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The effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of

Clavulanic acid, a β-lactamase inhibitor, is used together with β-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other β-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.

Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

Summary Streptomyces coelicolor produces an extracellular protease inhibitor protein, STI (Streptomyces trypsin inhibitor). We show that post-growth elimination of STI is needed for colonies to develop aerial mycelium efficiently. Inactivation of STI, and thus the normal progression of colony development, at least partly involves an extracellular protease specified by gene SCO5913. Two-hybrid analysis identified two possible targets of STI inhibition (the products of SCO1355 and SCO5447), both extracellular proteases containing a domain homologous with the P-domain of eukaryotic convertases, proteases that mediate the processing of many precursors with important cellular or developmental roles. At least the SCO1355 protease is needed for the normal progression of development. Two components of the proposed cascade are dependent on the tRNA for the rare UUA (leucine) codon, which is specified by the developmental gene bldA. A model is presented that links intracellular regulatory events with an extracellular protease cascade to facilitate normal development.

Three open reading frames denoted as orf21, orf22, and orf23 were identified from downstream of the currently recognized gene cluster for clavulanic acid biosynthesis in Streptomyces clavuligerus ATCC 27064. The new orfs were annotated after in silico analysis as genes encoding a putative sigma factor, a sensor kinase, and a response regulator. The roles of the individual genes were explored by disruption of the corresponding orfs, and the morphological and antibiotic production phenotypes of the resulting mutants were compared. In orf21 and orf22 mutants, no growth or morphological differences were noted, but modest reduction of cephamycin C (orf21), or both cephamycin C and clavulanic acid production (orf22) compared with wild-type, were observed. In orf23 mutant, cell growth and sporulation was retarded, and clavulanic acid and cephamycin C production were reduced to 40 and 47% of wild-type levels, respectively. Conversely, overexpression of orf23 caused precocious hyperproduction of spores on solid medium, and antibiotic production was increased above the levels seen in plasmid control cultures. Transcriptional analyses were also carried out on orf23 and showed that mutation had little effect on transcription of genes associated with the early stages of cephamycin C or clavulanic acid production but transcription of claR, which regulates the late stages of clavulanic acid production, was reduced in orf23 mutants. These observations suggest that the orf23 product may enable S. clavuligerus to respond to environmental changes by altering cell growth and differentiation. In addition, the effects of ORF23 on growth might indirectly regulate the biosynthesis of secondary metabolites such as clavulanic acid and cephamycin C.

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor . Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm -deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor .

The emergence and dissemination of extended-spectrum (ES) beta-lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by third-generation beta-lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C beta-lactamase with a wide spectrum of substrates. Unlike the well-studied class C ES beta-lactamase from Enterobacter cloacae GC1, the Omega-loop does not affect the active site conformation and the catalytic activity of CMY-10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 A resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of beta-lactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 beta-lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES beta-lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C beta-lactamases to extend their substrate spectrum.

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.

The effects of growth rate and nutrient uptake rate on the production of cephamycin C were determined in the parental strain, ΔrelA mutant, and Δrsh null mutant of S. clavuligerus . Production of cephamycin C was inversely related to mycelium growth and the phosphate feed rate was more critical for the production of cephmycin C. On the contrary, the production of cephamycin C was completely abolished in the ΔrelA mutant, but not in Δrsh mutant. The changes in the cephamycin C production by disruption of the relA and rsh genes are presumably associated with the consequent ability of the mutants to accumulate (p)ppGpp under nutrient starvation. Therefore, it is concluded that the stringent response of S. clavuligerus to starvation for nutrients is governed mainly by RelA rather than Rsh and that the response is more apparently regulated by the limitation of phosphate.

To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of Enterobacter species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the ampC genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four ampC genes ( bla EcloK992004.1 , bla EcloK995120.1 , bla EcloK99230 , and bla EareK9911729 ) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal ampC gene ( bla EcloMHN1 ) of Enterobacter cloacae MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two ampC genes ( bla EcloK9973 and bla EcloK9914325 ) showed close similarity to the chromosomal ampC gene ( bla EcloQ908R ) of E. cloacae Q908R (99.7% identity). The results from phylogenetic analysis suggested that six ampC genes could originate from bla EcloMHN1 or bla EcloQ908R .

The structure of the 28 kDa β-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 β-lactamase has been determined to 2.3 A resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed β-propeller with a unique blade motif consisting of only three antiparallel β-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first β-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1–BLIP and TEM-1–BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.

The diagnostic ability of the restriction fragment length dimorphism-polymerase chain reaction (RFLD-PCR) method was evaluated. Seven primer pairs, newly designed from 44 beta-lactamase genes encoding extended-spectrum beta-lactamases not related to TEM- and SHV-types, were used to differentiate OXA-2, FOX-3, CMY-3, IMP-1, and IMI-1 beta-lactamases. The RFLD-PCR was carried out successfully, and these genes were differentiated by the sizes of their PCR products and by the difference in restriction fragment length when each amplicon was digested with a unique restriction enzyme. This discriminatory detection of the genes was confirmed by sequencing the PCR products.

A new beta-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N-terminal sequence was NH(2)-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln-Phe-Gly. BLIP-I inhibited Bacto(R) Penase (Difco), and plasmid encoded TEM-1 beta-lactamase, whereas it did not inhibit Enterobacter cloacae beta-lactamases. The K(i) value of BLIP-I against TEM-1 beta-lactamase was determined to be 0.047 nm. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K(i) value of rBLIP-I against TEM-1 beta-lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K(i) of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild-type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation.

A soil isolate, which had been assigned to the genus Nocardia, was shown to have properties consistent with its classification in the genus Amycolatopsis. An almost complete nucleotide sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for members of the family Pseudonocardiaceae and related taxa and phylogenetic trees were inferred using three tree-making algorithms. The organism consistently formed a distinct monophyletic clade with the type strain of Amycolatopsis methanolica, but DNA-DNA relatedness data showed that the two strains belonged to distinct genomic species. The organism was also distinguished from the type strains of all validly described species of Amycolatopsis using a battery of phenotypic properties. The genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Amycolatopsis. The name proposed for the new species is Amycolatopsis thermoflava sp. nov. The type strain is IFO 14333T.

The effects of growth rate and nutrient feed rate on the production of actinorhodin (Act) and undecylprodigiosin (Red) were determined in Streptomyces coelicolor A3(2) and in a congenic relA null-mutant known to be deficient in ppGpp synthesis and antibiotic production under conditions of nitrogen limitation. In the relA+ strain, Act production was inversely related to specific growth rate in continuous cultures limited by glucose, ammonium, or phosphate, while Red biosynthesis was optimal at 0.05 h-1 regardless of the specific nutrient limitation. Production of Act and Red in the relA mutant was lower than that of the parental strain, particularly under conditions of glucose- and ammonium-limitation, indicating an important and general role for ppGpp in determining the onset of the antibiotic biosynthesis under conditions of nutrient limitation. At constant growth rate, but with varying nutrient feed rates, the specific rate of Act production was adversely influenced by increasing levels of glucose, ammonium, and phosphate, with phosphate having the greatest inhibitory effect. Under the same conditions, the specific rate of Red production was stimulated by increasing glucose levels, but markedly decreased by increased levels of phosphate.

A -lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF19 and its N-terminal amino acid sequence was determined. A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II. The bliB gene was isolated and sequenced. Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence. bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but -lactamase-inhibitory activity was recovered after refolding. In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the me/C1 promoter. The BLIP-II protein produced in recombinant strains of S. lividans was secreted into the culture supernatant in a biologically active form.

An actinomycete strain, 'Nocardai flavorosea' JCM 3332, was found to have properties consistent with its classification in the genus Nocardia. An almost complete gene sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organisms consistently formed a distinct clade with the type strain of Nocardia carnea. However, DNA relatedness experiments showed that the strain and N. carnea DSM 43397T belonged to two distinct genomic species. The organism was also distinguished from representative of all of the validly described species of Nocardia using a combination of phenotypic properties. These genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Nocardia. The name proposed for the new species of is Nocardia flavorosea sp. nov. The type strain is JCM 3332T.

The morphological differentiation of Streptomyces albidoflavus SMF301 in a batch culture was analyzed by pyrolysis-mass spectrometry (PyMS). Curie point pyrolysis mass spectra of whole cells at various growth phases were obtained. The PyMS spectra varied with growth phase and differentiation. It was possible to distinguish differentiation states with multivariate statistics and artificial neural network (ANN). Artificial neural networks (ANNs) were trained on PyMS data to predict the differentiation state using two different algorithms: backpropagation and a radial basis function classifier. Both the backpropagation and the radial-basis classifier succeeded in separating the differentiation state and identified the transient state.

Leupeptin-inactivating enzyme (LIE) was purified from Streptomyces exfoliatusSMF13 by ammoniumm sulphate fractionation of cell-free culture broth, ultrafiltration, anion-exchange chromatography on DEAE–Sephadex A-50 and gel filtration chromatography on Sephadex G-75. The molecular mass of the purified enzyme was measured as 34700 Da and the N-terminal amino acid sequence was APTPPDIPLANVPA. Acetyl-leucine, leucine and argininal were identified as the products of leupeptin inactivated by the LIE, indicating that leupeptin is inactivated by hydrolysis of peptide bond between leucine and leucine and between leucine and argininal of leupeptin (acetyl-leucine-leucine-argininal). Synthetic-peptide substrates specificity of LIE showed that LIE has absolute specificity for peptide bonds with leucine in the P1 position, suggesting that LIE is a leucine-specific protease. The optimum pH and temperature were pH 9.0 and 45 °C, respectively. LIE activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, o-phenanthroline and bestatin, but activated by Mg2+ and Ca2+, suggesting that the enzyme is a metalloprotease. Aerial-mycelium growth and aerial spore formation of S. exfoliatus SMF13 were inhibited by the addition of bestatin, an inhibitor of LIE. The inhibition of morphological differentiation was due to the inhibition of trypsin-like protease (TLP) activity, which is essential for aerial-mycelium formation and is inhibited specifically by remaining leupeptin that was not inactivated. These results show that LIEs play a role in controlling the amount of leupeptin during colony development. Therefore, it is suggested that the physiological function of LIE is to inactivate leupeptin when or where TLP activity is required for aerial-mycelium formation.

Summary: The effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of Streptomyces albidoflavus SMF301 were determined in chemostat cultures. Production of three types of proteases: chymotrypsin-like protease (CTP), trypsin-like protease (TLP) and metalloprotease (MTP) were analysed in relation to mycelium growth and spore formation. Production of CTP was closely linked to mycelium growth, whereas spore formation, TLP synthesis and MTP synthesis were inversely related to growth. Evaluation of various kinetic parameters [specific production rates of spores (q spo), TLP (q TLP), MTP (q MTP) and CTP (q CTP)] showed that mycelium growth rate and CTP production were optimal at 0.1 h−1, but submerged spore formation, TLP production and MTP production were optimal at 0.025 h−1. Changes in specific nutrient uptake rates [glucose (q glu), ammonium ion (q amn) and phosphate (q pho)] affected sporulation and protease production; limitation of carbon, nitrogen and phosphate stimulated spore, TLP and MTP production.

The number and content of the recombinant plasmid (pDML6) in Streptomyces lividans PD6 increased as the specific growth rate decreased. The specific expression rate of the cloned gene, calculated from cultivation in a glucose-limited chemostat, was closely related to the specific growth rate but not to the dosage of cloned gene.

Three proteases, namely chymotrypsin-like protease (CTP), trypsin-like protease (TLP) and metalloprotease (MTP), were identified in cultures of Streptomyces albidoflavus SMF301. The dynamics of protease production were determined and the roles of the proteases in morphological differentiation were deduced to be as follows: CTP is essential for hydrolysing the proteinaceous nitrogen source for mycelium growth; TLP plays a role in the formation of thickened mycelium in submerged culture and of aerial mycelium in solid culture; MTP may participate in the maturation of spores. The unique thickened mycelia in submerged culture are thought to be an intermediate form between mycelium and spores. TLP (molecular mass 32 kDa) and MTP (molecular mass 18 kDa) were purified and their enzymic properties were determined.

Among Streptomyces fradiae parent strain (NRRL 2702), aspartate auxotroph strain (SMF 305), and revenant strain (SMF 306), the revertant strain is the highest producer of tylosin and showed different repression patterns of tylosin production by ammonium ion from the parent strain. These results were elucidated by the facts that the revertant strain was superior to the parent or auxotroph strain in the biosynthesis of glutamine synthetase involved in ammonium assimilation and in the biosynthesis of threonine dehydratase (TDT) involved in providing precursors necessary for tylosin production, and ammonium ion inhibited the activity of TDT purified from the parent strain more than that of TDT from the revertant strain. TDT from the parent strain has been purified by DEAE cellulose, hydroxyapatite, Mono Q HR 5/5, and reversed-phase Protein C 4 chromatography. The molecular mass was 60 kDa by SDS-PAGE and 240 kDa by gel filtration. The N-terminal amino acid sequence of TDT was NH 3 -E-A-T-G-P-L-T-T-E-S-G-A-P-V. The activity of TDT was allosterically activated by adenosine monophosphate.

The physiological and nutritional factors regulating production of leupeptin, leupeptin-inactivating enzyme (LIE), and trypsin-like protease (TLP) in Streptomyces exfoliatus SMF13 were evaluated in batch and continuous cultures. Production of leupeptin in batch culture was closely associated with mycelial growth rate, but leupeptin was inactivated by LIE during the stationary phase. Production of LIE and TLP was induced when mycelial growth reached the stationary phase. Leupeptin production was reactivated by the external addition of glucose or leupeptin precursors (leucine or arginine) to cultures during the stationary phase, whereas the production of LIE and TLP was repressed. The production of leupeptin was related to mycelial growth rate, whereas the production of LIE and TLP was inversely related to mycelial growth rate in glucose-limited continuous culture. The production of leupeptin was enhanced under carbon-excess and phosphate-limited conditions, while the production of LIE and TLP was repressed. Production of LIE and TLP was strongly enhanced under the condition when both carbon and nitrogen were limited. Therefore, it is concluded that regulation of production of leupeptin is inversely related to that of LIE and TLP.

Rifamycin B production in batch culture of Nocardia mediterranei was compared with mycelium morphological changes. Rheological behaviour of batch culture medium was identified and various rheological parameters were evaluated in order to characterize quantitatively the morphological changes. Rifamycin B production kinetic parameters in the batch culture were also calculated and the parameters were compared with the rheological parameters. Consequently, specific rifamycin B production rate ( q rif ) was closely related to yield shear stress ( τ 0 ) and to morphological factor ( δ ∗ ). Both parameters are considered to be important for the production control of rifamycin B.

We report the first quantitative analysis of the relationship between environmental changes and sporulation of a streptomycete, Streptomyces albidoflavus SMF301, in submerged culture. A chemically defined medium was constructed for sporulation, over 10(9) spores ml-1 being formed in the submerged batch culture. Kinetic parameters calculated from batch and chemostat cultures showed that specific submerged spore formation rate (qspo) was inversely related to the specific mycelial growth rate (mu). The optimum growth rate for submerged spore formation was 0.05 h-1, when the maximum value of qspo was 1.0 x 10(6) spores g-1 h-1. The turnover rate of biomass at maximum growth yield was 0.029 h-1, when 5.6 x 10(6) spores were formed from 1 g mycelium. The present quantitative analysis of submerged spore formation using a controlled system opens the way for biochemical and molecular biological studies related to the morphological differentiation of Streptomyces spp.

SUMMARY: Streptomyces albidoflavus SMF301 produced abundant spores in submerged cultures (submerged spores) as well as on solid media (aerial spores). The content of carbon, hydrogen, nitrogen, and phosphorus in submerged and aerial spores was similar; however, the contents of metal ions (K+, Na+, Ca2+ and Mg2+) were very different. Glutamic acid, alanine, and glycine, all known to be cell-wall components, were the major amino acids in both types of spores. However, cysteine was more abundant in submerged spores than in aerial spores. The major fatty acid in aerial spores was n-C18 (61.74%), whereas in submerged spores it was ai-C16 (33.68%). The contents of ai-C14, and ai-C17 in submerged spores were also very much higher than in aerial spores. Unsaturated fatty acids were found in both kinds of spores but not in mycelium; they were particularly abundant in submerged spores. The composition of menaquinones in the two kinds of spores also varied. The resistance of aerial spores to lysozyme digestion, mild acid treatment, heating and desiccation was higher than that of submerged spores, but the submerged spores were more resistant to sonication.

The beta-lactamase inhibitory protein, BLIP-II, found in the culture supernatant of Streptomyces exfoliatus SMF19, shows no discernible sequence identity with other beta-lactamase inhibitory proteins identified in Streptomyces spp. A null mutant of the gene encoding BLIP-II (bliB::hygr) showed a bald appearance on solid media. Although BLIP-II was initially isolated from the supernatant of submerged cultures, sites of BLIP-II accumulation were seen in the cell envelope. The mutation of bliB was also associated with changes in the formation of septa and condensation of the chromosomal DNA associated with sporulation. The bliB mutant exhibited infrequent septa, showing dispersed chromosomal DNA throughout the mycelium, whereas the condensed chromosomes of the wild-type were separated by regularly spaced septa giving the appearance of a string of beads. Therefore, on the basis of these results, it is suggested that BLIP-II is a regulator of morphological differentiation in S. exfoliatus SMF19.

Beta-lactamase inhibitory protein (BLIP) binds a variety of beta-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein-protein interactions. Published studies have examined the binding of BLIP to diverse target beta-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme-inhibitor interface has remained unexplored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has beta-lactamase inhibitory activity very similar to that of BLIP; and (2) beta-lactamase-inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra-high-resolution structure), which is unable to inhibit any tested beta-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I.TEM-1 versus those formed at the interface of BLIP.TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein-protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation possible in the subnanomolar binding interaction with TEM-1. The corresponding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone-backbone/backbone-side-chain interactions with a protein partner can be negated by the presence of a few, strongly unfavorable interactions, especially electrostatic repulsions.

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis . However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σ F , suggesting that it is an antagonistic regulator of σ F . Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σ F during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis . It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.

The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by knock-in a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.

High oil prices, increasing focus on renewable carbohydrate-based feedstocks for fuels and chemicals, and the recent publication of its genome sequence, have provided continuing stimulus for studies on Zymomonas mobilis. However, despite its apparent advantages of higher yields and faster specific rates when compared to yeasts, no commercial scale fermentations currently exist which use Z. mobilis for the manufacture of fuel ethanol. This may change with the recent announcement of a Dupont/Broin partnership to develop a process for conversion of lignocellulosic residues, such as corn stover, to fuel ethanol using recombinant strains of Z. mobilis. The research leading to the construction of these strains, and their fermentation characteristics, are described in the present review. The review also addresses opportunities offered by Z. mobilis for higher value products through its metabolic engineering and use of specific high activity enzymes.

The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA , previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA , also known as bldH , a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.

This study is focused on the involvement of the unusual nucleotide (p)ppGpp during the morphological and physiological differentiation of Streptomyces clavuligerus. In particular, the functional and structural elements of two genes encoding the proteins RelA and Rsh were identified. The relA gene encodes an 843 aa protein (RelA), while the rsh gene encodes a 738 aa protein (Rsh). The relA and rsh genes were disrupted by the insertion of a hygromycin resistance gene and an apramycin resistance gene, respectively. The synthesis of ppGpp in the relA gene-disrupted mutant was completely eliminated under conditions of starvation for amino acids, whereas synthesis persisted, but was greatly reduced in the rsh gene-disrupted mutant. The relA gene-disrupted mutant had a bald appearance on agar plate cultures and retarded growth in submerged culture, while the rsh-disrupted mutant was unchanged in growth characteristics relative to the wild-type culture. The production of both clavulanic acid and cephamycin C were completely abolished in the relA-disrupted mutant. Thus, it is concluded that the relA gene rather than rsh is essential for morphological and physiological differentiation in S. clavuligerus and that RelA primarily governs the stringent response of S. clavuligerus to starvation for amino acids.

Among the 51 clinical isolates collected from a university hospital in Korea, nine isolates were resistant to cephamycins. Nine isolates were shown to produce CMY-11 and these also included three isolates producing TEM-1. The results from ERIC-PCR revealed that dissemination of CMY-11 was due to outbreaks of resistant species and to the intra-species spread of resistance to cephamycins in Korea. CMY-11 beta-lactamase genes from nine clinical isolates that were responsible for resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin and amoxicillin-clavulanic acid, were cloned and characterised. A sequence identical to the common regions in In6, In7 and a novel integron from pSAL-1 was found upstream from bla(CMY-11) gene at nucleotide 1-71. Eighteen nucleotides between position 71 and 72 were inserted into the bla(CMY-11) gene.

We report the description of a new plasmid-encoded AmpC-type beta-lactamase gene (bla(CMY-11)) from Escherichia coli K983802.1 that was isolated from a patient in South Korea suffering from a urinary tract infection. Antibiotic susceptibility testing, plasmid analysis, pI determination, transconjugation and Southern blot analysis were carried out to investigate the resistance mechanism to cefoxitin. PCR, sequencing and sequence analysis were used to identify and analyse the beta-lactamase gene (bla(CMY-11)) responsible for the cefoxitin resistance. CMY-11 and bla(CMY-11) are compared with other class C beta-lactamases and their genes to determine phylogenetic relationships. The cefoxitin-resistance phenotype of E. coli K983802.1 reflects the presence of a large plasmid [pYMG-2 (130 kb)]. A beta-lactamase with a pI value of 8.0 from a transconjugant of E. coli K983802.1 was identified by isoelectric focusing. A 1478 bp DNA fragment from pYMG-2 containing bla(CMY-11) was sequenced and an open reading frame coding for a 382 amino acid peptide (CMY-11) was found. Phylogenetic analysis clearly shows that bla(CMY-11) belongs to the group of ampC-related bla genes. It is likely that bla(CMY-11) evolved from bla(CMY-1) via bla(CMY-10).

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

Plasmid-mediated resistance mechanisms to beta-lactams, comprising mostly extended-spectrum beta-lactamase (ESBL) production, lead to resistance against even the most recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. In this work, the diagnostic ability of the restriction fragment length dimorphism (RFLD)-polymerase chain reaction (PCR) method in clinical samples was evaluated. Nine newly designed primer pairs were used to differentiate the genes encoding TEM-1a, SHV-12, MOX-1, MIR-1 and Toho-1 beta-lactamases. The RFLD-PCR was carried out successfully and these genes were differentiated by the sizes of their PCR product. This discriminatory detection of the genes was also confirmed by digestion with unique restriction enzyme sites and sequencing of the PCR products. The fragment sizes of PCR products digested with the enzymes were identical to the sizes calculated from nucleotide sequences of five beta-lactamase genes deposited in EMBL, GenBank and/or DDBJ databases and the sequences were also identical. In conclusion, the method and newly designed primers applied in this work can differentiate the ESBLs rapidly and effectively.

Medium composition and cultivation conditions were constructed for the optimum production of leupeptin by Streptomyces exfoliatus SMF13. The production of leupeptin was related to mycelial growth, being optimum in the cultivation with glucose-excess, phosphate-limited, and casamino acids medium. However, leupeptin-inactivating enzyme (LIE) was produced in the cultivation with glucose-limited, phosphate-excess, and Na-caseinate medium where mycelium degradation was accompanied. LIE was one of the most important factors in optimizing the leupeptin productivity. Kinetic parameters calculated from batch and chemostat cultivations revealed that q lpt was closely related to q s and μ, but q LIE was increased after μ, declined to near zero, and followed by k d . Optimum production process of leupeptin was determined with phosphate-limited continuous cultivation, which did not permit LIE production. The maximum productivity (0.24 g l −1 h −1 ) and production yield (1.64 g leupeptin per g glucose) of phosphate-limited chemostat cultivation were 2.4- and 4-times larger than those of batch cultivation, respectively. This is the first cultivation kinetic analysis for leupeptin production and its inactivation by LIE in relation to mycelium differentiation.

The growth rate and environmental effects on the stability of recombinant plasmid, pDML6 containing β-lactamase gene, and the cloned gene product formation in Streptomyces lividans were studied. A maximum production rate of the cloned gene product was obtained at a specific growth rate 0.106 h −1 in glucose-limited chemostat cultivations without genetic selection pressure. Optimum environmental conditions for the recombinant plasmid stability and maximum formation rates of the cloned gene product were determined using continuous cultivations at the optimum specific growth rate. The fractions of plasmid harboring mycelium in prolonged cultivation up to 50 generations were varied from 77 to 95%. The recombinant plasmid was stably maintained in the host cells grown in different temperatures (24 to 36°C) and pH (6.0 to 8.5). The formation of the cloned gene product was optimum at pH 7.0 and 27°C, at which the maximum enzyme production rate was 0.82 kU g −1 h −1 . Continuous cultivations varying the dissolved oxygen tension (10 to 80% air saturation) showed that the plasmids were maintained stably and the specific enzyme production rates were increased with increasing dissolved oxygen levels.