Early Stationary Growth Phase Research Articles

Cyclomorphosis in Scenedesmus subspicatus (Chlorococcales, Chlorophyta): stimulation of colony development at low temperature

The UTEX 2532 strain of Scenedesmus subspicatus R. Chodat was grown in batch culture at 10°C and 22°C. At 22°C only unicells developed. At the lower incubation temperature there was stimulation of colony production, with up to 67% 4-celled or 8-celled colonies. Colonies developed during the early stationary phase of growth. With the expression of both cold-water and warm-water ecomorphs, this ordered sequence of phenotypic plasticity can be termed a cyclomorphosis.Development of the complete range of phenotypic plasticity in certain species, and thus accurate identification of a laboratory culture such as S. subspicatus used in the present study, can be achieved only by use of a broader range of standard conditions of growth. At present, identification of preserved field samples from one site at different seasons is even more difficult.

The effect of arsenicals on alkaloid production by cell suspension cultures of Catharanthus roseus

AbstractThe effect of arsenic compounds on indole alkaloid production by cell suspension cultures of Catharanthus roseus was investigated. The analysis of indole alkaloids was achieved by using thermospray liquid chromatography‐mass spectrometry (LC MS) which facilitated the rapid screening of alkaloid composition in cultures treated with different arsenicals at different times in their growth cycle.Treatment with dimethylarsinate (DMA), a non‐selective herbicide, has a drastic inhibitory effect on alkaloid production although it is the least toxic arsenical to growth. Tryptamine, an early precursor in the biosynthesis of indole alkaloids, accumulates in cells treated with DMA, indicating that the initial step of condensation of tryptamine with secologanin is inhibited. Treatment with DMA during the early stationary phase of culture growth enhances the accumulation of some alkaloids, although some, such as catharanthine, are suppressed.The arsenicals arsenate and methylarsonate (MMA) have an inhibitory effect on alkaloid production when applied during the early growth stages. In contrast to MMA and DMA, arsenate has a stimulatory effect on catharanthine production when introduced to the culture during its early stationary phase.Thus the changes in the pattern of alkaloid accumulation on addition of arsenicals are dependent on the arsenic species and its concentration, as well as the time of application. This variable response indicates that each arsenical has a distinct mode of action on the secondary metabolic pathways of C. roseus.

Isoenzymes of phosphogluconate dehydrogenase (decarboxylating) from suspension-cultured Catharanthus roseus cells

Two isoenzymes of phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) were found in suspensioncultured Catharanthus roseus cells. They are located in the cytosol and the plastids (amyloplasts). These two isoenzymes were separated by fractionation on an anion-exchange column by HPLC. The two isoenzymes had similar kinetic properties and the same effectors. The K m values for 6-phosphogluconate were 15 and 18 μM and K m values for NADP + were 8 and 11 μM for the cytosolic and plastid isoenzymes, respectively. NADPH was a potent inhibitor of both isoenzymes. ATP and 5-phosphoribosyl-1-pyrophosphate also inhibited the activities of these isoenzymes. The maximum level of PGD was observed in cells at the lag phase of growth. The activity of cytosolic PGD relative to that of the plastid PGD was also high in these cells. In contrast, the activity of the plastid isoenzyme of PGD was ca two-fold higher than that of the cytosolic isoenzyme in the cells at the early stationary phase of growth. The role and regulation of these isoenzymes of PGD are discussed.

Production and regulation of potato-scab-inducing phytotoxins by Streptomyces scabies

Summary: Phytotoxins with potato-scab-inducing activity were produced by the pathogenic Streptomyces scabies strain RB2 when grown on oatmeal agar medium or in oatmeal broth medium. Five compounds isolated from cell filtrates were identified as thaxtomin compounds 1-5, previously reported as produced by S. scabies grown on potato slices. The optimum temperature for phytotoxin production in oatmeal broth medium was 28 °C. Production of thaxtomin A, the major product, was repressed at least 130-fold when S. scabies RB2 was grown in oatmeal broth medium supplemented with 0.5% glucose. Thaxtomin A production was also repressed by tryptophan and tyrosine, precursors of which may be involved in feedback inhibition of early steps in biosynthesis. Phytotoxins were secreted by the organism when the cells reached late exponential to early stationary phases of culture growth. The time during growth at which each thaxtomin compound was produced suggests a pathway for the latter steps in thaxtomin biosynthesis.

Inhibition of human neutrophil migration in vitro by low-molecular-mass products of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae commonly causes infections in the lower and upper respiratory tract, although the mechanisms of its colonization and persistence in the airways are unclear. Culture filtrates from six clinical isolates of this bacterium were assessed for their abilities to influence neutrophil function in vitro. Each culture filtrate was assessed on six separate occasions with neutrophils obtained from six different donors. During the log and early stationary phases of growth (0 to 18 h), culture filtrates contained primarily neutrophil chemokinetic activity but no activity affecting neutrophil migration toward the chemotactic factors N-formyl-L-methionyl-L-leucyl-L-phenylalanine and leukotriene B4. In contrast, filtrates obtained after 24 h of culture contained factors which inhibited neutrophil migration toward both of these chemotactic factors. This chemotaxis-inhibitory activity persisted between 24 and 72 h of bacterial culture, and it was not associated with the presence of either chemotactic or chemokinetic activity as assessed by checkerboard analysis. Gel filtration of pooled 72-h filtrates yielded three major peaks of chemotaxis-inhibitory activity. Endotoxin was present together with two other low-molecular-mass hydrophobic factors of approximately 8 and 2 kDa. These low-molecular-mass factors are chloroform insoluble and heat stable, and they are inactivated by protease, periodate, and diborane reduction. Activity was completely retained on a wheat germ agglutinin column, and it could be eluted with N-acetyl-D-glucosamine. These data suggest that inhibitory activity is associated with N-acetyl-D-glucosamine-containing glycopeptides, possibly derived from the bacterial cell wall. The production of these compounds may contribute to the persistence of this bacterium in vivo by inhibiting neutrophil chemotaxis in the microenvironment of the respiratory mucosa.

Induction of antibiotic production by protease in Bacillus brevis (ATCC 8185).

Bacillus brevis (ATCC 8185) produces an antibiotic peptide, linear gramicidin, in the early stationary growth phase. Since we observed that preculture in milk medium is essential for production of the antibiotic in broth medium, we studied the role of the preculture in the antibiotic production. We found that addition of the supernatant of the precultured milk medium was sufficient to induce gramicidin production in broth medium. Fresh milk medium had no effect. The effector substance in the overnight cultured milk medium was labile at both acidic and alkaline pH and was destroyed by heat. We also found that addition of a protease (for example, bovine pancreas chymotrypsin or Streptomyces griseus protease) instead of the supernatant could induce the gramicidin production. Addition of Mn2+ was not required for the protease-induced production of gramicidin. It is known that B. brevis cells secrete protease into milk medium. But neither use of the protease-pretreated broth medium nor addition of casamino acids to broth medium induced gramicidin production. These results suggest that B. brevis cells secrete a factor for linear gramicidin production, that the inducing factor is protease and that the target of the protease is a substance(s) produced by the bacteria.

Light-stimulated 14CO2 uptake and acetylene reduction by bacteriochlorophyll containing stem nodule isolate BTAi 1

Bradyrhizobial strain BTAi 1 nodulates both stems and roots of Aeschynomene spp. Previous work has shown that it contains bacteriochlorophyll a and forms photosynthetic reaction centers, and has provided indirect evidence of photosynthesis by bacteroids within stem nodules. Here we report physiological and biochemical characteristics of BTAi 1 ex planta, which also suggest the presence of photosynthetic activity. Light-stimulated uptake of 14CO2 by BTAi 1 was detected at all stages of growth. Inhibitors of photosynthesis, 1,10-orthophenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and the uncoupler NH4Cl, immediately suppressed light-driven 14CO2 uptake and increased O2 uptake. BTAi 1 is strictly aerobic and was unable to grow without organic C even in the light; also, it was unable to grow chemoautotrophically in an atmosphere enriched with H2 and CO2. In micro-aerobic conditions, strain BTAi 1 expressed acetylene reducing activity ex planta in an N-free medium. The highest rates of light-stimulated 14CO2 uptake and acetylene-reducing activity occurred during the exponential and early stationary phases of growth. Acetylene-reducing rates at a low glucose concentration were increased following a light-dark cycle in comparison with continuous dark conditions.

Characterization of catalase activities in a root-colonizing isolate of Pseudomonas putida

Pseudomonas putida, a saprophytic root-colonizing bacterium, produces multiple forms of catalase. Catalase A, which increases in specific activity during growth phase and after treatment with H2O2, is located in the cytoplasm and is inhibited by 3-amino-1,2,4-triazole, EDTA, and cyanide, but not by chloroform–methanol treatment. Catalase B, which is induced by external H2O2 or during stationary phase of growth, is membrane associated and is inhibited by chloroform–methanol, EDTA, and cyanide, but not by aminotriazole. Catalase A has a broad pH optimum, from pH 6.0 to 11.0, with two peaks, at pH 8.0 and 11.0. Catalase B is most active at pH 5.0–11.0. Mutant J-1, generated by ethyl methanesulfonate mutagenesis, lacked catalase A activity in extracts of cells harvested throughout lag to early stationary growth phase in liquid medium. Catalase B was produced by J-1 in stationary phase. Exposure of J-1 to H2O2 caused the production of both catalase A and catalase B. Mutant J-1 was more susceptible to cell death than the wild type upon direct exposure to 2.5 mM H2O2 but survived this treatment after exposure to lower (0.3 mM), nonlethal doses of H2O2. The ability to adapt to H2O2 may be related to the behaviour of J-1 on roots where active oxygen species are produced by root surface enzymes. J-1 colonized root surfaces at wild-type levels and produced catalases A and B after exposure to root surfaces for 12 h. Key words: Pseudomonas putida, catalase, root colonization.

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo- d-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm −1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.

Factors affecting the leukotoxin activity of Fusobacterium necrophorum

The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43°C), redox potentials (−352 to +375 mV), and iron concentrations (<0.2, 4.2, 42.1, and 361.4 μM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39°C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of −230 to −280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased ( P<0.05) the leukotoxicity of F. necrophorum. Low iron concentration (<0.2 μM) decreased ( P<0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity.

Influence of Cultural Conditions on Cytotoxin Production by Salmonella enteritidis

The ability of Salmonella enteritidis strain 11013 to produce cytotoxic activity against Vero cells was determined under different cultural conditions. The toxin, which was not neutralizable with antiserum to Shiga toxin or Escherichia coli verotoxin-1 or verotoxin-2, was principally cell-associated and was produced primarily during the early stationary phase of growth. Trypticase soy broth was the best of three media evaluated for toxin production. Bacteria produced toxin in the range of pH 4.5 to 8.0 and at 12 to 42°C, with the optimal pH and temperature for toxin production at pH 7.0 and 37°C, respectively. Release of cellular cytotoxin into growth media was induced by growing salmonellae at extremes of pH (4.5 or 8.0) or at high incubation temperature (42°C). The Vero cell CD50 of S. enteritidis lysates of cells grown under optimal conditions was a titer of 150 ± 50 per mg of lysate protein. Although the significance of ingesting preformed Salmonella cytotoxin in human disease is unknown, it can be implied from these results that toxin would not be produced in foods held refrigerated at ≤7°C or acidified at ≤pH 4.0.

Production of Proteases by Psychrotrophic Microorganisms

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-(L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52°C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.

Formate Production by Streptococcus thermophilus Cultures

Formate production of three strains of Streptococcus thermophilus was studied in whole milk and 1.1.1 medium at 37 and 42°C. Results showed that formate production was dependent on strain, culture medium, and temperature. The kinetics of formate production suggest that production takes place during the late exponential and early stationary growth phases both in milk and 1.1.1 medium.

Purification and characterization of the free form of the lactococcal extracellular proteinase and its autoproteolytic cleavage products

In addition to the cell wall proteinase, Lactococcus lactis subsp. cremoris produced significant amounts of a free extracellular proteinase. The free proteinase activity was highest in the late exponential and early stationary phase of growth, whereas the cell wall activity was highest in the last half of the exponential phase. Both proteinase forms had a pH optimum between 4-6 and 5-8, and they behaved similarly upon anion exchange and hydrophobic interaction chromatography, chromatofocusing and gel filtration, indicating that they were related. Purification to homogeneity, as judged by SDS-PAGE, resulted in a 50,000-80,000-fold increase in the specific activity of the free proteinase. It contained two major protein species (termed pro150 and pro115) with proteinase activity. As judged by SDS-PAGE, the Mr values of pro150 and pro115 were 150,000 and 115,000, respectively, and by chromatofocusing the isoelectric points were 4.3 and 4.1, respectively. Upon gel filtration, pro150 and pro115 had Mr values of 300,000 and 125,000, respectively, indicating that pro150 was a dimer and pro115 a monomer. Pro115 was an autodegradation product of pro150. Other distinct autodegradation products had Mr values of 90,000 (p90), 53,000 (p53), 37,000 (p37) and 30,000 (p30). These had little if any proteinase activity. Pro115, p90 and p53 had a common N-terminal sequence with that reported for the cell wall proteinase. Judging from its N-terminal sequence and Mr, p30 was derived from the C-terminal half of p53. Cleavage of pro150 to pro115 generated p37.

Repression of the cell wall protein gene operon in Bacillus brevis 47 by magnesium and calcium ions

Transcription from P2, one of the major promoters of the cell wall protein gene operon of Bacillus brevis 47, was markedly enhanced at the early stationary phase of growth. MgCl2, when added at 1 to 5 mM to the medium, inhibited this enhancement of transcription as well as shedding of the cell wall protein layers from the cell surface. MgSO4 or CaCl2 showed an effect similar to that of MgCl2. The possible coordination of the cell wall structure with regulation of the cell wall protein genes is discussed.

Production of Thermostable Clostridium thermohydrosulfuricum Xylose Isomerase in Bacillus brevis.

The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned into Bacillus brevis. Under control of the strong cell wall protein promoter, the gene was efficiently expressed during the early stationary phase of growth, when cell densities were high. The expressed gene product was a soluble cytoplasmic protein and made up more than 20% of the total cellular protein. A simple heat treatment at 85°C for 10 min gave a virtually pure enzyme. Final isomerase yields were about 0.5 g isomerase per liter culture. The purified isomerase has an optimum temperature at 85°C, and an optimum pH around 7. The isomerase is stable at 85°C for several hours, opening possibilities for new uses.

Expression of the major gas vesicle protein gene in the halophilic archaebacteriumHaloferax mediterranei is modulated by salt

In the moderately to extremely halophilic archaebacterium Haloferax mediterranei gas vacuoles are not observed before the stationary phase of growth, and only when the cells are grown in media containing more than 17% total salt. Under the electron microscope, isolated gas vesicles appear as cylindrical structures with conical ends that reach a maximal length of 1.5 microns; this morphology is different from the spindle-shaped gas vesicles found in the Halobacterium halobium wild type which expresses the plasmid-borne p-vac gene, but resembles that of gas vesicles isolated from H. halobium strains expressing the chromosomal c-vac gene. Both the p-vac and the c-vac genes encode very similar structural proteins accounting for the major part of the "membrane" of the respective gas vesicles. The homologous mc-vac gene was isolated from Hf. mediterranei using the p-vac gene as probe. The mc-vac coding region indicates numerous nucleotide differences compared to the p-vac anc c-vac genes; the encoded protein is, however, almost identical to the c-vac gene product. The start point of the 310 nucleotide mc-vac transcript determined by primer extension analysis and S1 mapping was located 20 bp upstream of the ATG start codon, which is at the same relative position as found for the other two vac mRNAs. During the growth cycle, mc-vac mRNA was detectable in Hf. mediterranei cells grown in 15% as well as 25% total salt, with a maximal level in the early stationary phase of growth. The relative abundance of mc-vac mRNA in cells grown at 25% salt was sevenfold higher than in cells grown in 15% total salt.

Relative effects of nitrogen or phosphorus depletion and light intensity on the pigmentation, chemical composition, and volume of Pyrenomonas salina (Cryptophyceae)

The nitrogen-rich phycobiliproteins are efficient light-harvesting pigments of cryptophytes, cyanobacteria, and rhodophytes. Whereas it is well established that certain cyanobacteria mobilize their phycobiliproteins in response to nitrogen deprivation, the effect of nitrogen stress on cryptophyte physiology has been studied only rarely. We compared the effects of nitrogen and phosphorus depletion on the biovolume, chemical composition, and pigment composition of the marine cryptophyte Pyrenomonas salina, grown in batch cultures under light-limiting and light-saturating conditions. Cellular phycoerythrin content declined during the late exponential growth phase in nitrogen-depleted cultures, and the cells were nearly devoid of phycoerythrin (< 3 % of the phycoerythrin content of nitrogen-replete cells) at the beginning of stationary growth. Phycoerythrin content also decreased during late exponential phase in phosphorus-depleted cultures, but these latter cells contained more than 10 times the phycoerythrin concentration of nitrogen-depleted cells at the same growth stage. Chlorophyll a content declined in nitrogen-depleted cultures, but at a slower rate than the decrease in phycoerythrin, such that the phycoerythrin to chlorophyll a ratio decreased by more than 95 O/O from the early exponential to early stationary growth phase. In nitrogen-depleted cultures grown at a low light intensity, the decline in phycoerythrin content preceded the decline in chlorophyll a content, and resulted in only slight changes in cellular nitrogen content and biovolume. These data indicate that phycoerythrin is preferentially lost from P. salina during nitrogen deficiency, and support the hypothesis that, similar to certain cyanobacteria, P. salina responds to nitrogen deprivation by mobilizing phycoerythrin in order to help sustain cellular nitrogen requirements.

Growth phase modulation of the productivity of α-galactosidase in budding yeast cultures

The complete utilization of raffinose and melibiose by Saccharomyces cerevisiae requires the synthesis and secretion of an α-galactosidase encoded by a single MEL1 gene. The influence of culture conditions on the productivity of α-galactosidase in batch growth of yeast cells has been investigated. A modulation of the MEL1 gene expression has been observed depending on growth condition, with a maximum of enzyme productivity during a transient physiological phase corresponding to the early stationary phase. Our experiments demonstrate that the highest α-galactosidase productivity observed in the early stationary phase of growth is not associated with a starvation condition, but it is coupled to a particular growth phase. The increase of specific activity of the enzyme occurs mainly for periplasmic and intracellular fractions while the amount of enzyme secreted in the medium remains fairly constant.

Expression of two gas vacuole protein genes in Halobacterium halobium and other related species.

The archaebacterium Halobacterium halobium contains two genes encoding gas vacuole proteins (vac). One resides on a large naturally occurring plasmid and encodes a protein of 76 amino acids (p-vac), while the other is a chromosomal gene that encodes a highly similar protein of 79 amino acids (c-vac). Northern analysis determined the c-vac and p-vac mRNA to be approximately 340 nucleotides in length, and S1 mapping of both transcripts indicated that the 5' terminus for each starts at the same relative nucleotide. Three other Halobacterium species producing gas vacuoles were investigated, H. spec. GN101, YC819-9, and SB3. All three contain only a chromosomal c-vac gene, and the 5' terminus of the 340 nucleotide mRNA starts at the same nucleotide as found for H. halobium. The c-vac gene region of H. spec. GN101 contains nine nucleotide exchanges, three of which occur in the coding region with no effect on the amino acid sequence. In contrast, the c-vac gene of H. spec. SB3 has an identical nucleotide sequence to the H. halobium c-vac gene. Gas vacuole production in each of these species was monitored during culture growth by phase contrast microscopy, and the vac mRNA level was determined for each time point. H. halobium p-vac deletion mutants, as well as the halobacterial species GN101 and YC819-9, start to synthesize gas vacuoles in early stationary growth phase with a maximal mRNA content in stationary phase. In contrast, H. halobium wild-type synthesizes gas vacuoles exclusively due to p-vac gene expression with a maximal mRNA level during logarithmic growth, and transcripts of the c-vac gene were not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)