Demonstration of a proteolytic activity on intracellular origin in Aeromonas hydrophila LP 50

Aeromonas hydrophila LP 50, isolated from packaged pasteurized milk, was grown in glucose-polypeptone medium at 30 degrees C. The proteolytic activity of A. hydrophila LP 50, optimum at the stationary phase of growth, is attributed to extracellular or membrane protease; no intracellular proteolytic activity was shown.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

Excretion of a protease by Serratia marcescens

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfurcontaining amino acids. The enzyme consists of one polypeptide chain. A molecular weight of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N--benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of diand tripeptides.Abbreviation SDS sodium dodecylsulfate     

Cation Transport in Escherichia coli : II. Intracellular chloride concentration

The intracellular Cl concentration in E. coli has been studied as a function of the Cl concentration in the growth medium and the age of the bacterial culture. The ratio of extracellular to intracellular Cl concentration is shown to be 3.0 in the logarithmic phase and 1.13 in the stationary phase, both ratios being independent of the extracellular Cl concentration. If it may be assumed that Cl is passively distributed in this organism, these results are consistent with a transmembrane P.D. of 29 mv, interior negative, during the logarithmic phase, and 3 mv, interior negative, in the stationary phase.     

Overproduction of extracellular protease activity by Streptomyces C5-A13 in fed-batch fermentation

Summary Streptomyces C5-A13, a non-sporulating, pleiotropic mutant of the anthracycline-producing strain, Streptomyces C5, overproduces extracellular proteolytic activity against the substrate azocasein. This extracellular protease activity was produced primarily during the stationary phase. This appears to be an effect related to growth rate rather than to glucose repression, because only very high concentrations of glucose appear to inhibit protease synthesis. Production of extracellular protease activity was stimulated by the presence of carbonate anions in the medium. The optimal concentration of soluble carbonate was 60–80 mM and the stimulation by carbonate was shown not to be due to a pH effect. Approximately 3200–3500 units of extracellular azocaseinase activity were produced per millititre of culture broth using partially optimized fed-batch fermentation processes. This value represents about ninefold greater activity than produced under shake flask conditions.     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.     

Protein degradation in extracts of exponential and stationary phase Vibrio cells

The degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 degrees C. Maximum stimulation was obtained with 3 mM-ATP and optimum degradation was at pH 8.0-8.5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.     

Production of thermostable lipolytic activity by thermus species

A quantitative screening for intra- and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.     

Exopolysaccharide production by the epipelic diatom Cylindrotheca closterium: effects of nutrient conditions

During the stationary phase of a batch culture of the epipelic diatom Cylindrotheca closterium, accumulation of exopolysaccharides and intracellular carbohydrates was observed. When nitrogen was added to the culture in the stationary phase, growth was resumed and the accumulation of exopolysaccharides was delayed. This indicated that nitrogen depletion caused cessation of growth, and stimulated exopolysaccharide accumulation. Exopolysaccharide accumulation was also stimulated when cells were either resuspended in medium lacking N or P, or when they were inoculated in medium with low concentrations of N or P. Growth was not immediately affected by low N or P concentrations. S depletion only resulted in exopolysaccharide accumulation when growth was affected. Si or Fe depletion did not stimulate exopolysaccharide accumulation, even when growth rates were lowered. Apparently, stimulation of exopolysaccharide accumulation is dependent on the type of nutrient depletion. Intracellular storage carbohydrates did not accumulate when cells were incubated at low N or P concentrations. Cells grown with ammonium as nitrogen source produced more carbohydrates (both extracellular and intracellular) than cells grown with nitrate as nitrogen source, indicating that both exopolysaccharides and intracellular carbohydrates accumulated as a result of overflow metabolism.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Adenosine 3′:5′-cyclic monophosphate concentrations and phosphodiesterase activities during axenic growth and differentiation of cells of the cellular slime mould Dictyostelium discoideum

During growth of myxamoebae of Dictyostelium discoideum (strain Ax-2) in axenic medium, the myxamoebae secrete cyclic AMP. As the cells leave the exponential phase of growth and enter the stationary phase, there is an approximate doubling of the intracellular cyclic AMP content, but the amount of extracellular cyclic AMP remains proportional, at all times, to the number of myxamoebae present. During development of axenically grown myxamoebae, there is first a rise in the intracellular concentration of cyclic AMP, followed by a rise in the amount of extracellular cyclic AMP, which reaches a peak at the time of aggregation and then declines. There is a second peak in the amount of extracellular cyclic AMP found at the time of fruiting-body formation, but this second peak is not associated with a rise in the intracellular cyclic AMP concentration. Controls thus exist over the synthesis and secretion of cyclic AMP. Evidence is presented for the belief that the activity of the adenylate cyclase enzyme controls the amount of cyclic AMP synthesized rather than the activity or amount of cyclic AMP phosphodiesterase present. Similar changes occur in extracellular cyclic AMP and phosphodiesterase concentrations during incubation of myxamoebae in buffered suspensions to those occuring during the first few hours of development of such cells on solid media, but the timing of these changes is different.     

Inactivation of allantoinase ofPseudomonas aeruginosa in vivo

The total and specific activity of allantoinase decreased in the stationary growth phase whenPseudomonas aeruginosa was cultured in a medium containing allantoin or allantoate as the sole source of carbon, nitrogen and energy. The enzyme was not instableper se. Inactivation was proven to be due to the synthesis of a protein, which exhibited no proteolytic activity toward allantoinase. The synthesis of a specific binding protein is postulated.     

Regulation of Proteolytic Enzyme Activity in Lactococcus lactis

Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were studied during batch and continuous cultivation. In both strains, the PrtP activity level was regulated by the peptide content of the medium. The highest activity level was found during growth in milk, and the lowest level was found during growth in the peptide-rich laboratory medium M17. Regulation of the intracellular peptidase activity appeared to be a strain-dependent phenomenon. In cells of strain MG1363, the activity levels of PepN and PepXP were regulated in a similar way to that observed for PrtP. In cells of strain SK1128, the levels of both peptidases were not significantly influenced by the peptide content of the medium. The presence of specific concentrations of the dipeptide prolylleucine could mimic the low activity levels of the regulated proteolytic enzymes, even to the activity level found on M17 medium. The effect of the presence of the dipeptide prolylleucine in the medium on the activity level of the regulated proteolytic enzymes was confirmed at fixed growth rates in chemostat cultures.     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Synthesis of exocellular proteins during the exponential and stationary phase of growth ofBacillus megaterium

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strainBacillus megaterium J-27 and asporogenic strain KM 1 was investigated. Both organisms excrete the enzyme into the medium during growth and during the stationary phase. In the asporogenic strain the excretion decreases at the end of the exponential phase. In the sporogenic strain it continues during the transition to the stationary phase at the original rate and proteolytic activity in the medium increases two to three times during 2 h after the end of the exponential phase. Both organisms synthesize relatively more exocellular proteins during the exponential phase than during the stationary phase. The proportion of exooellular protein synthesized during the exponential phase does not exceed 3 % of total proteins, during the stationary phase this proportion usually decreases to less than 1 %.     

Proteinase pattern in Trametes versicolor in response to carbon and nitrogen starvation

In stationary cultures of Trametes versicolor seven proteinase bands were revealed by electrophoresis in mycelium and five in the medium. Under conditions of nitrogen starvation the number of bands in mycelium was unchanged whereas one extracellular proteinase was missing. In the case of carbon starvation one new intracellular proteinase activity appeared and one extracellular activity disappeared. Moreover, in all starved cultures distinct differences in the intensity of particular bands were observed.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Identification and characterization of a novel serine protease, VvpS, that contains two functional domains and is essential for autolysis of Vibrio vulnificus

Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus, a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic β-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus. VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus.     

Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans