Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium.

When an Escherichia coli K-12 culture was starved for glucose, 50% of the cells lost viability in about 6 days. When a K-12 mutant lacking five distinct peptidase activities, CM89, was starved in the same manner, viability was lost much more rapidly; 50% of the cells lost viability in about 2 days, whereas a parent strain lacking only one peptidase activity lost 50% viability in about 4 days. Compared with the wild-type strain and with its parent strain CM17, CM89 was defective in both protein degradation and protein synthesis during carbon starvation. Similar results were obtained with glucose-starved Salmonella typhimurium LT2 and LT2-derived mutants lacking various peptidase activities. An S. typhimurium mutant lacking four peptidases, TN852, which was deficient in both protein degradation and synthesis during carbon starvation (Yen et al., J. Mol. Biol. 143:21-33, 1980), was roughly one-third as stable as the isogenic wild type. Isogenic S. typhimurium strains that lacked various combinations of three of four peptidases and that displayed protein degradation and synthesis rates intermediate between those of LT2 and TN852 (Yen et al., J. Mol. Biol. 143:21-33, 1980) displayed corresponding stabilities during carbon starvation. These results point to a role for protein degradation in the survival of bacteria during starvation for carbon.     

Characterization of alanyl aminopeptidase from insecticide resistant and susceptible strains of Musca domestica L.

To investigate the high activity of intracellular proteases in insecticide resistant strains of Musca domestica L., purification by anion‐exchange chromatography and gel filtration of one of the enzymes, alanyl aminopeptidase (Ala AP), in three strains of Musca domestica was carried out. The fractions collected by gel filtration of soluble homogenates of the three strains (571ab, 17bb and Cooper) showed a single peak of Ala AP activity. Partially purified Ala AP of the three strains showed high activity at pH 7.5. The presence or absence of Ca²⁺ in the assay medium did not produce any difference in activity of Ala AP in the 571ab and Cooper strains, but there was a significant difference in the 17bb strain. The activity of Ala AP in all three strains was essentially unaltered in the presence of inhibitors of serine (PMSF), cysteine (E‐64) proteases and carboxypeptidases (pepstatin). Ala AP hydrolyzed alanine amino methylcoumarin (Ala‐AMC) maximally, followed by phenyl alanine amino methylcoumarin (Phe‐AMC), leucyl amino methylcoumarin (Leu‐AMC) and ornithine amino methylcoumarin (Orn‐AMC). Ala AP from the three strains showed differential activity towards various substrates. The comparison of alanyl aminopeptidase's activity from different sources is discussed.     

Fertility-deficient E. coli K-12 mutants

Summary Fertility-deficient and f2-r mutants of E. coli K-12 were studied. The above mutants were isolated following the nitrogen-mustard treatment of the E. coli K-12 Hfr and E. coli F ¹ lac ⁺-strains. Isolation of these mutants from F ¹-strains showed that mutations occur in the F-factor no matter whether it was in autonomous state or integrated in a chromosome.The existence of mutants of two types, fertility-deficient and f2-r, was demonstrated. Type I mutants were characterized by the maintenance of a low level phage f2-adsorption activity and by a 10-fold decrease of their fertility as compared with the original strain when crossed in the liquid medium. In crosses in solid media the recombination frequency in the case of type I mutants used as donor was the same as with the original strain. Type II mutants were characterized by the entrie loss of their f2-phage adsorption ability, by a 1,000-fold decrease of fertility in liquid media, and by the inability to recombinate in solid media.     

Degree of Participation of Exogenous Thymidine in the Overall Deoxyribonucleic Acid Synthesis in Escherichia coli

Utilization of labeled exogenous deoxythymidine (TdR) for deoxyribonucleic acid (DNA) synthesis is widely used as a measure of DNA synthesis itself, on the assumption that the degree of participation (DP) of TdR in the overall synthesis of deoxythymidine monophosphate in DNA is constant under a variety of conditions. It is now found that in Escherichia coli DP (ratio of exogenous TdR incorporated into DNA to total TdR content of DNA) depends on many factors. In basal medium, DP is 38% in wild-type strain K-12SH and 48% in strain K-12SH28 mutated in the TdR phosphorylase gene. In thymine-requiring (T(-)) mutants having a functional TdR phosphorylase, DP is less than 100%. Shortening the doubling time of the cells by supplementing basal medium with all amino acids increases DP of K-12SH and K-12SH28 by 25%, and T(-) strains do reach 100%. Slowing cell growth by lowering the temperature to 30 C results in a decrease of DP by 15%. In the logarithmic phase, DP is higher than in the beginning of the stationary phase. It appears that exogenous TdR is more convenient for DNA synthesis during faster cell growth.     

Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum.

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.     

Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Konings, Appl. Environ. Microbiol. 56:526-532, 1990). The L. lactis pepN gene appeared to complement an E. coli strain carrying a mutation in its pepN gene. High-level expression of the pepN gene in E. coli was obtained by using the T7 system. The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots. Cloning of the pepN gene on a multicopy plasmid in L. lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein. Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N. The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein. The intracellular location of aminopeptidase N in L. lactis was confirmed by immunogold labeling of lactococcal cells.     

Novel Alkylsulfatases Required for Biodegradation of the Branched Primary Alkyl Sulfate Surfactant 2-Butyloctyl Sulfate

Recent reports show that contrary to common perception, branched alkyl sulfate surfactants are readily biodegradable in standard biodegradability tests. We report here the isolation of bacteria capable of biodegrading 2-butyloctyl sulfate and the identification of novel enzymes that initiate the process. Enrichment culturing from activated sewage sludge yielded several strains capable of growth on 2-butyloctyl sulfate. Of these, two were selected for further study and identified as members of the genus Pseudomonas. Strain AE-A was able to utilize either sodium dodecyl sulfate (SDS) or 2-butyloctyl sulfate as a carbon and energy source for growth, but strain AE-D utilized only the latter. Depending on growth conditions, strain AE-A produced up to three alkylsulfatases, as shown by polyacrylamide gel electrophoresis zymography. Growth on either SDS or 2-butyloctyl sulfate or in nutrient broth produced an apparently constitutive, nonspecific primary alkylsulfatase, AP1, weakly active on SDS and on 2-butyloctyl sulfate. Growth on 2-butyloctyl sulfate produced a second enzyme, AP2, active on 2-butyloctyl sulfate but not on SDS, and growth on SDS produced a third enzyme, AP3, active on SDS but not on 2-butyloctyl sulfate. In contrast, strain AE-D, when grown on 2-butyloctyl sulfate (no growth on SDS), produced a single enzyme, DP1, active on 2-butyloctyl sulfate but not on SDS. DP1 was not produced in broth cultures. DP1 was induced when residual 2-butyloctyl sulfate was present in the growth medium, but the enzyme disappeared when the substrate was exhausted. Gas chromatographic analysis of products of incubating 2-butyloctyl sulfate with DP1 in gels revealed the formation of 2-butyloctanol, showing the enzyme to be a true sulfatase. In contrast, Pseudomonas sp. strain C12B, well known for its ability to degrade linear SDS, was unable to grow on 2-butyloctyl sulfate, and its alkylsulfatases responsible for initiating the degradation of SDS by releasing the parent alcohol exhibited no hydrolytic activity on 2-butyloctyl sulfate. DP1 and the analogous AP2 are thus new alkylsulfatase enzymes with novel specificity toward 2-butyloctyl sulfate.     

Glutamate transport driven by an electrochemical gradient of sodium ions in Escherichia coli.

The role of Na+ in glutamate transport was studied in Escherichia coli B, strain 29-78, which possesses a very high activity of glutamate transport (L. Frank and I. Hopkins, J. Bacteriol., 1969). Energy-depleted cells were exposed to radioactive glutamate in the presence of a sodium gradient, a membrane potential, or both. One hundred- to 200-fold accumulation of the amino acid was attained in the presence of both electrical and chemical driving forces for the sodium ion. Somewhat lower accumulation values were obtained when either chemical or electrical driving forces were applied separately. A chemical driving force was produced by the addition of external Na+ to Na+-free cells. A membrane potential was established by a diffusion potential either of H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or of SCN-. These results support the hypothesis of a Na+-glutamate cotransport. Na+-driven glutamate transport was also observed in wild-type E. coli B but not in a strain of K-12.     

Evaluation of the antibiotic activity of extracellular compounds produced by the Pseudomonas strain against the Xanthomonas citri pv. citri 306 strain

In this work, we evaluated the antibiotic activity of metabolites produced by the Pseudomonas sp. LV strain and their effects on the cell morphology of the Xanthomonas citri pv. citri 306 strain (Xcc 306), which causes citrus canker lesions. The LV strain was cultivated, centrifuged, a cell-free supernatant was treated with dichloromethane and then concentrated, frozen in liquid nitrogen and lyophilized. The dichloromethane phase (DP) was fractionated by vacuum liquid chromatography (VLC) using six organic solvents with a crescent polarity. The antibiotic activity of the DP and all the fractions from VLC were tested against Xcc 306 and only the F3 fraction showed antimicrobial activity. The antibiotic activity of F3 was determined by minimum inhibitory concentration and the action on the cell morphology of Xcc 306 carried out in glass tubes with cell suspensions plus F3 solution sampled at three different times (one, three and six hours). The effects were analyzed by electron microscopy. Both the DP and F3 showed antibiotic activity against Xcc 306 in in vitro experiments. Electron microscopy showed that the F3 fraction completely disrupted the cell integrity after six hours. In a greenhouse experiment, the DP and F3 fraction (highly effective in in vitro experiments), reduced the formation of lesions by approximately 80% and 94%, respectively.     

Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid.

From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid. Peak I and peak IIa protein fractions behave like hydrophobic proteins.     

A secreted aminopeptidase of Pseudomonas aeruginosa. Identification, primary structure, and relationship to other aminopeptidases

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56). Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56) and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.     

Effect of ethylenediaminetetraacetate on phospholipids and outer membrane function in Escherichia coli.

Treatment of Escherichia coli K-12 strain S15, containing a normal amount of phospholipase A, with ethylenediaminetetraacetate (EDTA) resulted in an increase in sensitivity of the organism to actinomycin D. Strain S17, a mutant deficient in both detergent-resistant phospholipase A and detergent-sensitive phospholipase A, was considerably less sensitive to the antibiotic after the treatment. Both strains released lipopolysaccharide after EDTA treatment, indicating that this outer membrane component alone is not the barrier to actinomycin in these organisms. The phospholipase A-deficient strain released less alkaline phosphatase, a periplasmic enzyme. EDTA treatment of S15 resulted in the accumulation of free fatty acids, indicative of phospholipase A activation. Cells briefly treated with EDTA regained the barrier to actinomycin when incubated in growth media, and the cessation of the accumulation of free fatty acids was in approximate temporal agreement with restoration of the barrier. Cells in which phospholipase A was activated by brief exposure to EDTA synthesized relatively more phosphatidylethanolamine than did untreated cells in the initial period after dilution into growth media. These experiments suggest that the EDTA-induced loss of outer membrane barrier function of E. coli K-12 is mediated through the activation of phospholipase A.     

Subcellular localization and levels of aminopeptidases and dipeptidase in Saccharomyces cerevisiae.

Three aminopeptidases (L-aminoacyl L-peptide hydrolases, EC 3.4.11) and a single dipeptidase (L-aminoacyl L-amino acid hydrolase, EC 3.4.13) are present in homogenates of Saccharomyces cerevisiae. Bassed on differences in substrate specificity and the sensitivity to Zn2+ activation, methods were developed that allow the selective assay of these enzymes in crude cell extracts. Experiments with isolated vacuoles showed that aminopeptidase I is the only yeast peptidase located in the vacuolar compartment. Aminopeptidase II (the other major aminopeptidase of yeast) seems to be an external enzyme, located mainly outside the plasmalemma. The synthesis of aminopeptidase I is repressed in media containing more than 1% glucose. In the presence of ammonia as the sole nitrogen source its activity is enhanced 3--10-fold when compared to that in cells grown on peptone. In contrast, the levels of aminopeptidase II and dipeptidase are less markedly dependent on growth medium composition. It is concluded that aminopeptidase II facilitates amino acid uptake by degrading peptides extracellularly, whereas aminopeptidase I is involved in intracellular protein degradation.     

Separation and scavenging superoxide radical activity of chitooligomers with degree of polymerization 6-16

The separation of chitooligomers (COS) with well-defined degree of polymerization (DP) is of interest to further study their bioactivity. However, there has been no report on separation of chitooligomers with DP>6 and the activity of these oligomers is unknown. This paper focuses on separating COS with DP>6 and five fractions were separated from the prepared fully deacetylated chitooligomers mixture by CM Sepharose Fast Flow column and analyzed by HPLC, which mainly contained glucosamine oligomers with DP6-7 (41.31%, 50.22%), DP7-8 (22.47%, 70.13%), DP9-10 (53.06%, 27.99%), DP10-12 (18.45%, 49.36%, 22.31%), and DP>12, respectively. The superoxide radical scavenging activity of each fraction was investigated. The oligomers with DP ranging from 10 to 12 exhibited higher scavenging activity than other fractions and in combination with the DP distribution of fractions, it was further concluded that the chitooligomers with DP11 was likely to be optimal for scavenging superoxide radical activity.     

Calcium dependency of pregnant rat myometrium: comparison of circular and longitudinal muscle

The purpose of this study was to determine if the changes in spontaneous contractions of circular uterine muscle during pregnancy were related to alterations in calcium (Ca) sensitivity or dependence. Circular muscle (CM) and longitudinal muscle (LM) segments from rats on Days 16-17 of gestation and at term were compared with respect to: sensitivity of potassium (K)-induced contractions to changes in extracellular Ca, and rate and magnitude of decrease of K- and acetylcholine (ACh)-induced contractions in Ca-free medium and in methoxyverapamil (D-600). The effects of low Ca and D-600 on spontaneous electrical activity of CM were also studied. Ca sensitivity was no different in CM and LM and did not change between Day 16 and term. There was no difference in the Ca-dependence of K- or ACh-induced contractions during this time. Potassium contractions declined more rapidly than ACh contractions in Ca-free media, especially in CM. Spontaneous action potentials in CM were Ca-dependent and disappeared in low Ca or D-600 on Days 16-17 and at term. Therefore the changes in contractions of CM during pregnancy are not related directly to Ca sensitivity or dependence, but indirectly via Ca modulation of the action potentials.     

Structure activity relationships of stendomycin, a lipopeptide antibiotic from Streptomyces.

A strain of Streptomyces which produced stendomycin, a lipopeptide antibiotic, was grown in culture media containing various amino acids as nitrogen substrates. The nature of the fatty acid component of stendomycin was dependent on the nature of the amino acid present in the medium, but this did not affect antibiotic activity. Modifications in the peptide moiety resulted in a loss of antifungal activity.     

Enzymatic properties of the glycine d-alanine aminopeptidase of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.     

Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex

The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme.     

Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.