- Volume 142, Issue 1, 1996
Volume 142, Issue 1, 1996
- Physiology And Growth
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Catabolite repression in the hyperthermophilic bacterium Thermotoga neapolitana is independent of cAMP
More LessThermotoga neapolitana is a hyperthermophilic bacterium whose phylogenetic lineage includes the most primitive of the bacterial heterotrophs. It is not known whether Thermotoga exhibits preferences for growth substrates or regulates the synthesis of degradative enzymes. We have found that T. neapolitana exhibits diauxic growth in medium containing 300 μM glucose and 1 mM lactose. We measured the activity of β-galactosidase and β-glucosidase in extracts prepared from cells grown on defined media and found that cells grown on 0.5% lactose, galactose or cellobiose contained β-galactosidase specific activities of 1.19, 1.78 and 1.34 U (mg protein)-1, respectively. Cells grown on 0.5% glucose, maltose, fructose, sucrose, xylose, ribose or starch had no measurable β-galactosidase activity. β-Glucosidase activity was found only in cells grown on cellobiose. Cells grown on the combination of 0.5% lactose or galactose and 0.05% glucose had no detectable β-galactosidase activity, whereas up to 0.5% glucose did not prevent expression of β-galactosidase or β-glucosidase activity in cells induced with 0.5% cellobiose. These activities are catalysed by separate enzymes as determined by resolution of their activities on 6% native polyacrylamide gels. Therefore, only β-galactosidase synthesis induced by lactose is subject to catabolite repression. To determine the mechanism of catabolite repression, the levels of cAMP were measured in T. neapolitana cells grown on various defined media using an enzyme-immunoassay. The cAMP levels ranged from 44 to 280 fmol (mg protein)-1 irrespective of the carbon source used. By comparison, Escherichia coli grown on lactose contained 5.1 pmol (mg protein)-1. Like Gram-positive bacteria, T. neapolitana displays a cAMP-independent mechanism for catabolite repression and this may represent the more ancient mode of regulation.
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Nitrile hydrolysis by Rhodococcus erythropolis BL1, an acetonitrile-tolerant strain isolated from a marine sediment
More LessA number of physiologically different nitrile-hydrolysing bacteria were isolated from coastal marine sediments in Denmark by enrichment culture. One strain, BL1, identified as Rhodococcus erythropolis, grew on acetonitrile as sole carbon and nitrogen source in a defined medium. Growth occurred between 0 and 8% NaCl with an optimum around 2%, thus reflecting the marine origin of the isolate. Intact cells of R. erythropolis BL1 could hydrolyse a large variety of saturated and unsaturated aliphatic nitriles to their corresponding acids. Benzonitrile and benzylcyanide were not hydrolysed, whereas some aromatic compounds containing a -CN group attached to a C3 or C4 aliphatic side chain were accepted as substrates. The substrate spectrum of R. erythropolis BL1 was thus markedly different from those of other Grampositive nitrile-hydrolysing bacteria isolated from non-marine environments. Nitrile hydrolysis during growth and in resting cell suspensions usually occurred without intermediate accumulation of amide outside the cells. Detailed studies, however, showed that nitrile hydrolysis by strain BL1 was due to a nitrile hydratase/amidase enzyme system. Nitrile hydratase activity was found to be inducible whereas amidase activity was constitutive. The amidase activity of cells could, however, be enhanced manyfold by growth in media containing acetamide or acetonitrile. In most cases amides were hydrolysed at a much higher rate than the corresponding nitriles, which explained why amides were rarely detected in the surrounding medium during nitrile hydrolysis. R. erythropolis BL1 exhibited the highest tolerance towards acetonitrile ever reported for a nitrile-hydrolysing bacterium, as demonstrated by its ability to grow exponentially in the presence of 900 mM acetonitrile.
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Physiological responses of Pseudomonas putida KT2442 to phosphate starvation
More LessThe physiological responses of Pseudomonas putida KT2442 to phosphate starvation were examined with respect to cell morphology, qualitative demonstration of the accumulation of the intracellular storage component poly-3-hydroxyalkanoate (PHA), cellular ATP and ribosome content, and the rate of total protein synthesis. Upon prolonged incubation under phosphate-limiting conditions, the number of viable cells decreased by two to three orders of magnitude during the first 3 weeks. However, after this decline, viability of the cultures remained remarkably constant for many weeks. The cells remained rod-shaped under phosphate starvation conditions with a tendency to swell in parallel with the accumulation of PHA. Protein synthesis and ribosome concentration were gradually reduced, and ATP levels dropped to very low values after the onset of starvation; later, however, there was a return to near-normal ATP concentrations. Evidence was obtained that the strong selective pressure imposed by phosphate deprivation forces the selection of mutants with a competitive advantage. These mutants are able to grow, possibly utilizing nutrients derived from dead cells, and eventually take over the cultures. One frequently encountered mutant formed smaller colonies on rich solidified medium and displayed an altered cell morphology. This mutant was isolated and further characterized. By employing a bioluminescence-based marker system, we demonstrated that this mutant is able to replace wild-type cells in mixed culture experiments. Thus, long-term phosphate-deprived cultures represent dynamic regimes that can undergo population shifts.
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A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae
More LessRecombinant Saccharomyces cerevisiae harbouring the xylose reductase (XR) gene XYL1 from Pichia stipitis was grown in anoxic chemostat culture at two different dilution rates. At each dilution rate a transient experiment, encompassing a shift in the sugar content of the medium from glucose to glucose plus xylose was performed. The steady states at the beginning and the end of the transients were compared in terms of specific product fluxes from glucose metabolism. At both dilution rates, the specific glycerol flux decreased and the specific acetate and CO2 fluxes increased. The specific ethanol flux was not affected. At the lower dilution rate, the production of biomass decreased during the transient, but at the higher dilution rate it increased. The changes in product pattern can be explained as being due to the redox perturbation caused by the consumption of reduced cofactors in the XR-catalysed reaction. Regeneration of NAD partly through xylose reduction instead of glycerol production decreased the formation of glycerol. Additionally, xylose reduction activated those pathways which produce reduced cofactors, such as acetate formation and the pentose phosphate pathway, indicated by increased acetate and CO2 production. The dual cofactor specificity of XR, with a preference for NADPH over NADH, was evident from the effects of xylose reduction on product fluxes. Comparison of the xylose reduction rates at low and high glucose flux indicated that the supply of reduced cofactors partly controlled the reaction rate. At the higher dilution rate, control by some other factor such as xylose transport or XR activity increased. Calculation of carbon balances at the steady states showed that all substrate carbon was recovered in biomass or products. Based on the specific product fluxes, calculations of quantitative cofactor balances at the steady states was attempted. However, sensitivity calculations showed that analysis errors in the range of 5% caused substantial errors in the cofactor balance, without affecting the carbon balance.
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- Genome Analysis
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Molecular analysis of an operon in Bacillus subtilis encoding a novel ABC transporter with a role in exoprotein production, sporulation and competence
More LessThe levels of exoamylase and other exoenzymes of Bacillus subtilis are pleiotropically decreased by the ecs-26 (prs-26) and ecs-13 (prs-13) mutations. These mutations also cause a competence- and sporulation-deficient phenotype. In the present work, the ecs locus, which has been defined by the ecs-26 and ecs-13 mutations, was cloned and sequenced. Sequence analysis revealed a putative operon of three ORFs (ecsA, ecsB and ecsC). ecsA can encode a putative polypeptide of 248 amino acid residues containing an ATP-binding site. The polypeptide shows about 30% sequence similarity with the ATP-binding components of numerous membrane transporters of the ABC-type (ATP-binding cassette transporters or traffic ATPases). The ecs-26 mutation was found to result from a transition of one base pair changing the glycine164 of EcsA to a glutamic acid residue in the vicinity of the putative ATP-binding pocket. ecsB was predicted to encode a hydrophobic protein with six membrane-spanning helices in a pattern found in other hydrophobic components of ABC transporters. The properties deduced for the ecsA and ecsB gene products are consistent with the interpretation that ecs encodes a novel ABC-type membrane transporter of B. subtilis. The third ORF, ecsC, can encode a putative polypeptide of 237 amino acid residues. The polypeptide does not resemble components of ABC transporters.
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Physical mapping of 32 genetic markers on the Pseudomonas aeruginosa PAO1 chromosome
The Pseudomonas aeruginosa chromosome was fractionated with the enzymes Spel and Dpnl, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis.
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