Involvement of a chlorobenzoate-catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem.

A chlorobenzoate-catabolic transposon (Tn5271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp. strain BR60, into lake water and sediment flowthrough microcosms. Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl. The populations of the host, BR60, and organisms carrying Tn5271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method. Populations of Tn5271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems. The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration. Plasmid transfer in the 3-chlorobenzoate- and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp. strain BR60. Bacteria dominating 4-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets Tn5271, on a plasmid unrelated to pBRC60. The importance of plasmid transfer and transposition during chemical adaptation is discussed.     

Transfer and Expression of the Catabolic Plasmid pBRC60 in Wild Bacterial Recipients in a Freshwater Ecosystem

3-Chlorobenzoate (3Cba)-degrading bacteria were isolated from the waters and sediments of flowthrough mesocosms dosed with various concentrations of 3Cba and inoculated with a 3Cba-degrading Alcaligenes sp., strain BR60. Bacteria capable of 3Cba degradation which were distinct from BR60 were isolated. They carried pBRC60, a plasmid introduced with Alcaligenes sp. strain BR60 that carries a transposable element (Tn5271) encoding 3Cba degradation. The isolates expressed these genes in different ways. The majority of pBRC60 recipients were motile, yellow-pigmented, gram-negative rods related to the group III pseudomonads and to BR60 by substrate utilization pattern. They were capable of complete 3Cba degradation at both millimolar and micromolar concentrations. Two isolates, Pseudomonas fluorescens PR24B(pBRC60) and Pseudomonas sp. strain PR120(pBRC60), are more distantly related to BR60 and both produced chlorocatechol when exposed to 3Cba at millimolar concentrations in the presence of yeast extract. These species showed poor growth in liquid 3Cba minimal medium but could degrade 3Cba in continuous cultures dosed with micromolar levels of the chemical. Laboratory matings confirm that pBRC60 can transfer from BR60 to species in both the beta and gamma subgroups of the proteobacteria and that 3Cba gene expression is variable between species. Selection pressures acting on pBRC60 recipients are discussed.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection     

Chlorobenzoate catabolism and interactions between Alcaligenes and Pseudomonas species from Bloody Run Creek

A mixed community of bacteria from surface runoff waters of the Hyde Park industrial landfill was enriched on 3-chlorobenzoate. Alcaligenes and Pseudomonas species were dominant in the community. Alcaligenes sp. BR60 carried an unstable plasmid specifying 3-chlorobenzoate catabolism. Metabolites detected in culture supernatants included chlorocatechol and chloro-cis,cis-muconic acid. Oxygen uptake in the presence of 3- and 4-substituted methyl-catechols revealed a catechol-1,2-oxygenase activity specific for substituted catechols with very limited activity for catechol. The isolate grew very slowly on benzoate. Alcaligenes sp. BR60 was isolated in co-culture with Pseudomonas fluorescens NR52. The latter contained no detectable plasmids and did not grow on benzoate or any of the chlorobenzoates in pure culture. Growth of the co-culture in Bloody Run Creek water supplemented with 3-chlorobenzoate indicated that phosphate concentrations in the water severely limited biodegradation. Under phosphate limited conditions in continuous culture, Pseudomonas fluorescens NR52 effectively scavenged available phosphate when it was present at a ratio of 1 cell to 20 of Alcaligenes sp. BR60. Under these conditions the growth of Alcaligenes sp. BR60 on 3-chlorobenzoate was reduced 5 fold, the frequency of plasmid deletion mutants increased, and 96% of the contaminant remained in the outflow in the form of the starting material or metabolites. No evidence was found for conjugation of the plasmid determining chlorobenzoate catabolism in Alcaligenes sp. BR60 to P. fluorescens NR52.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - Ba benzoic acid     

Cloning and expression of the transposable chlorobenzoate-3,4-dioxygenase genes of Alcaligenes sp. strain BR60.

Growth on 3-chlorobenzoate was found to induce the enzymes of the protocatechuate meta ring fission pathway in Alcaligenes sp. strain BR60. The chlorobenzoate catabolic genes, designated cba, were localized to a 3.7-kb NotI-EcoRI fragment within the nonrepeated region of the composite transposon Tn5271. The cba genes were cloned onto two broad-host-range vectors and expressed in Escherichia coli and Alcaligenes sp. strain BR6024. In E. coli, expression of the cba genes with the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible tac promoter of the IncQ vector pMMB66HE resulted in the production of protocatechuate and chlorodihydroxybenzoate metabolites of 3-chlorobenzoate. Expression of this construct in one orientation resulted in the formation of two polypeptides 51 and 42 kDa in size. This result was confirmed by subcloning into pGEM3Zf and then incorporating L-35S-methionine into newly synthesized proteins, using the thermally regulated T7 polymerase-promoter system. Introduction of the NotI-EcoRI fragment into Alcaligenes sp. strain BR6024 (Cba-P), using the IncP broad-host-range, mobilizable plasmid pBW13, restored the 3-chlorobenzoate-degradative phenotype and resulted in the accumulation of protocatechuate and chlorodihydroxybenzoate intermediates. The data indicate that a two-component dioxygenase specified by Tn5271 oxidizes 3-chlorobenzoate at the 3,4- or 4,5-positions. This activity extends the range of pathways for chloroaromatic compounds known to be functional in the environment. The new pathway avoids the toxicity attributed to the accumulation of chlorocatechol metabolites in bacteria degrading chlorobenzoates.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Evidence for Aceticlastic Methanogenesis in the Presence of Sulfate in a Gas Condensate-Contaminated Aquifer

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-¹⁴C]acetate or [U-¹⁴C]acetate produced higher quantities of CH₄ compared to CO₂ in the presence or absence of sulfate.¹⁴CH₄ accounted for 70 to 100% of the total labeled gas in the [¹⁴C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.     

Experimental study of solute transport and extraction by a single root in soil

The fate of ¹⁴C-2,4,6-trinitrotoluene ([U-¹⁴C]TNT) in soil/plant systems was studied using onion (Allium cepa L.) plants with only a single root. It was found that the single roots grew exponentially and that the rate of water uptake of the onion plants increased exponentially, as well. The concentration of [U-¹⁴C] in the roots at first increased and then appeared to reach a steady state, while the [U-¹⁴C] concentration in the leaves was found to increase linearly with time. The [U-¹⁴C] concentration in the rhizosphere increased gradually, while in the bulk soil it decreased slowly. The accumulation of [U-¹⁴C] in the rhizosphere is likely to difference between movement into the rhizosphere (through advective mass flow of soil water by root uptake) and its uptake into the roots. The distribution of ¹⁴C in the soil/plant system was found to be 60–85% in the soil solid phase, 7–11% in the soil liquid phase, <1% in the soil air phase, <1% in the root compartment, and <0.01% in the leaf compartment. The maximum RCF (root concentration factor) value for TNT and its derivates was found to be about 20, and the maximum TSCF (transpiration stream concentration factor) was 0.18. These values can be changed by a variety of factors in soil-plant systems     

Sucrose suppression of chlorophyll synthesis in carrot tissue cultures: The role of invertase

Summary Free space invertase activities were determined in carrot callus strains CRT1 and CRT2 grown under conditions in which sucrose suppression of chlorophyll synthesis occurred in CRT1 but not CRT2. CRT2 possessed a high free space acid invertase activity (pH optimum 5.0 K_m for sucrose 3.1×10^-3M) while CRT1 lacked this enzyme. [U-¹⁴C] sucrose introduced into the free space of calluses was rapidly inverted by CRT2, but not by CRT1.Despite their different invertase levels, CRT1 and CRT2 showed similar sucrose uptake rates and took up [U-¹⁴C-glucosyl] sucrose and [5-T-glucosyl] sucrose from external bathing media essentially without prior inversion.It is concluded that acid invertase in callus tissue relieves the suppression of chlorophyll synthesis caused by sucrose in the free space. The invertase may in some circumstances hydrolyse sucrose before uptake, but is not an essential part of the sucrose uptake mechanism in carrot tissue cultures.     

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1000 g 3-chlorobenzoate g^-1 dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraderswere enumerated on minimal salts media containing 3-chlorobenzoate. Culturableheterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1000 g 3-chlorobenzoate g^-1 dry soil inoculated microcosms, degradersincreased from the initial inoculum and decreased following degradation of 3-CB.Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 × 10⁷ colony-forming-units g^-1 dry soil in the 500 g 3-chlorobenzoate g^-1 dry soil microcosms, but none were detected in the 1000 g 3-chlorobenzoate g^-1 dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species.In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1000 g 3-chlorobenzoate g^-1 dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.     

The phylogenetic distribution of a transposable dioxygenase from the Niagara River watershed

Horizontal gene transfer in the Bacteria has been demonstrated to occur under natural conditions. The ecological impact of gene transfer events depends on the new genetic material being expressed in recipient organisms, and on natural selection processes operating on these recipients. The phylogenetic distribution of cbaAB genes for chlorobenzoate 3,4-(4,5)-dioxygenase, which are carried within Tn5271 on the IncPβ plas-mid pBRC60, was investigated using isolates from freshwater microcosms and from the Niagara River watershed. The latter included isolates from surface water, groundwater and bioremediation reactor samples. The cbaAB genes have become integrated, through interspecific transfer, primarily into species of the β Proteobacteria (44/48 isolates). Only four isolates, identified as Pseudomonas fluorescens (3/48) and Xanthomonas maltophilia (1/48), belonged to the Γ Proteobacteria, despite the observation that pBRC60 was capable of mobilizing these genes into a wide range of β and Γ Proteobacteria in the laboratory. The natural host range correlated with the distribution of the meta-ring-fission pathway for metabolism of protocatechuates formed when the cbaAB genes were expressed (45/48 isolates). We proposed the hypothesis that natural selection has favoured recipients that successfully integrate the activity of the transferred dioxygenase with the conserved meta ring-fission pathway. The hypothesis was tested by transferring a plasmid construct containing the cbaAB genes into type strains representative of the β and γ Proteobacteria. The concept of applying mobile catabolic genes to probe the phylogenetic distribution of compatible degradative pathways is discussed.     

Molecular- and cultivation-based analyses of microbial communities in oil field water and in microcosms amended with nitrate to control H2S production

Nitrate injection into oil fields is an alternative to biocide addition for controlling sulfide production (‘souring’) caused by sulfate-reducing bacteria (SRB). This study examined the suitability of several cultivation-dependent and cultivation-independent methods to assess potential microbial activities (sulfidogenesis and nitrate reduction) and the impact of nitrate amendment on oil field microbiota. Microcosms containing produced waters from two Western Canadian oil fields exhibited sulfidogenesis that was inhibited by nitrate amendment. Most probable number (MPN) and fluorescent in situ hybridization (FISH) analyses of uncultivated produced waters showed low cell numbers (≤10³ MPN/ml) dominated by SRB (>95% relative abundance). MPN analysis also detected nitrate-reducing sulfide-oxidizing bacteria (NRSOB) and heterotrophic nitrate-reducing bacteria (HNRB) at numbers too low to be detected by FISH or denaturing gradient gel electrophoresis (DGGE). In microcosms containing produced water fortified with sulfate, near-stoichiometric concentrations of sulfide were produced. FISH analyses of the microcosms after 55 days of incubation revealed that Gammaproteobacteria increased from undetectable levels to 5–20% abundance, resulting in a decreased proportion of Deltaproteobacteria (50–60% abundance). DGGE analysis confirmed the presence of Delta- and Gammaproteobacteria and also detected Bacteroidetes. When sulfate-fortified produced waters were amended with nitrate, sulfidogenesis was inhibited and Deltaproteobacteria decreased to levels undetectable by FISH, with a concomitant increase in Gammaproteobacteria from below detection to 50–60% abundance. DGGE analysis of these microcosms yielded sequences of Gamma- and Epsilonproteobacteria related to presumptive HNRB and NRSOB (Halomonas, Marinobacterium, Marinobacter, Pseudomonas and Arcobacter), thus supporting chemical data indicating that nitrate-reducing bacteria out-compete SRB when nitrate is added.     

The use of 14C-labeled bacteria as a tracer of ingestion and metabolism of bacterial biomass by microbial grazers

A method for radiolabeling marine bacteria with d-[U-¹⁴C] glucose and a radiotracer method for measuring ingestion and metabolism of bacterial biomass by ciliated protozoa and other microzooplankton are presented. Problems associated with using live bacterial tracers, i.e., label retention, label recycling, tracer cell size and morphology, and intracellular distribution of label are evaluated.Bacterioplankton assemblages collected from field samples incorporated and retained label as efficiently as coastal isolates which were selected for glucose incorporation. Under grazing experimental conditions, labeled bacteria retained a high proportion of the label (hourly net loss = 1.2%). Bacterial recycling of released dissolved organic ¹⁴C (DO¹⁴C) was 0–2.2% of total ¹⁴C per h. Addition of labeled assemblages to nearshore water samples did not detectably alter mean cell size or size frequency distribution of the attendant bacterioplankton assemblages.In grazing experiments with cultured ciliates (Euplotes sp. and Uronema sp.), radioassay parameters varied as direct functions of predator and prey concentrations. Microautoradiographic analysis corroborated that ¹⁴C incorporation measured in the radioassay by filtration techniques primarily represented ingested bacterial biomass and that problems associated with attached and adsorbed labeled bacteria were minimized. Grazing experiments conducted with bacteria labeled with [U-¹⁴C]glucose yielded ingestion rates comparable to bacteria labeled with [U-¹⁴C]thymidine and additionally provided respiration and exudation rates.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Characterization of [14C]apiogalacturonans synthesized in a cell-free system from Lemna minor.

Radioactive polysaccharide was synthesized when uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (containing some uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate)) was incubated with a particulate enzyme preparation from Lemna minor. Characterization experiments established that the product: (i) was insoluble in methanol and water, (ii) contained d-[U-¹⁴C]apiose (75%) and d-[U-¹⁴C]xylose (25%), and (iii) was soluble in 1% ammonium oxalate. The material solubilized by ammonium oxalate (solubilized product): (i) was separated into five fractions by column chromatography with diethylaminoethyl-Sephadex (DEAE-Sephadex), (ii) contained [U-¹⁴C]apiobiose side chains that were removed by hydrolysis at pH 4, and (iii) was degraded by fungal pectinase. Both d-[U-¹⁴C]apiose residues of the [U-¹⁴C]apiobiose side chains were synthesized in vivo since radioactivity was distributed equally between the two residues. The presence of uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) during synthesis of radioactive polysaccharide resulted in: (i) an increase in the incorporation of radioactive d-[U-¹⁴C]apiose into solubilized product, (ii) an increase in the ratio of d-[U-¹⁴C]apiose to d-[U-¹⁴C]xylose present in solubilized product, (iii) an increase in the amount of [U-¹⁴C]apiobiose plus d-[U-¹⁴C]apiose released from the solubilized product by hydrolysis at pH 4, and (iv) a tighter binding of the solubilized product to DEAE-Sephadex. These results show that apiogalacturonans similar to or the same as those synthesized by the intact plant were synthesized in the particulate enzyme preparation isolated from L. minor. [¹⁴C]Apiogalacturonans completely free of d-[U-^l4C]xylose were not isolated. The [¹⁴C]apiogalacturonan with the least d-[U-¹⁴C]xylose still had 4.8% of its radioactivity present in d-[U-¹⁴C]xylose. The possibility remains that d-xylose is a normal constituent of the apiogalacturonans of the cell wall of L. minor.     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

Production Rate of Planktonic Bacteria in the North Basin of Lake Biwa, Japan

Vertical and seasonal variations in the cell number and production rate of planktonic bacteria were investigated at a pelagic site (water depth, ca. 72 m) of the north basin of Lake Biwa during April to October 1986. The [methyl-³H]thymidine uptake rate into a cold trichloroacetic acid-insoluble fraction and the frequency of dividing cells (FDCs) were measured for each sample as indices of the bacterial production rate. The seasonal data of bacterial number, thymidine uptake rate, and bacterial growth rate based on the FDCs were correlated with one another (rank correlation analysis, P < 0.05). These bacterial variables were not correlated positively with the chlorophyll a concentration. Vertically, the maxima of both bacterial number and the thymidine uptake rate were found in the euphotic zone. The direct counting of bacteria and the measurements of thymidine uptake rate combined with the size-fractionation method revealed that more than 90% of the bacterial biomass and production rate were attributed to unattached bacteria throughout the investigation period. The carbon flux estimates of bacterial production were less certain due to the variability of the conversion factor for the thymidine uptake method and that of the calibration for the FDC method, but even when the conservative range of bacterial net production rate was used (5 to 60 μg of carbon per liter per day), it can be suggested that bacterial net production in the investigated area was a significant fraction (ca. 30%) of the level of the primary production rate in the same water basin.