Utilization of Dissolved Nitrogen by Heterotrophic Bacterioplankton: Effect of Substrate C/N Ratio

The significance of dissolved combined amino acids (DCAA), dissolved free amino acids (DFAA), and dissolved DNA (D-DNA) as sources of C and N for marine bacteria in batch cultures with variable substrate C/N ratios was studied. Glucose, ammonium, alanine, and phosphate were added to the cultures to produce C/N ratios of 5, 10, and 15 and to ensure that phosphorus was not limiting. Maximum bacterial particulate organic carbon production (after 25 h of incubation) was inversely correlated with the C/N ratio: with the addition of identical amounts of carbon, the levels of production were 9.0-, 10.0-, and 11.1-fold higher at C/N ratios of 15, 10, and 5, respectively, relative to an unamended control. The bacterial growth efficiency increased from 22% (control cultures) to 44 to 53% in the cultures with manipulated C/N ratios (C/N-manipulated cultures). Net carbon incorporation from DCAA, DFAA, and D-DNA supported on average 19, 4, and 3% (control cultures and cultures to which only phosphate was added [+P cultures]) and 5, 4, and 0.3% of the particulate organic carbon production (C/N-manipulated cultures), respectively. In the C/N-manipulated cultures, a 2.6- to 3.4-fold-higher level of incorporation of DCAA, relative to that in the control cultures, occurred. Incorporation of D-DNA increased with the substrate C/N ratio, suggesting that D-DNA mainly was a source of N to the bacteria. Organic N (DCAA, DFAA, and D-DNA) sustained 14 to 49% of the net bacterial N production. NH₄⁺ was the dominant N source and constituted 55 to 99% of the total N uptake. NO₃^- contributed up to 23% to the total N uptake but was released in two cultures. The studied N compounds sustained nearly all of the bacterial N demand. Our results show that the C/N ratio of dissolved organic matter available to bacteria has a significant influence on the incorporation of individual compounds like DCAA and D-DNA.     

Utilization of Dissolved Nitrogen by Heterotrophic Bacterioplankton: a Comparison of Three Ecosystems

The contributions of different organic and inorganic nitrogen and organic carbon sources to heterotrophic bacterioplankton in batch cultures of oceanic, estuarine, and eutrophic riverine environments were compared. The importance of the studied compounds was surprisingly similar among the three ecosystems. Dissolved combined amino acids (DCAA) were most significant, sustaining from 10 to 45% of the bacterial carbon demands and from 42 to 112% of the bacterial nitrogen demands. Dissolved free amino acids (DFAA) supplied 2 to 7% of the carbon and 6 to 24% of the nitrogen incorporated into the bacterial biomass, while dissolved DNA (D-DNA) sustained less than 5 and 12% of the carbon and nitrogen requirements, respectively. Ammonium was the second most important source of nitrogen, meeting from 13 to 45% of the bacterial demand in the oceanic and estuarine cultures and up to 270% of the demand in riverine cultures. Nitrate was taken up in the oceanic cultures (uptake equaled up to 46% of the nitrogen demand) but was released in the two others. Assimilation of DCAA, DFAA, and D-DNA combined supplied 43% of the carbon demand of the bacteria in the oceanic cultures, while approximately 25% of the carbon requirements were met by the three substrates at the two other sites. Assimilation of nitrogen from DCAA, DFAA, D-DNA, NH₄⁺, and NO₃^-, on the other hand, exceeded production of particulate organic nitrogen in one culture at 27 h and in all cultures over the entire incubation period (50 h). These results suggest that the studied nutrient sources may fully support the nitrogen needs but only partially support the carbon needs of microbial communities of geographically different ecosystems. Furthermore, a comparison of the initial concentrations of the different substrates indicated that relative pool sizes of the substrates seemed to influence which substrates were primarily being utilized by the bacteria.     

d-Aminoaciduria in mutant mice lackingd-amino-acid oxidase activity

Summary Urine of mutant ddY/DAO^– mice lackingd-amino-acid oxidase activity contained more serine and proline than that of normal ddY/DAO⁺ mice.d-Amino-acid oxidase treatment of urinary amino acids decreased the serine and proline, suggesting that they containedd-isomers. An HPLC analysis confirmed the presence ofd-serine. Urinary serine and proline contents were not decreased when the ddY/DAO^– mice were fed a diet which did not contain supplementaryd-methionine or when they were given water containing antibiotics. These results suggest that thed-serine andd-proline do not derive from thed-methionine supplemented in the diet or from intestinal bacteria. In urine of the ddY/DAO^– mice, a substance which seemed to bed-methionine sulfoxide and/ord-methionine sulfone was present. It is probably a metabolite of thed-methionine supplemented in the diet. Thed-aminoaciduria in the mutant mice lackingd-amino-acid oxidase activity indicates that this enzyme is involved in the metabolism of thed-amino acids in normal mice.     

D-Amino acids in protein de novo design. II. Protein-diastereomerism versus protein-enantiomerism

Summary With a few exceptions, proteins in our biosphere are based exclusively onl-amino acids. The inversion of configuration of all the stereogenic centers in a protein leads to anall-d compound with ‘mirror image’ properties and ‘mirror image’ structure. We propose to use the termprotein-enantiomerism to describe the relationship between two proteins that have the same sequence but whose amino acids have opposite configuration. We will use the termprotein-diastereomerism to define the relationship between two proteins that have the same sequence in which some amino acids have opposite configurations. A classification of type I, II, III, and IV protein-diastereomerism is proposed. By extension, a diastereoprotein is a protein where some amino acids have the same configuration (l ord) while others have the opposite one (d orl). A particular case of diastereoproteins aremesoproteins, also analyzed in this article. In addition to the goal of making proteins resistant to protease degradation, the use ofd-amino acids in protein de novo design may give rise to proteins with structures, and perhaps properties, very different to those of nativeall-l-proteins.     

Colloidal and dissolved organic matter in lake water: Carbohydrate and amino acid composition,and ability to support bacterial growth

Bacterial utilization of dissolved organic matter (DOM) was studied in water from a humic and a clearwater oligotrophic lake. Indigenous bacteria were inoculated into either 0.2 m natural filtered lake water, or lake water enriched fivefold with colloidal DOM >100 kD but below 0.2 m. Consumption of DOM was followed from changes in concentrations of total dissolved organic carbon (DOC), dissolved combined and free carbohydrates and amino acids (DCCHO and DFCHO, and DCAA and DFAA, respectively) and by uptake of monosaccharide and amino acid radioisotopes. DCCHO and DCAA made up 8% (humic lake) to 33–44% (clear-water lake) of the natural DOC pools, while DFCHO and DFAA contributed at most 1.7% to the DOC pools. Addition of >100 kD DOM increased the DOC concentrations by 50% (clearwater lake) to 92% (humic lake), but it only resulted in a higher bacterial production (by 63%) in the humic lake. During the incubations 13 to 37% of the DOC was assimilated by the bacteria, at estimated growth efficiencies of 4–8%. Despite the measured reduction of DOC, statistically significant changes of specific organic compounds, especially of DCCHO and DCAA, generally did not occur. Probably the presence of high molecular weight DOC interfered with the applied analytical procedures. Addition of radiotracers indicated, however, that DFAA sustained 17–58% and 29–100% of the bacterial carbon and nitrogen requirements, respectively, and that glucose met 1–3% of the bacterial carbon requirements. Thus, our experiments indicate that radiotracers, rather than measurements of concentration changes, should be used in studies of bacterial utilization of DOC in freshwaters with a high content of humic or high molecular weight organic matter.     

Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids

Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603^T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603^T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.     

Regulation of bacterial proteolytic activity at natural concentrations in dissolved organic matter in a maritime pond]

The regulation of the bacterial exoproteolytic activity, at natural substrate concentrations, was studied during the survey of an Atlantic coastal marine pond (France). The regulation of this activity occurs at two different levels: on the one hand, at the cellular level, the ectoenzyme synthesis is regulated by hydrolysis substrates, dissolved combined amino acids (DCAA), and end products, dissolved free amino acids (DFAA), in terms of the relative amounts available to the cell, and on the other hand, at the ecosystem level, i.e. the hydrolytic activity, by the total amounts of DCAA and DFAA in situ. The DFAA acts as an inhibitor in enzymatic synthesis; in contrast, dissolved proteins induce the enzymatic synthesis and the exoproteolytic activity. These results, obtained in natural concentration conditions, confirm the functioning in situ of the ectoenzymatic activity regulation model of Chróst, until now only validated in an enriched experimental medium.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

D-amino acid N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free D-amino acids

d-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on d-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyltransferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of d-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except l-lysine and d-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of d-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.     

Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.     

Contribution of Bacterial Cell Wall Components to DOM in Alkaline,Hypersaline Mono Lake,California

Soda lakes are often characterized by high densities of prokaryotes and high concentrations of dissolved organic carbon. Since bacterial cell walls are less degradable than most other cell constituents, accumulation of cell wall material may occur in these lakes and contribute to the DOM pool, but composition of DOM in soda lakes has rarely been examined. Here we report concentrations of DOM components likely originating from bacterial cell walls, including D amino acids, glucosamine (GluA) and muramic acid (MurA), in depth profiles of stratified, alkaline, hypersaline Mono Lake, CA. Concentrations of cell wall components were related to total pools of dissolved free and combined amino acids (DFAA and DCAA), and bacterial density and production. In the free pool, total DFAA ranged from 50 to 3250 nM and typically increased with depth, while GluA (5 to 140 nM) and MurA (< 0.5 nM and only detected in 2005) fluctuated with depth. In the combined pool, DCAA varied between 5000 and 15000 nM and did not show clear depth-related trends. GluA ranged from 1000 to 5000 nM and tended to increase in the hypolimnion, while MurA varied between 25 and 75 nM. Free D isomers in the DFAA pool either made up < 13% (Asp and Ser) or varied from 10 to 57% (Glu and Ala). In the combined pool, D isomers of Asp, Glu, Ser and Ala made up 24-48% of these DCAA and typically showed minor changes with depth. In 2005, lysozyme activity had highest rates in the surface and correlated negatively with most D isomers among the combined amino acids. Our observations demonstrate that the pool of dissolved combined amino compounds in the lake was about 5-fold higher than in other eutrophic lakes and that a substantial portion of these amino compounds originated from bacterial cell walls.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

The VANA glycopeptide resistance protein is related to d-alanyl-d-alanine ligase cell wall biosynthesis enzymes

Summary Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 by DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37400. VANA was structurally related to the d-alanyl-d-alanine (d-ala-d-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive d-ala-d-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

l-lactate or pyruvate stimulated growth of novikoff rat hepatoma cells

Summary Novikoff rat hepatoma cells (subline N1S1-67) grew when 30mm l-lactate or pyruvate was substituted ford-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lackingl-lactate, pyruvate ord-glucose, cell growth of 0.42 generation was obtained. Growth withl-lactate was dependent on thel-lactate concentration up to 30mm at which the greatest increase in cell number occurred. Significant growth did not occur whend-lactate, glycerol, acetate, α-ketoglutarate, succinate or malate, each at 30mm, was substituted ford-glucose. Growth in the medium containingl-lactate was not due to the utilization ofd-glucose or some other substrate carried into the culture with the inoculum. Medium contamination byd-glucose was insufficient to explain the growth obtained in the medium containingl-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration ofl-lactate in the medium remained unchanged. The increase in cell number cannot be explained byl-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoproteins in the medium.l-Lactate does not serve as a significant carbon or energy source in the growth of these cells. This investigation was supported by grants from the National Institute of Allergy and Infectious Disease, the National Science Foundation, and the United States Public Health Service.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Studies of aluminum in rat brain

The effects of high aluminum concentrations in rat brain were studied using¹⁴C autoradiography to measure the uptake of [¹⁴C]2deoxy-d-glucose ([¹⁴C]2dG) and microbeam proteon-induced X-ray emission (microPIXE) with a 20-μm resolution to measure concentrations of magnesium, aluminum, potassium, and calcium. The aluminum was introduced intracisternally in the form of aluminum tartrate (Al-T), and control animals were given sodium tartrate (Na-T). The¹⁴C was administered intravenously. The animals receiving Al-T developed seizure disorders and had pathological changes, which included cerebral cortical atrophy. The results showed that there was a decreased uptake of [¹⁴C]2dG in cortical regions in which increased aluminum levels were measured, i.e., there was a correlation between the aluminum in the rat brain and decreased brain glucose metabolism. A minimum detection limit of about 16 ppm (mass fraction) or 3×10⁹ Al atoms was obtained for Al under the conditions employed.