Competition and antibiosis in the biological control of potato scab.

Nonpathogenic, antibiotic-producing streptomycetes have been shown to reduce potato scab when added to disease-conducive soil. Spontaneous mutants of the pathogenic Streptomyces scabies RB4 that are resistant to at least one antibiotic activity produced by the nonpathogenic suppressive isolates Streptomyces diastatochromogenes strain PonSSII and S. scabies PonR have been isolated. To determine the importance of antibiosis in this biocontrol system, these mutants were investigated for their ability to cause disease in the presence of the two pathogen antagonists in a greenhouse assay. Disease caused by one of the mutant strains was reduced in the presence of both suppressive isolates, whereas disease caused by the other five mutants was not significantly reduced by either suppressive strain. In addition, a nonpathogenic mutant of S. scabies RB4 was isolated, which produced no detectable in vitro antibiotic activity and reduced disease caused by its pathogenic parent strain when the pathogen and mutant were coinoculated into soil. Population densities of the pathogen were consistently lower than those of the suppressive strains when individual strains were inoculated into soil. When a pathogen was coinoculated with a suppressive strain, the total streptomycete population density in the pot was always less than that observed when the suppressive isolate was inoculated alone. When the pathogens were inoculated individually into soil, a positive correlation was seen between population density and disease severity. In coinoculation experiments with pathogen and suppressive strains, higher total streptomycete population densities were correlated with lower amounts of disease.

A single-use luciferase-based mercury biosensor using Escherichia coli HB101 immobilized in a latex copolymer film.

A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 microm thick whereas the bottom layer of copolymer plus E. coli cells was 30 microm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl2 in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl2. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at -20 degrees C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl2.

A patch coating method for preparing biocatalytic films of Escherichia coli.

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.

Quantitative relationships among thaxtomin A production, potato scab severity, and fatty acid composition in Streptomyces.

Thaxtomin A production in culture, potato common scab severity (percentage of tuber surface infected or number of lesions per tuber), and fatty acid profiles were determined for 78 Streptomyces isolates. Only pathogenic Streptomyces spp. (n = 17) produced thaxtomin A in culture. Thaxtomin A production in culture (microgram/mL) was significantly positively correlated with the percentage of tuber surface infected (R = 0.60; p = 0.017) but not with the number of lesions per tuber (R = 0.37; p = 0.17). An increase of 1 microgram/mL in thaxtomin A production corresponded to an 11% increase in disease severity (percentage of tuber surface infected). The data indicate that quantitative information on the ability of a particular pathogen isolate or population to produce thaxtomin A may be critical to understanding and predicting the disease potential of that population. Using cluster analysis of fatty acid data, 94% of 67 unknown field isolates grouped with other field isolates having the same pathogenicity (plus or minus).

Isolation and characterization of an antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII.

An antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII has been isolated and partially characterized. The antibiotic is produced throughout culture growth, with maximum amounts accumulating in the broth when the culture is in the early stationary phase of growth. The activity declines within about 30 h after the culture enters stationary phase. Purification techniques included chromatography on Amberlite XAD-2, DEAE Sephadex and SP Sephadex in addition to C18 HPLC with an average yield of 75%. This antibiotic only inhibits pathogenic strains of S. scabies that cause scab disease on potato and other tuberous vegetables and does not affect S. griseus, S. venezuelae, Actinomyces bovis, Nocardia asteroides, Clostridium perfringens, Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, Micrococcus luteus, Serratia marcescens and Escherichia coli. The antibiotic has a molecular weight of 500 or less, and is stable for weeks at acidic pH but is very labile at alkaline pH conditions.

Isolation of a mRNA instability sequence that is cis-dominant to the ompA stability determinant in Escherichia coli.

Transcriptional fusions with ompA and bla have been used to identify a novel mRNA instability element. A 287-nucleotide (nt) sequence containing a repetitive extragenic palindrome (REP) from the chloramphenicol acetyltransferase (cat) gene was inserted into the 3' untranslated region (UTR) of the ompA gene. In one orientation, the insert had no effect on the half-life of the ompA-cat chimeric transcript. In the other orientation, however, the sequence functioned as a destabilizing element and was dominant to the 5'-UTR ompA and REP stability elements. The orientation-dependent effect of the instability sequence suggests that sequence rather than structure alone is important to the function of the instability determinant. In addition, the instability sequence also destabilized an ompA-bla fusion construct when fused to its 3'-UTR region. A sensitive RNA ligation/PCR amplification technique was developed and used to analyze RNA decay intermediates. The results indicated that degradation of the chimeric transcript initiated within the 287-nt inserted sequence.

Use of repetitive intergenic DNA sequences to classify pathogenic and disease-suppressive Streptomyces strains.

PCR DNA fingerprinting using repetitive intergenic DNA sequences (rep-PCR) was investigated as a means of differentiating between closely related strains of Streptomyces which were, in some cases, indistinguishable by other classification methods. Our results demonstrated that the majority of strains had unique rep-PCR DNA fingerprints and established that the technique could be a very useful tool in rapidly determining strain identity.

Mutational analysis of the Streptomyces scabies esterase signal peptide.

Ten site-directed mutations affecting the predicted 39-amino-acid signal peptide of the Streptomyces scabies esterase were used to examine start-codon usage and esterase secretion in S. lividans. The first of two in-frame AUG codons was preferred for translation initiation. Removal of 2 of the 4 positively charged amino acids at the amino terminus of the signal peptide reduced esterase expression more than 100-fold; however, deletion of all 4 charged residues reduced expression by only 2- to 5-fold. Deletion of 4 or 8 amino acids from the hydrophobic core of the signal peptide reduced esterase production more than 200-fold, and a signal peptide processing site deletion completely disrupted esterase expression. For all constructs in which a mutation in the signal sequence decreased esterase production, esterase mRNA levels were also reduced, suggesting that a defect in secretion or processing affected esterase transcript abundance.

Cloning and sequencing of a secY homolog from Streptomyces scabies.

The complete DNA sequence of the Streptomyces scabies (Ss) secY homolog and partial sequences of adjacent upstream and downstream open reading frames (ORFs) have been determined. The nucleotide sequence of a 2-kb region predicts a polypeptide of 437 amino acids in length with homology to the SecY protein family. The Ss secY homolog lies upstream from a sequence that has homology to the adenylate kinase gene (adk) family. The translational stop codon of the putative SecY ORF overlaps the predicted start codon for the Adk ORF. Another ORF that lies upstream from the secY homolog has sequence similarity to the genes that code for the L15 r-protein. Within the 243-bp intergenic region between the L15 and SecY coding sequences, the presence of a streptomycete-like promoter sequence and an 18-bp inverted repeat suggests that the secY homolog and the adjacent downstream sequences may be transcribed independently of the L15 coding sequence. Transcript analysis indicates that the secY homolog is expressed in both Ss and Streptomyces lividans. The proposed gene and transcript organization of the L15-SecY-Adk coding regions in the Ss clone resembles that of Micrococcus luteus which, like the streptomycetes, has a G+C-rich genome.

A novel variant of the catalytic triad in the Streptomyces scabies esterase.

The crystal structure of a novel esterase from Streptomyces scabies, a causal agent of the potato scab disease, was solved at 2.1 A resolution. The tertiary fold of the enzyme is substantially different from that of the alpha/beta hydrolase family and unique among all known hydrolases. The active site contains a dyad of Ser 14 and His 283, closely resembling two of the three components of typical Ser-His-Asp(Glu) triads from other serine hydrolases. Proper orientation of the active site imidazol is maintained by a hydrogen bond between the N delta-H group and a main chain oxygen. Thus, the enzyme constitutes the first known natural variation of the chymotrypsin-like triad in which a carboxylic acid is replaced by a neutral hydrogen-bond acceptor.

CDP-6-deoxy-delta 3,4-glucoseen reductase from Yersinia pseudotuberculosis: enzyme purification and characterization of the cloned gene.

The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V.P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.

Crystallization and preliminary crystallographic data of a Streptomyces scabies extracellular esterase.

X-ray quality single crystals of an extracellular esterase from pathogenic Streptomyces scabies were obtained by the hanging drop method. The crystals are monoclinic (space group C2, a = 161.1 A, b = 51.2 A, c = 124.2 A, beta = 100.6 degrees) with two molecules related by a noncrystallographic dyad in the asymmetric unit, with a solvent content of approximately 64%. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube CuK alpha radiation. The study has been initiated in order to elucidate the mechanism of this unusual non-serine-dependent esterase, and to gain better understanding of the molecular basis of the pathogenesis of the scab disease.

Identification of a protein-binding sequence involved in expression of an esterase gene from Streptomyces scabies.

Expression of an esterase gene from Streptomyces scabies is regulated by zinc in both Streptomyces scabies and Streptomyces lividans. A specific protein-binding site was identified on an esterase promoter fragment by using an S-30 extract from S. scabies. The location of the protein-binding site was determined by gel shift assays of promoter deletion fragments and by DNase I footprinting analysis. The protein-binding site maps from nucleotides -59 to -81 relative to the start of transcription. An esterase gene construct cloned and expressed in S. lividans was used to assess the importance of the protein-binding site. Deletion of the 23-bp protein-binding site resulted in a 10-fold decrease in esterase production when cells were grown in zinc-inducing conditions. The protein-binding site may represent a region involved in positive regulation of the S. scabies esterase gene.

Regulation and secretion of an extracellular esterase from Streptomyces scabies.

Production of a heat-stable, extracellular esterase by Streptomyces scabies is regulated by zinc ions. The esterase-encoding gene (est) from S. scabies was cloned and expressed in Streptomyces lividans. In S. lividans, expression of the est gene is also regulated by Zn2+, and the esterase is efficiently secreted in this organism. The sequence of the est gene suggests that a 39-amino acid signal peptide is removed during secretion of this protein. Deletion analysis has indicated that the hydrophobic domain of the signal peptide is required for secretion. Gel retardation assays and DNaseI footprinting using an S-30 protein extract from S. scabies have previously identified a specific 23-bp protein-binding site upstream from the est coding sequence. Deletion of this protein-binding sequence significantly decreased expression of the est gene.

Characterization of cat messenger RNA decay suggests that turnover occurs by endonucleolytic cleavage in a 3' to 5' direction.

Turnover of the chloramphenicol acetyltransferase (cat) messenger RNA in Escherichia coli was investigated by analysis of cellular mRNA decay intermediates and the transcript sequence. Analysis of the sequence has revealed the presence of a repetitive extragenic palindromic (REP) element at the 3' end of the transcript as well as several 5'-UUAU-3' sequences, two elements which have roles in modulating turnover of other E. coli mRNAs. For cat mRNA, however, removal of the REP sequence has no effect on the half-life of the transcript, indicating that the REP sequence does not stabilize the upstream region of this message. Results from mapping of the mRNA decay products by several techniques suggest that the message is instead subject to endonucleolytic attack at five sites 5' of the REP element. The sequence UUAU is present at three of these five sites. It also appears that the cat mRNA is sequentially cleaved in a 3' to 5' direction during turnover of this mRNA in vivo. A model for cat mRNA turnover is discussed.

Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.

The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702. In S. lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S. scabies. The 36-kDa secreted form of the esterase was purified from S. lividans. N-terminal amino acid sequencing indicated that the processing site utilized in S. lividans for the removal of the signal sequence was the same as that recognized for processing in S. scabies. Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts. A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase. When this deleted form of the esterase was expressed in S. lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion. The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli. By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space.

Effect of gene amplification on mercuric ion reduction activity of Escherichia coli.

The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.

A novel transcriptional response by the cat gene during slow growth of Escherichia coli.

A novel response to growth rate was found with expression of the chloramphenicol acetyltransferase (cat) gene in Escherichia coli. The amount of cat mRNA relative to total RNA increased about 11-fold as growth rates decreased 5- to 6-fold, without an increase in translation. The accumulation of cat mRNA was in contrast to decreased cellular concentrations of total RNA, trxA, ompA, or 23S rRNA as the growth rate decreased and was not due to changes in gene dosage or mRNA stability. Stability of the cat mRNA does not appear to be regulated by growth rate. No significant change in either chemical or functional stability was observed within a five- to sixfold range of growth rates when chemostat-grown cells were used. However, cat mRNA stability was affected by growth medium composition. The half-life of cat mRNA decreased about threefold, with an approximate fourfold increase in generation time due to changes in growth medium. Transcriptional studies have indicated that accumulation of cat mRNA at slow growth rates is the result of a specific transcriptional response to changes in cellular generation times. We propose that increases in the cellular concentration of a specific message at slow growth rates may reflect an additional type of survival response in E. coli.

A model for mercuric ion reduction in recombinant Escherichia coli.

Plasmid-encoded mercuric reduction involves transfer of Hg(2+) across the cellular envelope and reduction to Hg(0) by the cytoplasmic mercuric reductase using NADPH. A mathematical model was developed for the binding and transfer of Hg(2+) by transport proteins and the subsequent reduction of Hg(2+). The values of the model parameters were determined using experimental data. The derived rate expressions were similar to the previously experimentally determined ones. The model predicted that a differential amplification of the transport protein relative to mercuric reductase expression levels may enhance the Hg(2+) reduction rate in whole cells.