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Biomedical subjects

L O Ingram

Publications and source records attributed to L O Ingram.

At least 145 records · Page 8Linked to original sources
PubMed1973

Mechanism for the regulation of cell division in Agmenellum.

We describe a nonlethal temperature-conditional mutant of Agmenellum quadruplicatum which allows dissociation of the processes of growth and cell division. With this system, evidence has been obtained for the regulation of one step in the process of cell division by a small effector molecule. The effector molecule is apparently released into the surrounding medium and can be obtained from lyophilized spent medium by extraction with 80% ethanol. The addition of this extract to serpentine filaments of the SN29 mutant strain stimulates cell division in these filaments and leads to the production of cells approximating normal dimensions within one generation time. The degree of stimulation of cell division is directly related to the amount of extract added. A general hypothesis is presented for the positive regulation of the initiation of cell wall and cell membrane invagination in this organism.

Bacteria↗
PubMed1973

Novel mutant impaired in cell division: evidence for a positive regulating factor.

A novel, conditional, cell-division mutant from Agmenellum quadruplicatum strain BG1 is described. During rapid growth in dilute suspensions, cell division lags behind mass increase and the cells form filaments. These filaments spontaneously divide into unit cell lengths as the culture density increases. Other conditions that favor the accumulation of metabolic products in the medium antagonize filament formations. An 80% ethanol-water extract of dried, spent medium also restores the ability of filaments to divide into cells of unit length. Our results suggest that at least one chemical factor acting as a positive effector is involved in cell division.

Bacteriological Techniques↗
PubMed1973

Role of reduced exogenous organic compounds in the physiology of the blue-green bacteria (algae): photoheterotrophic growth of a "heterotrophic" blue-green bacterium.

Nostoc sp. (strain Mac) was shown to be capable of using glucose, fructose, or sucrose as a sole source of carbon and energy in the dark. In the light in the absence of exogenously supplied CO(2), this strain exhibited a more versatile metabolism. In addition to the three sugars above, glycerol and acetate served as sole sources of carbon. This photoheterotrophic growth in the absence of exogenously supplied CO(2) appears to involve O(2)-evolving photosynthesis. The action spectrum for photoheterotrophic growth on acetate closely resembles the action spectrum for photosynthesis. The physiology of photoheterotrophic growth was further investigated through determinations of stable carbon isotope ratios and measurements of gas exchanges. These investigations suggest that respired CO(2) from substrate oxidation is assimilated by the photosynthetic machinery.

Acetates↗
PubMed1973

Role of reduced exogenous organic compounds in the physiology of the blue-green bacteria (algae): photoheterotrophic growth of an "autotrophic" blue-green bacterium.

The unicellular blue-green bacterium Agmenellum quadruplicatum strain BG-1 was found to be capable of rapid photoheterotrophic growth but unable to grow in the dark on a variety of reduced organic substrates. The generation time on glycerol was 12 h, and on CO(2), 3 h. Glycerol carbon was converted into cellular carbon with a very high efficiency. This high efficiency of carbon conversion, the action spectrum for growth on glycerol, cell pigmentation, gas exchange measurements, and immediate ability of photoheterotrophically grown cells to evolve O(2) (upon the addition of CO(2)) suggest the involvement of both photosystems I and II of photosynthesis during photoheterotrophic growth.

Carbon Dioxide↗
PubMed1973

Occurrence of cell lytic enzymes in blue-green bacteria.

Detergent extracts of three blue-green bacteria (Agmenellum quadruplicatum strain BG1, Anacystis nidulans strain TX20, and Nostoc sp. strain MAC) contained enzymes capable of lysing suspensions of Micrococcus lysodeikticus. The enzyme preparation from A. quadruplicatum released soluble reducing fragments from purified peptidoglycan. The lytic activity exhibited a pH optimum between 6 and 7, was relatively heat stable, and was susceptible to attack by proteolytic enzymes. These results extend the range of bacterial types exhibiting cell lytic activity as well as confirm the existence of the lytic system commonly observed in "water blooms".

Bacteria↗
PubMed1972

Documentation of auxotrophic mutation in blue-green bacteria: characterization of a tryptophan auxotroph in Agmenellum quadruplicatum.

A tryptophan-requiring auxotroph of Agmenellum quadruplicatum strain BG1, a species of blue-green bacteria, was isolated by means of a nitrosoguanidine-penicillin procedure. Its growth characteristics were determined, and the enzymological block was identified in the A activity of tryptophan synthetase. Starvation of the auxotroph for tryptophan resulted in the derepression of the synthesis of all five enzymes. The first four enzymes derepressed 2- to 3-fold, and tryptophan synthetase B derepressed 20-fold. In the parental prototroph, BG1, anthranilate synthetase was active in crude extracts with ammonia as the amino donor reactant, but not with glutamine.

Anthranilate Synthase↗
PubMed1972

Cell division mutations in the blue-green bacterium Agmenellum quadruplicatum strain BG1: a comparison of the cell wall.

The peptidoglycan from two types of filamentous cell division mutants of Agmenellum quadruplicatum strain BG1 has been compared to that of the parent organism. Small variations in the total peptidoglycan composition on a dry-weight basis were found in the mutants. The reduced level of peptidoglycan in the serpentine mutant is consistent with a general decrease in the ratio of surface area to volume as compared to the parent organism. The increased peptidoglycan content in the septate mutant confirms previous ultrastructural observations of the greatly enlarged peptidoglycan septum between adjacent cells. A comparison of peptidoglycan composition and cross-linking in the two types of filamentous mutants of A. quadruplicatum and in drug-induced phenocopies suggests that structural alterations of the peptidoglycan are not involved in the apparent impairments of cellular division. Furthermore, data concerning the relative susceptibilities of the parent and mutants to antibiotics indicate that neither mutant exhibits a gross alteration of permeability.

Amino Acids↗
PubMed1972

Selective inhibition of deoxyribonucleic acid synthesis by 2-deoxyadenosine in the blue-green bacterium Agmenellum quadruplicatum.

Concentrations of deoxyadenosine which have little effect on net ribonucleic acid (RNA) synthesis or on increase in cell mass selectively inhibit deoxyribonucleic acid (DNA) synthesis in Agmenellum quadruplicatum. Exogenously supplied deoxyadenosine, at concentrations above 10 mug/ml, stimulates DNA degradation. These results are correlated with a rapid loss in viability. Over a narrow concentration range (6-15 mug/ml), deoxyadenosine impairs the division process and induces the formation of elongated cells. Low exogenous concentrations of deoxyadenosine are readily incorporated into both DNA and RNA, with the major portion as DNA.

Cell Count↗
PubMed1970

Characteristics of a stable, filamentous mutant of a coccoid blue-green alga.

Filamentous mutants were induced in a coccoid blue-green alga, Agmenellum quadruplicatum strain BG1, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutants fall into two general classes: filaments with cross walls and filaments without cross walls. All mutants of these general types derived from BG1 are stable and have growth rates the same as or very similar to the wild type under a variety of conditions. Detailed examination of one mutant, 53SB2, revealed no difference in deoxyribonucleic acid content nor in base ratios. Mutant 53SB2 did not revert to the normal cell size and shape when grown under different physical conditions nor upon the addition of potential reversing agents to the basal medium. It is our general experience that filamentous mutants such as those described here in BG1 are commonly induced in other coccoid blue-green algae after NTG treatment.

Cell Nucleus↗
PubMedDate not supplied

Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose.

Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.

Amino Acid Sequence↗
PubMedDate not supplied

Use of UV absorbance To monitor furans in dilute acid hydrolysates of biomass.

A simple method based on UV spectra was developed for the estimation of total furans (furfural and hydroxymethylfurfural) in hemicellulose hydrolysates. UV spectra of hemicellulose hydrolysate contained a single dominant peak at around 278 nm. Approximately two-thirds of this peak can be attributed to furan absorbance (furfural and hydroxymethylfurfural). At 284 nm, both furfural and hydroxymethylfurfural have equal absorbance on a weight basis. A comparison of HPLC determinations for different samples of hydrolysate was used to develop a simple equation that allows the accurate prediction of total furans based on the difference in absorbance at 284 and 320 nm. This method may prove useful for quality control applications during the production of biomass syrups using a dilute acid hydrolysis process and during treatments for the amelioration of toxins. Although furans represent only a portion of the toxins present in hemicellulose hydrolysates, the abundance of furans appears to serve as a useful marker to predict relative toxicity.

Acids↗
PubMedDate not supplied

Detoxification of dilute acid hydrolysates of lignocellulose with lime.

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis is accompanied by the production of inhibitors that retard microbial fermentation. Treatment of hot hydrolysate with Ca(OH)(2) (overliming) is an effective method for detoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our results indicate that the optimal lime addition for detoxification varies and depends on the concentration of mineral acids and organic acids in each hydrolysate. This optimum was shown to be readily predicted on the basis of the titration of hydrolysate with 2 N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average composition of 15 hydrolysates prior to treatment was as follows (per L): 95.24 +/- 7.29 g sugar, 5.3 +/- 2.99 g acetic acid, 1.305 +/- 0.288 g total furans (furfural and hydroxymethylfurfural), and 2.86 +/- 0.34 g phenolic compounds. Optimal overliming resulted in a 51 +/- 9% reduction of total furans, a 41 +/- 6% reduction in phenolic compounds, and a 8.7 +/- 4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarity of microorganisms, it is possible that the titration method described here may also prove useful for detoxification and fermentation processes using other microbial biocatalysts.

Acids↗
PubMedDate not supplied

Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose.

The simplicity of the fermentation process (anaerobic with pH, temperature, and agitation control) in ethanologenic Escherichia coli KO11 and LY01 makes this an attractive system to investigate the utility of gene arrays for biotechnology applications. By using this system, gene expression, glycolytic flux, and growth rate have been compared in glucose-grown and xylose-grown cells. Although the initial metabolic steps differ, ethanol yields from both sugars were essentially identical on a weight basis, and little carbon was diverted to biosynthesis. Expression of only 27 genes changed by more than 2-fold in both strains. These included induction of xylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR and the cyclic AMP-CRP system and repression of Mlc-regulated genes encoding glucose uptake (ptsHIcrr, ptsG) and mannose uptake (manXYZ) during growth on xylose. However, expression of genes encoding central carbon metabolism and biosynthesis differed by less than 2-fold. Simple statistical methods were used to investigate these more subtle changes. The reproducibility (coefficient of variation of 12%) of expression measurements (mRNA as cDNA) was found to be similar to that typically observed for in vitro measurements of enzyme activities. Using Student's t test, many smaller but significant sugar-dependent changes were identified (p < 0.05 in both strains). A total of 276 genes were more highly expressed during growth on xylose; 307 genes were more highly expressed with glucose. Slower growth (lower ATP yield) on xylose was accompanied by decreased expression of 62 genes concerned with the biosynthesis of small molecules (amino acids, nucleotides, cofactors, and lipids), transcription, and translation; 5 such genes were expressed at a higher level. In xylose-grown cells, 90 genes associated with the transport, catabolism, and regulation of pathways for alternative carbon sources were expressed at higher levels than in glucose-grown cells, consistent with a relaxation of control by the cyclic AMP-CRP regulatory system. Changes in expression of genes encoding the Embden-Meyerhof-Parnas (EMP) pathway were in excellent agreement with calculated changes in flux for individual metabolites. Flux through all but one step, pyruvate kinase, was predicted to be higher during glucose fermentation. Expression levels (glucose/xylose) were higher in glucose-grown cells for all EMP genes except the isoenzymes encoding pyruvate kinase (pykA and pykF). Expression of both isoenzymes was generally higher during xylose fermentation but statistically higher in both strains only for pykF encoding the isoenzyme activated by fructose-6-phosphate, a key metabolite connecting pentose metabolism to the EMP pathway. The coordinated changes in expression of genes encoding the EMP pathway suggest the presence of a common regulatory system and that flux control within the EMP pathway may be broadly distributed. In contrast, expression levels for genes encoding the Pentose-Phosphate pathway did not differ significantly between glucose-grown and xylose-grown cells.

Carbohydrate Metabolism↗
PubMedDate not supplied

Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant).

Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p < 0.10) between KO11 and LY01 under each of six different growth conditions (p < 0.000001). Statistical evaluation of differentially expressed genes according to various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from choline and choline uptake, respectively). Addition of glycine (10 mM) increased ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11. Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of the cell envelope components were also noted, which may contribute to increased ethanol tolerance.

Directed Molecular Evolution↗
PubMedDate not supplied

Development of industrial-medium-required elimination of the 2,3-butanediol fermentation pathway to maintain ethanol yield in an ethanologenic strain of Klebsiella oxytoca.

Fermentation efficiency and nutrient costs are both significant factors in process economics for the microbial conversion of cellulosic biomass to commodity chemicals such as ethanol. In this study, we have developed a more industrial medium (OUM1) composed of 0.5% corn steep liquor (dry weight basis) supplemented with mineral salts (0.2%), urea (0.06%), and glucose (9%). Although the growth of strain P2 was vigorous in this medium, approximately 14% of substrate carbon was diverted into 2,3-butanediol and acetoin under the low pH conditions needed for optimal cellulase activity during simultaneous saccharification. Deleting the central region of the budAB genes encoding alpha-acetolactate synthase and alpha-acetolactate decarboxylase eliminated the butanediol and acetoin coproducts and increased ethanol yields by 12%. In OUM1 medium at pH 5.2, strain BW21 produced over 4% ethanol in 48 h (0.47 g ethanol per g glucose). Average productivity (48 h), ethanol titer, and ethanol yield for BW21 in OUM1 medium (pH 5.2) exceeded that of the parent (strain P2) in rich laboratory medium (Luria broth).

Bioreactors↗
PubMedDate not supplied

Ultrasound stimulates ethanol production during the simultaneous saccharification and fermentation of mixed waste office paper.

The commercial production of ethanol from cellulose by simultaneous saccharification and fermentation (SSF) is prevented in part by the high cost of fungal cellulase enzymes. Intermittent exposure of SSF processes to ultrasonic energy under selected conditions (5 FPU of cellulase/g of substrate; 15 min of exposure/240 min cycle during the latter half of SSF) was found to increase ethanol production from mixed waste office paper by approximately 20%, producing 36.6 g/L ethanol after 96 h (70% of the maximum theoretical yield). Without ultrasound, 10 FPU of cellulase/g of substrate was required to achieve similar results. Continuous exposure of the organism to ultrasonic energy was bacteriostatic and decreased ethanol production but may be useful for the controlling bacterial growth in other processes.

Carbohydrate Metabolism↗