Exocellular Protein Research Articles

Kinetics of nutrient removal and expression of extracellular polymeric substances of the microalgae, Chlorella sp. and Micractinium sp., in wastewater treatment

Two species of green algae, Chlorella sp. and Micractinium sp., were cultivated in primary effluent wastewater and high-strength wastewater (a mixture of anaerobic digestion centrate and primary effluent) to study nutrient removal and EPS (extracellular polymeric substances) expression during their growth. The high N concentration and P-limited condition in the mixed wastewater (total N=197 mg/L; N/P mass ratio=56) led to about 3 times greater specific N removal rate than the primary effluent set, indicating that algal cells growing in N-rich wastewater had N over-uptake. Both Chlorella and Micractinium grown in the high-strength wastewater also produced larger amounts of protein EPS, possibly accounting for higher N uptake in those cultivation sets. These results suggest that different types of wastewater could cause different nutrient removal kinetics and EPS expression by algae, which may subsequently influence harvesting and anaerobic digestion of their biomass.

The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell‐surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase

Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non-electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N- and C-termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti-TiBP polyclonal antibodies by whole cell and protein enzyme-linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were not recognized by these antibodies. Similar results were obtained by fluorescence and scanning electron microscopy. Our postulation that DLDH is located on the surface of Rh. ruber GIN1, serving as a TiO2 binding protein, is in accordance with literary evidence on DLDH in other organisms, Bacteria, Archea, and Eukaryots that suggests it is associated with the outer membranes or cell surfaces. As an exocellular protein DLDH assumes various tasks which are not related to its classical role as a 2-oxoacid dehydrogenase, including serving as an adhesion/binding protein in certain bacteria.

A unique polypeptide from the C-terminus of the exocellular esterase of Acinetobacter venetianus RAG-1 modulates the emulsifying activity of the polymeric bioemulsifier apoemulsan

An exocellular esterase from the oil-degrading Acinetobacter venetianus RAG-1 was previously shown to enhance the emulsification and emulsion stabilization properties of the amphipathic, aminopolysaccharide bioemulsifier, emulsan [Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608-15]. This enhancement was specific for the RAG-1 esterase and was independent of catalytic activity. In this report, fragments from both the N'- and C'-termini were cloned as fusions to the C-terminus of the maltose-binding protein (MBP) and were tested for enhancement activity in the presence of the deproteinated form of emulsan, apoemulsan. The activity could be localized to the C-terminal third of the protein which exhibited the same activity as the intact enzyme. MBP itself was completely inactive and could be cleaved from the fusion without affecting the subsequent emulsification. However, the enhancement completely depended on the presence of a unique C-terminal 20 amino acid peptide not found in any other protein in the databases. In addition, progressive removal of amino acids from the N-terminus of the active MBP polypeptide resulted in a concomitant loss of activity, indicating that enhancement is also proportional to the size of the peptide fragment. The middle third and the C-terminal third of the enzyme each contained a copy of the conserved Cardin-Weintraub consensus sequence for protein binding to heparin. These sequences were not detected in homologous esterases from a closely related strain, Acinetobacter calcoaceticus BD413.

Novel polysaccharide–protein-based amphipathic formulations

Previous results showed that the cell-surface esterase from Acinetobacter venetianus RAG-1 enhances the emulsification properties of the polymeric bioemulsifier emulsan and its deproteinated derivative apoemulsan (Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608-2615). Here we show that in the presence of the his-tagged recombinant esterase from RAG-1, 18 different polysaccharides from microbial, plant, insect and synthetic sources formed hexadecane-in-water emulsions. Emulsifying activities were distributed over a 13-fold range from over 4800 U/mg protein/mg polysaccharide in the case of apoemulsan to 370 U/mg protein/mg polysaccharide in the case of alginic acid. The stability of the emulsions ranged between 95 and 58%. Emulsions formed in the presence of seven of the polysaccharides exhibited stabilities of over 80%. The esterase from A. calcoaceticus BD4, which shows sequence homology to the RAG-1 esterase, was inactive in emulsification enhancement. The sequence of the RAG-1 esterase was shown to contain two conserved peptide sequences previously shown to be implicated in carbohydrate/polysaccharide binding. A hypothetical model illustrating a possible mode of interaction between the esterase, the apoemulsan and the oil droplet is presented. The complex is presumed to generate a series of "coated" oil droplets which are restricted in their ability to coalesce resulting in a relatively stable emulsion.

Enhanced granulation by natural ionic polymer additives in UASB reactor treating low-strength wastewater

Effect of natural ionic polymer additives on granulation in lab-scale UASB reactors treating low-strength synthetic wastewater (COD 750–850 mg/L) was examined. The organic loading rate was 1.477±0.118 kg COD/m 3/day. Under identical conditions four similar reactors were operated in parallel with the following additives: control with no additive, anionic part of Reetha ( Sapindus trifoliata) extract, cationic part of Reetha extract, and Chitosan. By the end of the study period, Chitosan as an additive produced largest granules with mean size of 0.15 mm closely followed by the cationic fraction of the Reetha extract with mean size of 0.144 mm, and anionic fractions of the Reetha extract with 0.139 mm. Control reactor with no additives had the smallest size granules with mean size of 0.128 mm. The fraction of granules in the sludge bed of size >0.1 mm showed similar trend. The largest granule size observed in the reactors with additives was 4–5 mm as compared to 2 mm in the control reactor. Cationic polymers were more effective additives for enhancing sludge granulation. Exo-cellular protein, lipid, sugar and total polymer increased with granulation in the reactors. A COD removal efficiency of 95–98% was achieved in all the reactors.

Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis

To develop a strategy to increase the stability of transcripts of structural genes expressed under the control of sacR, the leader region of Bacillus subtilis levansucrase gene. Insertion of Shine Dalgarno like sequences in the 5'-untranslated sacR region controlling the expression of sacB. Depending on the number of stabilizing sequences inserted and the position of these sequences with respect to the translation start codon, it was observed that the mRNA stability and the final protein production could be increased or decreased. This mRNA stabilization can be used to increase exocellular protein production in the degU32 (Hy) mutant. This approach can be applied to the expression of heterologous genes of biotechnological interest.

Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica

In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A. The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate. Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, [ 32P]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities. The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE. An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue. Optimum pH value is 6.1, apparent K m for phosphotyrosine was calculated to be 760 μM and Hill coefficient 3.2 indicating a rather high cooperativity. These results showed that the involvement of alkaline and/or acid phosphatase was unlikely. In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.

Characterization of Exocellular Protein and Its Role in Bioflocculation

The relationship between exocellular biopolymer concentration and cation concentration was examined using laboratory scale activated sludge reactors with bactopeptone as a feed. An increase in the divalent cation concentration in the feed to the reactors was associated with an increase in the bound exocellular protein concentration, and high sodium concentrations resulted in a decrease in the bound protein concentration. The changes in bound biopolymer were explained according to the cation bridging model. Incubation of a laboratory activated sludge with a proteolytic enzyme resulted in deflocculation of the suspension as measured by an increase in the number of particles in the 5–40 μm range, which suggested that the exocellular protein was strongly involved in the aggregation of bacteria into flocs. SDS PAGE results revealed the presence of a single protein in the exocellular biopolymer extract from municipal, industrial, and laboratory activated sludge samples. The molecular weight of the protein was approximately 15 daltons. Amino acid analysis and amino acid sequencing results suggested the protein was a lectinlike protein, and binding site inhibition studies demonstrated that the protein had lectinlike activity.

Cellular response to a multiple-metal stress in Pseudomonas fluorescens

Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with millimolar amounts of aluminum (5 mM), iron (5 mM), zinc (3 mM), calcium (2 mM) and gallium (1 mM). A slight decrease in growth rate and a 22% diminution in cellular yield were observed as compared to the control medium. Citrate, the sole source of carbon to which the test metals were complexed, was completely utilized. Although at stationary phase of growth most of the metals were immobilized in an exocellular lipid-rich residue, ultracentrifugation and dialysis studies revealed that metals were associated with phosphatidylethanolamine (PE) from early stages of growth. As growth progressed the metal content of the soluble cellular extract increased reaching an optimum at 35 h of incubation. However, no detectable amounts of metals in this cellular component were discerned at stationary phase of growth. There appeared to be no marked variation in exocellular protein and carbohydrate production in control and metal-stressed cultures. Transmission electron microscopic studies revealed metal rich bodies associated with the cytoplasm. Scanning electron microscopic analyses of the dialyzate aided in the identification of the metal-rich bodies associated with elongated structures comprised of carbon, oxygen and phosphorus. PE appeared to be an important organic constituent of the gelatinous residue.

Influence of phosphate on aluminum tolerance inPseudomonas fluorescens

When grown in a minimal mineral medium containing 0.64 mM phosphate, Pseudomonas fluorescens attained a cellular yield comparable to that observed in a similar medium with 64 mM phosphate. However, the inclusion of aluminum in these two media evoked disparate responses to aluminium stress. In the latter culture, aluminum was immobilized as a gelatinous lipid-rich residue (Appanna et al., FEMS Microbiol. (1994) 119, 295–302), while in the former medium the trivalent metal was sequestered in soluble metabolite(s) localized in the spent fluid. In the medium with 0.64 mM phosphate, the cellular yield decreased by 10–40% in the presence of 3–15 mM aluminum. Citrate, the sole source of carbon to which the metal was complexed, was completely utilized and no significant change in exocellular protein or carbohydrate was evident. While only a trace amount of aluminum was recorded in the soluble cellular fraction, only 11% of the test metal was immobilized as a lipid residue. However, most of the metal was sequestered in metabolite(s) localized in the spent fluid. It appears that mechanism(s) initiated by the microbe to achieve aluminum homeostasis is dependent on the phosphate concentration in the medium.

Enzyme-linked immunosorbent assay for the detection of antibodies to exocellular proteins of Staphylococcus aureus in bone infection.

An enzyme-linked immunosorbent assay (ELISA) was developed for measurement of the antibody response to exocellular protein antigens of Staphylococcus aureus. The wells of a microtitre plate were coated with the exocellular proteins present in the tryptic soya broth growth medium from a stationary phase culture of a bone infection strain of S. aureus. The wells were then reacted sequentially with patient sera, protein A-peroxidase conjugate and chromogenic substrate. Serum from patients with S. aureus bone infection gave a significantly higher IgG titre than sera from patients with Staphylococcus epidermidis or Streptococcus sanguis bone infection or healthy uninfected individuals. The assay therefore appears to have potential in the serodiagnosis of S. aureus bone infection.

Medium development for xanthan production

Statistical designs were used to determine optimum levels of medium nutrients for xanthan production in shake flask fermentations. Information about the effects of each medium component on xanthan fermentation and the metabolism of Xanthomonas campestris was also obtained. The use of high carbon to nitrogen ratios in the defined medium resulted in a major increase in the broth consistency index. Maximal production was achieved in nitrogen limitation, at a carbon to nitrogen ratio of 23. Mineral sources such as CaCO 3 and KH 2 PO 4 proved to be important factors influencing polysaccharide production and quality. A maximum concentration of 11·15 g/litre of xanthan was obtained during the optimization of the defined medium. An increase of xanthan production was accompanied by a decrease of exocellular protein. The addition to the defined medium of yeast extract at a concentration above 0·75 g/litre and peptone above 2·5 g/litre resulted in inhibition of production due to a drop in the carbon to nitrogen ratio. However, if the complex nutrients were added in lower quantities, a slight stimulation of xanthan formation was obtained, 12 g/litre of polysaccharide being achieved.

Purification and properties of an extracellular invertase from Azotobacter chroococcum

Analysis of distribution of invertase (EC 3.2.1.26) activity in Azotobacter chroococcum ATCC 4412 and A. vinelandii UW cells revealed that A. chroococcum invertase is an exocellular protein, whereas the A. vinelandii enzyme is located inside the cell. Invertase has been purified by a simple and rapid procedure from cell-free supernatants of A. chroococcum batch cultures. The enzyme preparation appeared homogeneous on SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and was identified as a glycoprotein. The exoinvertase was freed of its carbohydrate component, without loss of catalytic activity, upon treatment with β- N-acetylglucosaminidase (EC 3.2.1.30). Invertase molecular weight was estimated to be 57 kDa by analytical gel filtration and 59 KDa by SDS-PAGE. It appears, therefore, to be composed of a single subunit. The amino acid analysis of A. chroococcum invertase revealed a high proportion of acidic residues and lack of arginine and histidine. 5 m m pyridoxal-5′-phosphate or pyridoxamine nearly abolished the invertase activity. Activation energy of the enzyme for sucrose hydrolysis was 18 kcal mol −1. The specific activity of A. chroococcum invertase, expressed as turnover number, was 100 s −1.

Bioreactor operation for the production of exotoxin A by Pseudomonas aeruginosa

Biotechnology and BioengineeringVolume 34, Issue 9 p. 1214-1220 Communications to the EditorFree Access Bioreactor operation for the production of exotoxin A by Pseudomonas aeruginosa Ilse I. Blumentals, Ilse I. Blumentals Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorRobert M. Kelly, Robert M. Kelly Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorJoseph Shiloach, Corresponding Author Joseph Shiloach Biotechnology Unit, Bldg 6, B1-33, NIDDK The National Institutes of Health Bethesda, Maryland 20892Biotechnology Unit, Bldg 6, B1-33, NIDDK The National Institutes of Health Bethesda, Maryland 20892Search for more papers by this author Ilse I. Blumentals, Ilse I. Blumentals Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorRobert M. Kelly, Robert M. Kelly Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorJoseph Shiloach, Corresponding Author Joseph Shiloach Biotechnology Unit, Bldg 6, B1-33, NIDDK The National Institutes of Health Bethesda, Maryland 20892Biotechnology Unit, Bldg 6, B1-33, NIDDK The National Institutes of Health Bethesda, Maryland 20892Search for more papers by this author First published: November 1989 https://doi.org/10.1002/bit.260340913AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 B. H. Iglewski and D. Kabat, Proc. Natl. Acad. Sci. USA., 72, 2284 (1975). 2 L. F. Hanne, T. R. Howe, and B. H. Iglewski, J. Bacteriol., 154, 383 (1983). 3 S. H. Leppla, O. C. Martin, and L. A. Muehl, Biochem. Biophys. Res. Commun., 40, 1437 (1978). 4 S. Lory and R. J. Collier, Infect. Immun., 28, 494 (1980). 5 M. L. Vasil, C. Chamberlain, and C. C. R. Grant, Infect. Immun., 52, 538 (1986). 6 M. J. Bjorn, B. H. Iglewski, S. K. Ives, J. C. Sadoff, and M. L. Vasil, Infect. Immun., 19, 789 (1978). 7 R. M. DeBell, Infect. Immun., 24, 132 (1979). 8 P. V. Liu, J. Infect. Dis., 128, 506 (1973). 9 I. I. Blumentals, R. M. Kelly, M. Gorziglia, J. B. Kaufman, and J. Shiloach, Appl. Environ. Microbiol., 53, 2013 (1987). 10 C. C. R. Grant and M. L. Vasil, J. Bacteriol., 168, 1112 (1986). 11 S. Lory, J. Bacteriol., 168, 1451 (1986). 12 G. L. Gray and M. L. Vasil, J. Bacteriol, 147, 275 (1981). 13 R. C. Hedstrom, C. R. Funk, J. B. Kaper, O. R. Pavlovskis, and D. R. Galloway, Infect. Immun., 51, 37 (1986). 14 V. L. Miller and J. J. Mekalanos, Proc. Natl. Acad. Sci. USA. 81, 3471 (1984). 15 V. L. Miller and J. J. Mekalanos, J. Bacteriol., 163, 580 (1985). 16 M. L. Vasil, D. Kabat, and B. H. Iglewski, Infect. Immun., 16, 353 (1977). 17 D. W. Chung and P. J. Collier, Infect. Immun., 16, 832 (1977). 18 C. L. Paddon and R. W. Hartley, Gene. 40, 231 (1985). 19 T. Maniatis, E. F. Fritsch, and J. Sambrook, Molecular Cloning, A Laboratory Manual. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982). 20 A. Uhlrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter, and H. M. Goodman, Science, 196, 1313 (1977). 21 C. H. Fiske and Y. Subbarow, J. Biol. Chem., 66, 375 (1925). 22 P. H. Clarke, M. A. Houldsworth, and M. D. Lilly, J. Gen. Microbiol., 51, 225 (1968). 23 A. Body, P. H. Clarke, M. A. Houldsworth and M. D. Lilly, J. Gen. Microbiol., 48, 137 (1967). 24 R. W. Smith and A. C. R. Dean, J. Gen. Microbiol., 72, 32 (1972). 25 Z. Fencl and J. Pazlarova, Folia Microbiol., 27, 340 (1982). 26 J. P. Kotze and A. Kistner, Can. J. Microbiol., 20, 861 (1974). 27 Z. Fenel, J. Ričica, and J. Kodešovä, J. Appl. Chem. Biotechnol., 22, 405 (1972). Volume34, Issue9November 1989Pages 1214-1220 ReferencesRelatedInformation

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.

Comparative Analysis of the 5′-End Regions of Two Repressible Acid Phosphatase Genes in Saccharomyces cerevisiae

The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.

Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.

Levansucrase of Bacillus subtilis: Conclusive evidence that its production and export are unrelated to fatty-acid synthesis but modulated by membrane-modifying agents.

Cerulenin action was studied in Bacillus subtilis strains which overproduce several exocellular enzymes. Under conditions in which fatty acid synthesis is partially or completely inhibited by the drug cerulenin, we observed that cell growth and endocellular protein synthesis are scarcely affected during an initial phase, whereas the synthesis of two exocellular enzymes, levansucrase and α-amylase, is markedly reduced. However, careful investigation showed that fatty acid synthesis and exocellular protein production are not inhibited in the same range of cerulenin concentration. Moreover, at a drug concentration which completely inhibits fatty acid synthesis both the half-life of levansucrase messenger RNA and the lag time of the induction process of this enzyme in an inducible strain are unmodified. To account for these data we suggest that cerulenin inhibition of exoenzyme synthesis in these B. subtilis strains is the result of its physiochemical interaction with the membrane rather than the result of its interference with intracellular lipid synthesis. This hypothesis is substantiated by the results obtained from the study of the effects of two membrane-modifying agents on B. subtilis exocellular enzyme production: ethanol and dodecanoic acid. These compounds are not metabolized by the cells and they inhibit levansucrase synthesis. Dodecanoic acid affects levansucrase synthesis to the same extent as cerulenin and within the same concentration range but it does not affect the incorporation of acetate into lipids and is reversibly bound to the cells. A quantitative interpretation of the inhibition caused by these three compounds is proposed, based on the hypothesis that they act at the level of the membrane sites of exocellular enzyme synthesis.

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

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strain Bacillus 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 exocellular 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 %.

Demonstration of a cell-associated, inactive precursor of an exocellular protease produced by Pseudomonas aeruginosa.

The enzymatically active form of protease 1, the major exocellular protein produced by Pseudomonas aeruginosa strain 34362, has been shown to exist exclusively exocellularly with no significant cell-associated activity. However, the presence of a cell-associated, enzymatically inactive protein which is serologically cross-reactive with, and convertible to, active enzyme has been demonstrated. One method of conversion of "precursor" to active enzyme is via limited proteolysis. Two assay systems for precursor were developed, one a radioimmune assay, and the other a proteolytic activation procedure. Localization studies suggest that the association while more tenacious than classical periplasmic enzymes is still an ionic rather than a covalent one. Kinetics of production studies showed to precursor to be synthesized early in the growth cycle and to accumulate prior to the rapid release of the active enzyme. Molecular weight studies showed only slight changes produced upon activation.