Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non‐ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N‐methylated cyclic dipeptides such as thaxtomins. Based on high‐performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.

The modABC gene products constitute the molybdate-specific transport system in Escherichia coli. Another operon coding for two proteins which diverges from the modABCD operon has been identified. The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa The presence of molybdenum is required for the activity of several enzymes found in animals, plants, and bacteria, such as sulfite oxidase, xanthine dehydrogenase, nitrate reductase, formate dehydrogenase, and nitrogenase (46). In these organisms, the molybdenum is present in the molybdoenzymes in the form of a pterin-containing molybdenum cofactor (MoCo), with the exception of that found in nitrogenase, which has an ironmolybdenum cofactor (FeMoCo) (1,16,34).In Escherichia coli, the successful production of molybdoenzymes relies upon the efficient uptake of molybdate via the molybdate-specific transporter encoded by the modABCD operon (formerly known as the chlD locus) (18,26,35,42). The E. coli molybdate transport machinery, in which modA encodes a molybdate-specific periplasmic binding protein, modB encodes an integral membrane channel-forming protein, and modC encodes an ATP-binding energizer, closely resembles the established ATP-binding cassette (ABC) transporter motif (15, 26). Homologous molybdate transport systems have also been described for Azotobacter vinelandii, Rhodobacter capsulatus, and Haemophilus influenzae (9,23,45).Initial studies of the characterization of mod mutants revealed that strains harboring these mutations were incapable of producing nitrate reductase or formate dehydrogenase activity without molybdate supplementation (11,14,27,36,39,41,43). There are data to suggest that when mod mutants are grown in molybdate-supplemented media, molybdate enters the cells by means of the sulfate transport system as well as other nonspecific anion transporters (22,36). Some of the mod mutants were also analyzed for their molybdate uptake kinetics and were shown to transport molybdate at a much lower rate than mod ϩ strains did (5, 14). Further investigations of mod mutants have demonstrated that the modABCD operon is regulated by the intracellular concentration of molybdate. Specifically, high levels of molybdate reduced the level of transcription of the transport genes (27,35,36). This reduction in the level of transcription of the modABCD operon is most likely mediated by a molybdate-activated repressor protein.An analysis of the modABCD DNA sequence revealed a region in between the transcription and translation start sites of the modA gene which contains an 8-base inverted repeat (TAAC ⅐ GTTA) flanked by two CAT (CA) boxes (26,35,36). There are indications that either the CAT boxes or the inverted repeat or both are involved in the binding of the mod operon repressor (35). The inverted repeat found in this region is analogous to the inverted-repeat sequences implicated as the target binding sites for both the Met and Trp repressor (MetJ and TrpR) proteins (21,32,33).In our laboratory, a derivative of strain SE2069 ...

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H ؉ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.All living systems generate the needed energy for cell maintenance and growth by catalyzing a set of coupled oxidationreduction reactions. A critical component of these oxidationreduction reactions is the redox balance of the sum of all metabolic reactions. With external input of an oxidant, such as dioxygen, the carbon and energy source can be completely oxidized to carbon dioxide with concomitant reduction of the terminal electron acceptor. However, under strict anaerobic conditions and in the absence of an external input of an oxidant, such as nitrate, metabolic intermediates serve the role of oxidant to maintain the overall redox balance. For many heterotrophic fermentative bacteria, the primary oxidant is pyruvate or derivatives of pyruvate, the terminal product of glycolysis. The redox state of total carbon in all final end products produced by the anaerobic cell should equal the redox state ...

Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the effects of particular mutations on the regulation of the mod operon as well as the fdhF and hyc operons which code for the components of the formate hydrogenlyase (FHL) complex. ⌽(hyclacZ ؉ ) mod double mutants produced ␤-galactosidase activity only when they were cultured in medium supplemented with molybdate. This requirement was specific for molybdate and was independent of the moa, mob, and moe gene products needed for molybdopterin guanine dinucleotide (MGD) synthesis, as well as Mog protein. The concentration of molybdate required for FHL production by mod mutants was dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the production of half-maximal FHL activity was increased approximately 20 times by the addition of 40 mM of sulfate. mod mutants growing in low-sulfur medium transported molybdate through the sulfate transport system, as seen by the requirement of the cysA gene product for this transport. In wild-type E. coli, the mod operon is expressed at very low levels, and a mod ؉ merodiploid E. coli carrying a modA-lacZ fusion produced less than 20 units of ␤-galactosidase activity. This level was increased by over 175 times by a mutation in the modA, modB, or modC gene. The addition of molybdate to the growth medium of a mod mutant lowered ⌽(modA-lacZ ؉ ) expression. Repression of the mod operon was sensitive to molybdate but was insensitive to mutations in the MGD synthetic pathway. These physiological and genetic experiments show that molybdate can be transported by one of the following three anion transport systems in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering the cytoplasm, molybdate branches out to mod regulation, fdhF and hyc activation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis. Synthesis of active molybdoenzymes byEscherichia coli is dependent on the various gene products required for the transport of molybdate into cells (5,9,12,25,29,39,40,43), metabolic conversion of molybdate to an appropriate form, and incorporation into the pterin component of molybdopterin guanine dinucleotide (MGD) during maturation of the apoprotein to active enzyme (6,8,34,46; see references 13, 38, and 46 for reviews). Several mutant strains of E. coli which are defective in one or more of these steps have been isolated and described. Mutants defective in the first step of this process, molybdate transport, were isolated as chlorate-resistant derivatives (chlD [renamed as mod]) (41) whose phenotype can be suppressed by supplementation of the growth medium with molybdate (5,9,12,13,29,40,43,45). The rates of molybdate transport and internal molybdate concentrations of some of these mutants were determined (5, 12, 40). Recent studies have established that the mod operon contains four genes, of which three r...

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3 of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as 1 H-and 13 C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other wellcharacterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.

Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.

The levels of DNA-DNA hybridization among members of the three major groups of Streptomyces spp. that cause potato scab did not exceed 20% for any pair. The majority of the Streptomyces scabies strains which we examined exhibited greater than 70% relatedness to the type strain, although values as low as 21% were obtained. The levels of homology between S . scabies and nonpathogenic type strains belonging to the Diastatochromogenes group ranged from 10 to 42 9% , while the reciprocal values obtained with labeled DNAs from Streptomyces bottropensis ATCC 25435T (T = type strain) and Streptomyces eurythermus ATCC 14975T ranged from 37 to 74% and from 2 to 24%, respectively. In contrast to S . scabies, the levels of relatedness between Streptomyces acidiscabies isolates and their type strain were high (83 to 111%). The levels of DNA relatedness within the Streptomyces aZbidoJlavus group were uniformly low. These results are consistent with phenotypic data which indicate that pathogens in the three groups are not related. The genetic diversity of strains referred to as S . scabies exceeds the genetic diversity found at the species level, and some of these strains appear to be related to phenotypically similar nonpathogens.Scab diseases of potatoes (Solanum tuberosum) and certain other root crops are caused by streptomycetes, including the major species Streptomyces scabies, which was first described by Thaxter in 1891 (11, 22), the acid-tolerant species Streptomyces acidiscabies (12), and other strains whose characteristics are consistent with those of the Streptomyces albidojlavus group of Williams et al. (11, 24). S. scabies is most closely related to the Diastatochromogenes group of Elesawy and Szabo (2), while the S. albidoflavus strains have been associated with Streptomyces griseus and Streptomyces tendae (7). The results of studies of 42 phenotypic traits (11, 12) have suggested that these three pathogen groups are not closely related and that the levels of strain diversity are lowest for S . acidiscabies and greatest for the S . albidoflavus group. The purposes of this study were to confirm these relationships, to determine the genomic relationship of S . scabies to the Diastatochromogenes group, and to interpret the significance of common pathogenicity mechanisms among potato scab pathogens. MATERIALS AND METHODSStrains. The isolates which we used are listed in Table 1 DNA extraction. Total DNAs were isolated from Streptomyces strains by using the method of Hintermann et al. (6), with some modifications. Mycelia grown in Trypticase soy broth shake cultures were homogenized, centrifuged, rinsed, and suspended in 5 ml of cold TS buffer (50 mM Tris, 0.75 M sucrose; pH 8.0). Lysozyme hydrochloride and EDTA were added to concentrations of 5 mg/ml and 0.1 M, respectively.After incubation at 37°C for 1 h, 1 mg pronase E per ml was added, and incubation was continued for 15 min. Sodium dodecyl sulfate was added to a concentration of 1%, and the suspensions were incubated at 37°C until full lysis was achieved (30 to 6...

Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.