism under these conditions. Evidence is presented that two mechanisms are active for the stabilization of the intracellular adenylate energy charge: specific excretion and adenylate degradation.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.     

Comparison of Two Cellulomonas Strains and Their Interaction with Azospirillum brasilense in Degradation of Wheat Straw and Associated Nitrogen Fixation

A mutant strain of Cellulomonas sp. CS1-17 was compared with Cellulomonas gelida 2480 as the cellulolytic component of a mixed culture which was responsible for the breakdown of wheat straw to support asymbiotic nitrogen fixation by Azospirillum brasilense Sp7 (ATCC 29145). Cellulomonas sp. strain CSI-17 was more efficient than was C. gelida in cellulose breakdown at lower oxygen concentrations and, in mixed culture with A. brasilense, it supported higher nitrogenase activity (C₂H₂ reduction) and nitrogen fixation with straw as the carbon source. Based on gravimetric determinations of straw breakdown and total N determinations, the efficiency of nitrogen fixation was 72 and 63 mg of N per g of straw utilized for the mixtures containing Cellulomonas sp. and C. gelida, respectively. Both Cellulomonas spp. and Azospirillum spp. exhibited a wide range of pH tolerance. When introduced into sterilized soil, the Cellulomonas sp.-Azospirillum brasilense association was more effective in nitrogen fixation at a pH of 7.0 than at the native soil pH (5.6). This was also true of the indigenous diazotrophic microflora of this soil. The potential implications of this work to the field situation are discussed.     

Biosynthesis of bacterial glycogen: Kinetic studies of a glucose-1-P adenylyltransferase (EC 2.7.7.27) from a glycogen-excess mutant of Escherichia coli B

An Escherichia coli B mutant, CL1136 accumulates glycogen at 3.4 to 4 times the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to α- and β-amylolysis, chain length determination and I₂-complex absorption spectra. The CL1136 mutant contains normal glycogen synthase and branching enzyme activity but has an ADPglucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and in contrast to the present strain enzyme studied previously, is highly active in the absence of the allosteric activator. The response of the CL1136 enzyme to energy charge has been determined and this enzyme shows appreciable activity at low energy charge values where the E. coli B enzyme is inactive. The response to energy charge for the CL1136 and E. coli B enzymes are correlated with the rates of glycogen accumulation observed in the microorganisms. The regulation of glycogen synthesis in E. coli is to a great extent at the level of ADPglucose pyrophosphorylase; varying concentrations of fructose-P₂ and energy charge determine the rate of ADPglucose and glycogen synthesis. Both the allosteric regulation of ADPglucose pyrophosphorylase as well as the genetic regulations of the synthesis of glycogen biosynthetic enzymes (glycogen synthase and ADPglucose pyrophosphorylase) are involved in the regulation of glycogen accumulation in E. coli B.     

Photoproduction of H2 from Cellulose by an Anaerobic Bacterial Coculture

Cellulomonas sp. strain ATCC 21399 is a facultatively anaerobic, cellulose-degrading microorganism that does not evolve hydrogen but produces organic acids during cellulose fermentation. Rhodopseudomonas capsulata cannot utilize cellulose, but grows photoheterotrophically under anaerobic conditions on organic acids or sugars. This report describes an anaerobic coculture of the Cellulomonas strain with wild-type R. capsulata or a mutant strain lacking uptake hydrogenase, which photoevolves molecular hydrogen by the nitrogenase system of R. capsulata with cellulose as the sole carbon source. In coculture, the hydrogenase-negative mutant produced 4.6 to 6.2 mol of H₂ per mol of glucose equivalent, compared with 1.2 to 4.3 mol for the wild type.     

Accumulation,mobilization and turn-over of glycogen in the blue-green bacterium Anacystis nidulans

1. Accumulation of glycogen up to a constant amount per cell was observed during the post-exponential phase of growth, in the presence of an excess of a utilizable carbon source. Cell multiplication was reproducibly controlled by growth of the organism in a nitrogen-limiting medium under photoautotrophic conditions (presence of light, air plus CO₂).
2. Temporary starvation, i.e. by removal of light or by the addition to an illuminated culture of DCMU, 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea, a specific inhibitor of photosystem II, lead to a mobilization of glycogen in the cell. Furthermore, Anacystis nidulans, having accumulated glycogen by virtue of preculture under nitrogen-limiting conditions, will resume cell division when the culture medium is complemented with a nitrogen source. The ability of the organism to use glycogen as an endogenous carbon source for growth was observed by addition of a nitrogen source to nitrogen-starving cells and simultaneous removal of CO₂.
3. During the period of constant amount of glycogen per cell the reserve polysaccharide was subject to turnover as demonstrated with a pulse chase-labelling technique. The demonstration of a turnover—for the first time with a bacterial species—indicated a strict balance in the relative rate of synthesis and degradation.

     

Continuous Cultivation for Apparent Optimization of Defined Media for Cellulomonas sp. and Bacillus cereus

Steady-state continuous culture was used to optimize lean chemically defined media for a Cellulomonas sp. and Bacillus cereus strain T. Both organisms were extremely sensitive to variations in trace-metal concentrations. However, medium optimization by this technique proved rapid, and multifactor screening was easily conducted by using a minimum of instrumentation. The optimized media supported critical dilution rates of 0.571 and 0.467 h⁻¹ for Cellulomonas and Bacillus, respectively. These values approximated maximum growth rate values observed in batch culture.     

Nonribosomal Nature of Novel, Stable Ribonucleic Acid Species Accumulated by Blue-Green Bacteria

The facultatively chemoheterotrophic blue-green bacterium Aphanocapsa 6714 accumulates two novel, stable ribonucleic acid species when deprived of sources of carbon and energy. At least one of these species is nonribosomal.     

Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing α-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and β-glucoside hydrolyzing activities. All strains synthesized β-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on α-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10–13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. β-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and β-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi. These studies were supported byPakistan Atomic Energy Commission. Some chemicals were purchased from funds allocated byUnited States Agency for International Development, Washington (DC, USA), under PSTC proposal 6.163.     

Mode of Loss of Phosphatidyl-ethanolamine in Autoxidation

This report identifies and describes the chemical structure of granular material that accumulates in the cytoplasm of Selenomonas ruminantium grown in glucose or lactate medium. The granular material was identified to be glycogen. Its molecular weight was about 2 x 10⁷ daltons. Conversion into maltose with α-amylase, β-amylase, and isoamylase was 61%, 37%, and 15%, respectively. The glycogen was digested completely by joint action of β-amylase and isoamylase, and its conversion into maltose was 103%. The average chain length of the glycogen was 23.5. The maximum absorption of the iodine complex of the glycogen was at 520 nm. These results led us to conclude that the chemical structure of this glycogen was similar to that of plant amylopectin, unlike normal Microbiol or animal glycogen so far known. When S. ruminantium was grown in glucose medium, the amount of glycogen in cells reached about 260 µg/mg dry weight of cells during late exponential phase and early stationary phase.     

Differential Expression of Xylanases and Endoglucanases in the Hybrid Derived from Intergeneric Protoplast Fusion between a Cellulomonas sp. and Bacillus subtilis

A stable hybrid obtained by protoplast fusion between a Cellulomonas sp. and Bacillus subtilis exhibits an altered pattern of enzyme induction with different cellulosic substrates. Unlike in the Cellulomonas sp., xylanase was induced in the hybrid organism specifically by xylan, and endoglucanase was induced by carboxymethyl cellulose. The amount and specific activity of xylanase produced by the hybrid were more than those produced by the Cellulomonas sp. β-Glucosidase which is cell bound or intracellular in the Cellulomonas sp. was secreted by the hybrid organism, and relative amounts of extracellular β-glucosidase were high. Furthermore, this extracellular β-glucosidase activity was dependent on the nature of the cellulosic substrate. Endoglucanases synthesized in the hybrid differed in their electrophoretic mobilities as compared with the parental enzymes.     

Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose

A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet.     

The curdlan-type exopolysaccharide produced by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> KU forms part of an extracellular glycocalyx involved in cellulose degradation

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.     

Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium

d-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO₂ as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L⁻¹ and a production rate of 0.15 g mannitol L⁻¹ day⁻¹. This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO₂ in cyanobacteria.     

Production of single-cell protein with cellulomonas sp. on hempstalk wastes

Strains of Cellulomonas isolated from enrichment cultures with cellulose powder were cultivated with hempstalk wastes as the sole carbon source in a mineral medium. The yields of protein were not significantly different using the crude or alkali pretreated substrate in flasks experiments. Up to 12.5% protein was obtained on the untreated substrate with the best strain cultivated in a small fermenter.     

Cellulose Decomposition and Associated Nitrogen Fixation by Mixed Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans

Mixed cultures of Cellulomonas gelida plus Azospirillum lipoferum or Azospirillum brasilense and C. gelida plus Bacillus macerans were shown to degrade cellulose and straw and to utilize the energy-yielding products to fix atmospheric nitrogen. This cooperative process was followed over 30 days in sand-based cultures in which the breakdown of 20% of the cellulose and 28 to 30% of the straw resulted in the fixation of 12 to 14.6 mg of N per g of cellulose and 17 to 19 mg of N per g of g straw consumed. Cellulomonas species have certain advantages over aerobic cellulose-degrading fungi in being able to degrade cellulose at oxygen concentrations as low as 1% O₂ (vol/vol) which would allow a close association between cellulose-degrading and microaerobic diazotrophic microorganisms. Cultures inoculated with initially different proportions of A. brasilense and C. gelida all reached a stable ratio of approximately 1 Azospirillum/3 Cellulomonas cells.     

S9A Serine Protease Engender Antigenic Gluten Catabolic Competence to the Human Gut Microbe

The human gut microbiome has a significant role in host physiology; however its role in gluten catabolism is debatable. Present study explores the role of human gut microbes in gluten catabolism and a native human gut microbe Cellulomonas sp. HM71 was identified. SSU rDNA analysis has described human gut microbiome structure and also confirmed the permanent residentship of Cellulomonas sp. HM71. Catabolic potential of Cellulomonas sp. HM71 to cleave antigenic gluten peptides indicates presence of candidate gene encoding biocatalytic machinery. Genome analysis has identified the presence of gene encoding S9A serine protease family—prolyl endopeptidase, with Ser591, Asp664 and His685 signature residues. Cellulomonas sp. HM71 prolyl endopeptidase activity was found optimal at pH 7.0 and 37 °C with a K_M of 35.53 μmol and specifically cleaves at proline residue. Current study describes the gluten catabolism potential of Cellulomonas sp. HM71 depicting possible role of human gut microbes in gluten catabolism to confer resistance mechanisms for the onset of celiac diseases in populations with gluten diet.     

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     

Hexavalent chromium reduction by Cellulomonas sp. strain ES6: the influence of carbon source, iron minerals, and electron shuttling compounds

The reduction of hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III), can be an important aspect of remediation processes at contaminated sites. Cellulomonas species are found at several Cr(VI) contaminated and uncontaminated locations at the Department of Energy site in Hanford, Washington. Members of this genus have demonstrated the ability to effectively reduce Cr(VI) to Cr(III) fermentatively and therefore play a potential role in Cr(VI) remediation at this site. Batch studies were conducted with Cellulomonas sp. strain ES6 to assess the influence of various carbon sources, iron minerals, and electron shuttling compounds on Cr(VI) reduction rates as these chemical species are likely to be present in, or added to, the environment during in situ bioremediation. Results indicated that the type of carbon source as well as the type of electron shuttle present influenced Cr(VI) reduction rates. Molasses stimulated Cr(VI) reduction more effectively than pure sucrose, presumably due to presence of more easily utilizable sugars, electron shuttling compounds or compounds with direct Cr(VI) reduction capabilities. Cr(VI) reduction rates increased with increasing concentration of anthraquinone-2,6-disulfonate (AQDS) regardless of the carbon source. The presence of iron minerals and their concentrations did not significantly influence Cr(VI) reduction rates. However, strain ES6 or AQDS could directly reduce surface-associated Fe(III) to Fe(II), which was capable of reducing Cr(VI) at a near instantaneous rate. These results suggest the rate limiting step in these systems was the transfer of electrons from strain ES6 to the intermediate or terminal electron acceptor whether that was Cr(VI), Fe(III), or AQDS.