Gene sequence, purification and characterization of N-acetyl-beta-glucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene (cht60) encoding N-acetyl-beta-glucosaminidase (Cht; EC 3.2.1.30) from the marine bacterium Alteromonas sp. strain O-7 was cloned into pUC18 in Escherichia coli JM109. The nucleotide (nt) sequence of cht60 was determined. A 1797-bp open reading frame encoded a polypeptide of 598 amino acids (aa) (M(r) 64,535). The aa sequence of the cloned enzyme (Cht60) deduced from the nt sequence showed no significant sequence homologies with available aa sequences from databases. Cht60 was purified from the periplasmic fraction of E. coli cells carrying pCHT982. The enzyme was most active towards p-nitrophenyl-N-acetyl-beta-D-glucosaminide(PNP-beta-GlcNAc) and diacetylchitobiose. The optimum pH and temperature of the enzyme were pH 7.5 and 37 degrees C, respectively. The N-terminal 11 aa residues of Cht60 were sequenced, and the location of the signal peptide cleavage site was clarified.

Cloning and sequence of an alkaline serine protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7.

The gene (aprII) encoding alkaline serine protease (AprII; subtilase) from Alteromonas sp. strain O-7 was cloned in plasmid pUC19 and transformed into Escherichia coli JM109. The nucleotide (nt) sequence of aprII has been determined. A single open reading frame (ORF) encoded a protein consisting of 621 amino acids (aa) with a M(r) of 63,958. The results of aa sequence analysis indicated that AprII is produced as a large precursor consisting of four domains: the signal sequence, the N-terminal pro-region (AprII-N), the mature AprII (AprII-M) and the C-terminal pro-region (AprII-C). The aa sequence of AprII-M shows high sequence homology with those of class-II subtilases. Two conserved sequences were found in AprII-N which might play a critical role in the maintenance of chaperone-like activity. Repeated aa sequences were observed in AprII-C (AprII-C1 and AprII-C2). The aa sequences of AprII-C1 and AprII-C2 show high sequence homology with those of the C-terminal pro-region of the other known proteases.

Studies on new aminoglycoside antibiotics, istamycins, from an actinomycete isolated from a marine environment. I. The use of plasmid profiles in screening antibiotic-producing streptomycetes.

Plasmid profiles were used to screen streptomycetes for production of new antibiotics. Among about 100 strains isolated from sea muds, an isolate designated SS-939 was revealed to harbor several plasmids of different sizes, and to produce istamycins, new aminoglycoside antibiotics. Based on the characteristics of the strain, a new Streptomyces species is proposed: S. tenjimariensis.

Studies on new aminoglycoside antibiotics, istamycins, from an actinomycete isolated from a marine environment. II. Possible involvement of plasmid in istamycin production.

Acriflavine treatment of an istamycin-producing Streptomyces tenjimariensis strain designated SS-939 resulted in a high frequency of isolates with reduced istamycin production. Some of these were shown to have lost a particular plasmid present in the parent strain. Istamycin production by these isolates was largely restored by the addition of 2-deoxystreptamine (DOS) to the medium whereas the effect of DOS was small in the strain SS-939. Sodium palmitate also stimulated production, especially when added together with DOS. These stimulative effects by DOS and palmitate, however, were not exhibited in the presence of glucose (1.0%).

Studies on marine microorganisms. IV. A new antibiotic SS-228 Y produced by Chainia isolated from shallow sea mud.

A new antibiotic named SS-228 Y, which inhibits growth of Gram-positive bacteria, Ehrlich carcinoma in mice, and dopamine-beta-hydroxylase, was obtained from a species of Chainia isolated from shallow sea mud in Sagami Bay. It was yellowish brown powder having the molecular formula C19H1406. From the physical and chemical properties, SS-228 Y was concluded to b a new antibiotic having structure of peri-hydroxyquinone moiety.

Studies of the mode of action of amiclenomycin.

Amiclenomycin (AM) was found to be a strong inhibitor of KAPA-DAPA aminotransferase of Brevibacterium divaricatum. This transamination was suggested to follow Ping Pong Bi Bi mechanism. Inhibition of this transamination by AM is of a noncompetitive type in a Lineweaver-Burk plot of initial velocity, but not in a Dixon plot. The activity of KAPA-DAPA aminotransferase drops abruptly after preincubation with AM, but its activity is restored by dialysis against 10 mM potassium phosphate buffer (pH 7.0). Inhibition by AM is decreased by an increase of KAPA in the reaction mixture, but not by an increase of S-adenosyl-L-methionine (SAM) or pyridoxal-5'-phosphate (PALP). These facts indicate that AM exerts its inhibitory action against KAPA-DAPA aminotransferase by binding to the enzyme, probably to the KAPA-DAPA binding site.

Biosynthetic similarity between Streptomyces tenjimariensis and Micromonospora olivasterospora which produce fortimicin-group antibiotics.

The profile of bioconversion products of istamycin (IS) components by a blocked IS mutant of Streptomyces tenjimariensis that lost IS-productivity suggested a possible biosynthetic pathway of IS similar to that of fortimicin (FT) by Micromonospora olivasterospora. Both organisms are resistant to the antibiotics produced by each other. Based on these similarities, they were examined for their capability to convert an FT-intermediate (FT-B) and IS-intermediates (IS-A0 and -B0) through their biosynthetic pathways. S. tenjimariensis formed 1-epi-FT-B, 2''-N-formimidoyl-FT-A (= dactimicin) and 1-epidactimicin (a new antibiotic) from FT-B. On the other hand, M. olivasterospora converted IS-A0 and -B0 to 2''-N-formimidoyl-IS-A (= IS-A3) and -B (= IS-B3), respectively. Thus, the similarity in antibiotic biosynthesis was confirmed between these FT-group antibiotic-producing organisms. It was also found that the major fermentation product of M. olivasterospora is not FT-A (astromicin) but dactimicin.

Plasmid variability in the istamycin producing strains of Streptomyces tenjimariensis.

Three strains of istamycin-producing Streptomyces tenjimariensis were isolated over a period of time from soils at the same location and were found to have three different types of plasmid profiles. Protoplast fusion between two of these strains provided a clone harboring a smaller plasmid not present in the parent strains. None of the plasmids had restriction sites for EcoR I and Hind III. Most of the plasmids had one or two restriction sites for BamH I, Bcl I, Bgl II, Kpn I, Pst I and Pvu II, and more than two restriction sites for Sal I and Sst II. Plasmid restriction maps and Southern hybridization experiments revealed that pST2, pST12 and pST22 were identical, as were pST10 and pST20. In addition, it was revealed that pST1, pST1, pST11 and pST21 were related to each other.

Studies on new aminoglycoside antibiotics, istamycins, from an actinomycete isolated from a marine environment. III. Nutritional effects on istamycin production and additional chemical and biological properties of istamycins.

Streptomyces tenjimariensis SS-939 produced istamycins in a medium containing starch as the carbon source and soy bean meal as the nitrogen source. Istamycin production decreased substantially when starch was substituted with mono- or di- saccharides such as glucose, glycerol and maltose. A marked decrease of istamycin production was also observed when a rapidly used nitrogen source such as yeast extract, peptone or casamino acid was employed instead of soy bean meal. Addition of palmitate at a concentration of 0.2% doubled istamycin production. Istamycins A and B were found to be as active as fortimicin A and sporaricin A against Gram-positive and Gram-negative bacteria including aminoglycoside-resistant strains.

Studies on marine microorganisms. V. A new antibiotic, aplasmomycin, produced by a streptomycete isolated from shallow sea mud.

A new antibiotic, aplasmomycin, which inhibits growth of Gram-positive bacteria including myobacteria in vitro, and plasmodia in vivo was obtained from a strain of Streptomyces griseus isolated from shallow sea sediment in Sagami Bay. The antibiotic forms colorless needle-like crystals and has a molecular formula of C41H60O14Na. Based on its physical and chemical properties, aplasmomycin was concluded to be a new antibiotic. The antibiotic was produced in selected media devised to relate to a marine environment.

Biological studies of amiclenomycin.

The action of amiclenomycin (AM) in inhibiting growth of microorganisms is specific against mycobacteria in vitro, but the antibiotic does not show a therapeutic effect against tubercle bacilli in vivo. The action of AM is reversed by biotin, desthiobiotin (DTB) and 7,8-diaminopelargonic acid (DAPA), but not by 7-keto-8-aminopelargonic acid (KAPA), pimelic acid and glutaric acid. In the presence of AM, cultures of Mycobacterium smegmatis and Bacillus sphaericus accumulated KAPA, whereas the formation of DTB decreased. Therefore, AM is thought to inhibit KAPA-DAPA transamination in biotin biosynthesis. In M. smegmatic and B. sphaericus the conversions of KAPA to DAPA and of DTB to biotin were rate limiting in biotin synthesis. Accordingly, the synergistic antibiotic activity of AM, inhibiting the former, and actithiazic acid, inhibiting the latter reaction, would be simply explained.

Mechanism of resistance to aminoglycoside antibiotics in nebramycin-producing Streptomyces tenebrarius.

Streptomyces tenebrarius ISP 5477, which produces nebramycins, was highly resistant to the following aminoglycoside antibiotics: neamine, ribostamycin, butirosin A, neomycin B, paromomycin, kanamycin A, dibekacin, gentamicin C complex, lividomycin A, istamycin B and streptomycin. Polyphenylalanine synthesis on the ribosomes of this strain was highly resistant to neamine, ribostamycin, butirosin A, kanamycins A, B and C, dibekacin, gentamicin C complex and istamycin B, moderately resistant to lividomycin A and streptomycin, but sensitive to neomycin B and paromomycin. Moreover, cell free extract of the strain contained phosphotransferase and N-acetyltransferase. The former enzyme was confirmed to be an aminoglycoside 6-phosphotransferase which inactivated streptomycin; the latter inactivated kanamycins B and C, dibekacin, neamine, neomycin B, paromomycin, lividomycin A, butirosin A and ribostamycin, but did not inactivate kanamycin A, gentamicin C complex and sagamicin, suggesting an aminoglycoside 2'-acetyltransferase. These results indicated that the high resistance of S. tenebrarius ISP 5477 to a wide range of aminoglycoside antibiotics is due to ribosomal resistance and to the inactivating enzymes, aminoglycoside N-acetyltransferase(s) and aminoglycoside 6-phosphotransferase.

Resistance mechanisms of kanamycin-, neomycin-, and streptomycin-producing streptomycetes to aminoglycoside antibiotics.

Streptomyces kanamyceticus ISP5500, S. fradiae ISP5063 and S. griseus ISP5236, which produce kanamycin, neomycin or streptomycin respectively, were highly resistant to the antibiotics they produced. Polyphenylalanine synthesis in cell free systems was also resistant to the action of the antibiotics. Reciprocal exchange between ribosomes and S150 fractions from the three strains revealed that the S150 fraction of each strain had an enzyme activity that inactivated the appropriate antibiotic whereas the ribosomes were susceptible to the antibiotics. It was concluded that the resistance of the in vitro polyphenylalanine synthesizing systems of these antibiotics was due to the presence of inactivating enzymes. Furthermore, S. fradiae and S. kanamyceticus were highly resistant to aminocyclitol-containing aminoglycoside antibiotics other than those produced by the two strains. In these cases, the inactivating enzymes were found to have a major role in the resistance mechanism. However, the resistance of S. kanamyceticus ISP5500 to streptomycin seems to be due to resistance at the ribosomal level.

Self-resistance of a Streptomyces which produces istamycins.

Streptomyces tenjimariensis SS-939, a producer of istamycins, is highly resistant to its own antibiotics and grows in Tryptic Soy Broth containing istamycin A or B at 3,000 microgram/ml. No istamycin-inactivating enzyme was detected in extracts of strain SS-939. Polyphenylalanine synthesis in an in vitro system, consisting of the S-150 fraction and ribosomes prepared from strain SS-939, was not inhibited by 200 microgram/ml of istamycins. Using reciprocally reconstituted systems consisting of S-150 fractions and ribosomes from strain SS-939 and those from Streptomyces griseus ISP5236 (istamycin-sensitive strain), ribosomes of strain SS-939 were found to be resistant to istamycins. Thus, ribosomes have the main role in the self-resistance mechanism of S. tenjimariensis SS-939.

New antibiotic-producing streptomycetes, selected by antibiotic resistance as a marker. II. Features of a new antibiotic-producing clone obtained after fusion treatment.

A new antibiotic-producing Streptomyces strain SK2-52 obtained by a protoplast fusion treatment between Streptomyces griseus NP1-1 and S. tenjimariensis NM16 showed taxonomical features identical with those of S. griseus. The strain resistant to wider range of aminoglycoside antibiotics than the parental strains. This multiple resistance corresponded to the activities of streptomycin kinase and acetyltransferase which were probably derived from S. griseus NP1-1. Clones with fast-growth and reduced antibiotic productivity frequently segregated from strain SK2-52, while their antibiotic resistance was stable. The results suggest that the fusion treatment caused a genetic change in S. griseus which enhanced the expression of genes for unique multiple resistance to aminoglycoside antibiotics and also induced new antibiotic production.

Bagougeramines A and B, new nucleoside antibiotics produced by a strain of Bacillus circulans. I. Taxonomy of the producing organism and isolation and biological properties of the antibiotics.

A bacterial isolate from soil, designated as TB-2125 had a unique pattern of multiple resistance to aminoglycoside antibiotics (AG) and produced new nucleoside antibiotics. Taxonomic properties of this strain fell into those of Bacillus circulans, providing unique characteristics such as strict susceptibility to acidic pH, motility of colony as well as multiple AG-resistance. Two new antibiotics which were named bagougeramines A and B had a broad antimicrobial activity and a specific activity against the two spotted spider mite.

Bagougeramines A and B, new nucleoside antibiotics produced by a strain of Bacillus circulans. II. Physico-chemical properties and structure determination.

Bagougeramines A and B obtained as sulfates were soluble in water and positive to Sakaguchi, chlorine-tolidine and ninhydrin color reactions. Their structures were determined by acid hydrolysis and spectroscopic analysis. Structurally they were closely related to gougerotin and they contained the guanidino-D-alanine instead of the serine residue in gougerotin. Bagougeramine B had the spermidine instead of the 6'-NH2 in structure of bagougeramine A.

Altemicidin, a new acaricidal and antitumor substance. I. Taxonomy, fermentation, isolation and physico-chemical and biological properties.

Screening of new insecticidal and acaricidal antibiotics was carried out with reference to anti-brine shrimp activity from actinomycete strains isolated from marine environments. Of 200 actinomycete isolates, one isolate was found to produce a new substance, altemicidin. The strain was isolated from sea mud collected at Gamo, Miyagi Prefecture, Japan, and identified as Streptomyces sioyaensis SA-1758. Altemicidin was purified by Diaion CHP-20P and Sephadex LH-20 column chromatographies. The molecular formula was determined as C13H20N4O7S by elemental analysis, MS and 13C NMR spectrum. Altemicidin showed not only acaricidal activity but also antitumor activity. The compound showed no antimicrobial activity except the inhibitory activity to Xanthomonas strains.

Multiple resistance to aminoglycoside antibiotics in actinomycetes.

Actinomycetes were characterized in terms of resistance to 11 different aminoglycoside antibiotics (AGs). Strains freshly isolated in AG containing media showed wide varieties of multiple AG resistance, while the majority of ISP (International Streptomyces Project) cultures and the actinomycete strains isolated in an AG free medium were susceptible to all or most of the AGs tested. Marked characteristics were noted in multiple AG resistance of gray and yellow colored actinomycetes and AG-producing strains. In gray colored isolates, multiple resistance to kanamycin A, dibekacin, ribostamycin, butirosin A, istamycin A and neamine was often observed. Yellow colored isolates having multiple AG resistance were mostly resistant to neamine, ribostamycin and streptomycin and, to a lesser extent, istamycin A, dibekacin and butirosin A. Most of the AG producers tested showed unique multiple AG resistance patterns.

Structure of aplasmomycin.

A new antibiotic, aplasmomycin, was isolated from a broth cultivated with a marine isolate of actinomycete, and inhibits Gram-positive bacteria in vitro and Plasmodium berghei in vivo. It is a natural ionophore and the structure of the Ag-salt was solved by an X-ray crystallographic analysis. It has symmetric structure having boron in the centre of the molecule.