Streptomyces Colonies Research Articles

Redefining development in Streptomyces venezuelae: integrating exploration into the classical sporulating life cycle.

Streptomyces bacteria have evolved diverse developmental and metabolic strategies to thrive in dynamic environmental niches. Here, we report the amalgamation of previously disparate developmental pathways, showing that colony expansion via exploration can proceed in tandem with colony sporulation. This developmental integration extends beyond phenotype to include shared genetic elements, with sporulation-specific repressors being required for successful exploration. Comparing this new exploration mode with previously identified strategies has revealed key differences (e.g., no need for environmental alkalinization), and simultaneously allowed us to define unifying requirements for Streptomyces exploration. The "reproductive exploration" phenomenon reported here represents a unique bet-hedging strategy, with the Streptomyces colony engaging in an aggressive colonization strategy while transporting a protected genetic repository.

First Report of Streptomyces niveiscabiei Causing Potato Common Scab in Shanxi and Gansu, China.

Multiple species of Streptomyces cause common scab disease in potato (Solanum tuberosum) (Kämpfer et al. 1991). Potato tubers (cv. Jinshu1 #5 and Longshu #6) with severe pitted common scab symptoms were observed from two farms in Chaozhou in Shanxi Province and in Tianzhu in Gansu Province during the national disease survey of bacterial diseases of potatoes in 2021. The disease incidence was around 30% on the 6.7 ha of the Chaozhou farm and 10% on the 0.7 ha on the Tianzhu farm. Three tubers with scab symptoms were surface disinfested with 3% sodium hypochlorite for 1 min. The symptomatic tissue was then ground in sterile water. Serially diluted ground samples were cultured on Streptomyces ISP Medium 5 agar plates (Shirling and Gottlieb 1966) and incubated at 280C for 5 days. Eight pure Streptomyces colonies were obtained and sequenced for identification using the universal 16S rRNA gene primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (Monciardini et al. 2002) by colony PCR. Blast results of the sequences against the NCBI GenBank for the eight isolates, ZRIMU1508, ZRIMU1510, ZRIMU1511, ZRIMU1512, ZRIMU1514, ZRIMU1515, ZRIMU1516 and ZRIMU1530 (Accession numbers: OP941573 - OP941580), showed more than 99% sequence identity to S. niveiscabiei NRRLB-24457T type strain. Additionally, 12 housekeeping gene sequences, acnA (OP997624 - OP997625), atpD (OP997622 - OP997623), dnaN (OP997620 - OP997621), gap (OP997618 - OP997619), gyrA (OP997614 - OP997615), gyrB (OP997612 - OP997613), infB (OP997610- OP997611), mdh (OP997608 - OP997609), recA (OP997602 - OP997603), rplB (OP997600 - OP997601), rpoB (OP997598- OP997599), and trpB (OP997594 - OP997595), were extracted from the genome sequences of two strains, ZRIMU1510 and ZRIMU1530, and uploaded to GenBank. Genes for pathogenicity, txtA (OP997593 - OP997594), tomA (OP997596 - OP997597) and Nec1(OP997606 - OP997607), were also identified from the genome sequence and uploaded to GenBank. The housekeeping genes and the pathogenicity genes showed over 98% identity with S. niveiscabiei. Phylogenetic trees were constructed using concatenated housekeeping gene sequences (Kumar et al. 1994) and the cladogram showed that the isolates ZRIMU1510 and ZRIMU1530 grouped with the type strain NRRLB-24457T. Pathogenicity tests were done by drench application of 100 ml spore suspensions (104 CFU/ml) of ZRIMU1530, ZRIMU1510, or phosphate buffer into pots with potato plants (cv. Favorita) grown in potting mix. Five tubers were planted and inoculated with each pathogen or phosphate buffer as the negative control. The plants were then placed in a greenhouse with 12 h of light per day, irrigated regularly, and harvested after 3 months. The newly formed tubers were checked for disease symptoms. Tubers from pots inoculated with ZRIMU1530 and ZRIMU1510 exhibited typical symptoms of common scab with raised corky lesions with deep pits, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed to be identical to the original isolates by 16S rRNA gene sequences, thus completing Koch's postulates. The pot experiment was conducted twice: first in May 2022 and second in February 2023. To our knowledge, this is the first report of S. niveiscabiei causing common scab of potato in Shanxi and Gansu, China. S. niveiscabiei was first reported in Korea (Park et al. 2003) and this report will draw attention to the study and management of scab pathogens in China.

AKTIVITAS ANTITUBERKULOSIS EKSTRAK KULTUR CAIR Streptomyces ENDOFIT RIMPANG Kaempferia galanga TERHADAP Mycobacterium tuberculosis RESISTEN

The objective of this research was to examine the antituberculosis activity of drug resistant stains using liquid culture extracts Streptomyces Kaempferia galanga rhizomes. The endophytes Streptomyces, there were KJ.3A, KJ3B, KJ.4, and KJ.11. Two resistant strain of Mycocacterium tuberculosis were used, there were HE strain which resistant to isoniazid dan etambutol and SR stainswhich resistant to streptomisin and rifampisin. The liquid culture extracts of Streptomyces endophytes was obtained from centrifugation method by growing Streptomyces colony into YMB media Antituberculosis activity was using Lowen-stein Jensen (LJ) methode after incubation for 8 weeks. The result showed that the four liquid culture extracts Streptomyces endophytes have not been able to inhibit the growth of Mycobacterium tuberculosis in both HE and SR strains

Cryptic specialized metabolites drive Streptomyces exploration and provide a competitive advantage during growth with other microbes

Streptomyces bacteria have a complex life cycle that is intricately linked with their remarkable metabolic capabilities. Exploration is a recently discovered developmental innovation of these bacteria, that involves the rapid expansion of a structured colony on solid surfaces. Nutrient availability impacts exploration dynamics, and we have found that glycerol can dramatically increase exploration rates and alter the metabolic output of exploring colonies. We show here that glycerol-mediated growth acceleration is accompanied by distinct transcriptional signatures and by the activation of otherwise cryptic metabolites including the orange-pigmented coproporphyrin, the antibiotic chloramphenicol, and the uncommon, alternative siderophore foroxymithine. Exploring cultures are also known to produce the well-characterized desferrioxamine siderophore. Mutational studies of single and double siderophore mutants revealed functional redundancy when strains were cultured on their own; however, loss of the alternative foroxymithine siderophore imposed a more profound fitness penalty than loss of desferrioxamine during coculture with the yeast Saccharomyces cerevisiae. Notably, the two siderophores displayed distinct localization patterns, with desferrioxamine being confined within the colony area, and foroxymithine diffusing well beyond the colony boundary. The relative fitness advantage conferred by the alternative foroxymithine siderophore was abolished when the siderophore piracy capabilities of S. cerevisiae were eliminated (S. cerevisiae encodes a ferrioxamine-specific transporter). Our work suggests that exploring Streptomyces colonies can engage in nutrient-targeted metabolic arms races, deploying alternative siderophores that allow them to successfully outcompete other microbes for the limited bioavailable iron during coculture.

Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.

Molecular characterization of covalently closed circular plasmid DNA in local Streptomyces strains isolated from soil with detection of antibiotic production and antibacterial activity

Various of Streptomyces species have two kinds of plasmids, circular plasmids (8 to 31 kb) and linear plasmids (12 to 1700 kb). Covalently closed circular (CCC) plasmids are profuse in the genus of Streptomyces and involved in production and resistance of antibiotics by genetic controlling. We collected fifty clinical soil samples from different regions in Al-Najaf Al-Ashraf province/Iraq. The samples included five from Al-Ghadeer Quarter, five from Al-Karama Quarter, 10 from Kufa University, five from Al-Ameer Quarter, four from Al-Forat Quarter, 10 from North Quarters and eleven from desert roads in Al-Najaf. Diluted samples were cultured on Yeast extract Malt extract (YEME) agar medium as a selective medium; then the presumptive Streptomyces colonies were subcultured on Tryptone Yeast extract (TYE) agar, then incubation at 37ᵒC for 7 days. Seven biochemical tests for identification of Streptomyces isolates these are: Catalase test, Oxidase test, Urase test, Kligler Iron Agar test (KIA), Simmon᾽s Citrate test, addition to MacConkey agar test and Mannitol Salt agar test. Five antibiotic discs were used for detection of antibiotic sensitivity of the Streptomyces isolates; these are: Tetracycline, Gentamycin, Vancomycin, Ampicillin, Erythromycin. The sensitivity of the antibiotics was observed by recorded the diameter of inhibition zone around the discs. Two test bacteria (Staphylococcus aureus and E. coli) were used for the determination of antibacterial activity. Plasmid isolation was done by the alkaline lysis method. This method is characterized by the rapid isolation of DNA from Streptomyces. Then, detection of Plasmid DNA occurred by using agarose gel electrophoresis.

VARIABILITY OF CAROTENOID-SYNTHETIC STRAINS STREPTOMYCES GLOBISPORUS 1912 AFTER DEEP CULTIVATION AND STORAGE

Aim. To determine the variability of the trait of carotenoids biosynthesis in two mutant strains Streptomyces globisporus 1912 after deep cultivation and storage. Relevance. Study of the spontaneous variability of strains-producers of carotenoids after deep cultivation and long-term storage is necessary for understanding the mechanisms of intrapopulation inheritance and the possibility of their control. Methods. Visually the analysis of phenotype of streptomyces colonies, statistical analysis was performed with Windows Software Excel 2007. Results. The appearance of non-productive/non-active variants (yellow, creamy and colorless) in populations of strains S. globisporus 4Lcp-Hp7 and S. globisporus 7Crt after cultivation in corn-soy liquid medium was 10-3 and 10-2, at 1–2 orders of magnitude higher than their standard cleavage with frequent transplantation of subcultures. The variability during storage had high indexes, which were dependent on storage temperature. Non-productive variants of strains S. globisporus 4Lcp-Hp7 and S. globisporus 7Crt at storage temperature of 4 °C were about 50% and 10% of the population, respectively. There were near 10% non-active colonies in the population of strain S. globisporus 4Lcp-Hp7, and 20–30% ones in the case strain S. globisporus 7Crt at storage temperatures of 21 °C and 28 °C. After one-year storage near 52% and 66% colonies of the populations of lyophilized cultures S. globisporus 4Lcp-Hp7 and S. globisporus 7Crt supported the high level of synthesis of corresponding carotenoids. Conclusions. It has been shown, that the trait of carotenoids biosynthesis in the mutant strains S. globisporus 4Lcp-Hp7 and S. globisporus 7Crt was showing variability with the high frequency after deep cultivation (10-3 and 10-2 non-active colonies) and during long storage on slants and in lyophilized state (10–50%).

First Report of Streptomyces turgidiscabies Causing Potato Common Scab in Spain

Potato common scab (CS) is an important crop problem worldwide. Previously, Streptomyces europaeiscabies has been considered as a predominant pathogen in the west European region. Potato (Solanum tuberosum) samples having visible CS symptoms were collected from Galicia, Spain, during 2015. Potato samples were heavily infected with CS symptoms from superficial netted scab to pitted lesions. Samples were washed with tap water before and after disinfecting with 2.8% sodium hypochlorite for 1 min. Scab lesions on the surface of infected potatoes were used to isolate Streptomyces colonies (Wanner 2006). During the survey, more than 25 Streptomyces isolates were identified as pathogenic to potatoes from different regions of Spain (data not shown). Streptomyces isolate strain AC-51 was identified after morphological and physiological characterization as S. turgidiscabies (Bouchek-Mechiche et al. 2006). Pure colonies of Streptomyces were cultured in tryptic soy broth (TSB). A Streptomyces pellet was used for genomic DNA extraction by Fast DNA kit (MP Biomedicals, U.S.A.). Purified DNA was amplified for 16S rRNA gene by using pH/pA primers (Edwards et al. 1989). The resultant 16S rDNA sequences were submitted to GenBank. NCBI BLAST analysis of S. turgidiscabies strain AC-51 (LN908786) showed 97% similarity with S. turgidiscabies (FJ546740). Streptomyces spp. was further characterized by performing a positive PCR reaction with S. turgidiscabiei-specific primers Tur-R/F (Tagawa et al. 2008) but had negative reaction with individual species-specific primers from S. scabies, S. europaeiscabies, S. stelliscabies, S. bottropensis, and S. acidiscabies (Wanner 2006). A pathogenicity island consisting of txtAB, nec1, and tomA genes was confirmed in S. turgidiscabies strain AC-51 by using different PCR primers (Bukhalid et al. 1998; Wanner 2006). The pathogenicity of strain AC-51 was tested on potatoes by pot assay in a greenhouse facility. Potato tubers (cv. Kennebec) were planted in potting soil (Compo Sana, Germany) with an average temperature maintained between 25 and 28°C. Cell suspension of strain AC-51 was prepared at concentration of 106CFU/ml. Three-week-old potato plants were drenched with 100 ml of prepared inoculum in duplicate experiments. To validate the experimental results, nonpathogenic Streptomyces sp. (KU560918) and pathogenic S. scabies RL-34 (kindly provided by Dr. J. Leiminger, DSMZ41658) were used in the same concentration as negative and positive controls, respectively. Three-month-old plants were harvested and CS symptoms on potato surface were recorded. Disease symptoms were observed from moderate to high pitted scab lesions among the pots infected with S. turgidiscabies strain AC-51 and S. scabies (Wanner 2006), while no CS symptoms were observed with nonpathogenic Streptomyces spp. Disease severity was recorded as mean surface area covered by CS symptoms as 32% for strain AC-51, 38% for S. scabies, and 0% for nonpathogenic Streptomyces spp. Pathogenic S. turgidiscabies strain AC-51 was again isolated from infected tubers in greenhouse study to confirm Koch’s postulates. To our knowledge, this is the first report of S. turgidiscabies in Galicia, Spain. This study will provide a basic knowledge about the pathogens which will helpful to develop preventive strategies against this disease.

Vplyv hnojenia na biologick� aktivitu p�dneho spolocenstva streptomyc�t

The search for new natural mechanisms to inhibit the growth of phytopathogenic microorganisms has become widely widespread. Therefore, the main objective of the present study was determination of antimicrobial activities of actinomycetes isolated from agricultural soil, which was fertilized mainly by organic fertilizers, against 8 selected phytopathogenic strains. Among the actinomycetes, Streptomyces species have been extensively studied, because they have been recognized as an important source of secondary metabolites, which can suppress the growth of undesirable pests in crops. The results indicated that the richest source of Streptomyces colonies was soil fertilized with compost (103 x104 CFU*g-1 dry soil). On the basis of morphological signs, total of 65 isolates were selected and examined for antimicrobial activities. Isolates exhibited the best activity against Gram negative bacterium Clavibacter michiganensis subsp. sepedonicus, disease agent of “ring rot” of potatoes and against fungus Fusarium poae, disease agent of Fusarium head blight of wheat. Twelve isolates exhibited promising broad-spectrum activity against tested organisms. On the basis of results, six of them were selected for further screening. Comparison of polyphasic studies with available literature led to identification of biological active strains S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) and S. avidinii (13SPC4). After quantification analysis of various enzymes, tested isolates produced alkaline phosphatase, leucinearylamidase, valinearylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, glucosidase in high values (>40 nmol) and were positive for nitrate reduction, hydrolysis of gelatin, urease, and esculin. These isolates can be used in the development of new biopesticides anf biofertilizers with antibacterial and antifungal effect.

Effect of fertilization on biological activity of community of soil streptomycetes

The search for new natural mechanisms to inhibit the growth of phytopathogenic microorganisms has become widely widespread. Therefore, the main objective of the present study was determination of antimicrobial activities of actinomycetes isolated from agricultural soil, which was fertilized mainly by organic fertilizers, against 8 selected phytopathogenic strains. Among the actinomycetes, Streptomyces species have been extensively studied, because they have been recognized as an important source of secondary metabolites, which can suppress the growth of undesirable pests in crops. The results indicated that the richest source of Streptomyces colonies was soil fertilized with compost (103 x104 CFU*g-1 dry soil). On the basis of morphological signs, total of 65 isolates were selected and examined for antimicrobial activities. Isolates exhibited the best activity against Gram negative bacterium Clavibacter michiganensis subsp. sepedonicus, disease agent of “ring rot” of potatoes and against fungus Fusarium poae, disease agent of Fusarium head blight of wheat. Twelve isolates exhibited promising broad-spectrum activity against tested organisms. On the basis of results, six of them were selected for further screening. Comparison of polyphasic studies with available literature led to identification of biological active strains S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) and S. avidinii (13SPC4). After quantification analysis of various enzymes, tested isolates produced alkaline phosphatase, leucinearylamidase, valinearylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, glucosidase in high values (>40 nmol) and were positive for nitrate reduction, hydrolysis of gelatin, urease, and esculin. These isolates can be used in the development of new biopesticides anf biofertilizers with antibacterial and antifungal effect.

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments

Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65°C for 30min and then incubated at 27°C for 3weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55°C, treatment at 65°C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90% Micromonospora. In addition, treatment at 65°C did not affect the diversity of Micromonospora species compared with treatment at 55°C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65°C for 30min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.

Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R(2) = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 10(1) to 10(6) pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 10(3) to 10(6) per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

A novel compartment, the ‘subapical stem’ of the aerial hyphae, is the location of a sigN‐dependent, developmentally distinct transcription in Streptomyces coelicolor

Streptomyces coelicolor has nine SigB-like RNA polymerase sigma factors, several of them implicated in morphological differentiation and/or responses to different stresses. One of the nine, SigN, is the focus of this article. A constructed sigN null mutant was delayed in development and exhibited a bald phenotype when grown on minimal medium containing glucose as carbon source. One of two distinct sigN promoters, sigNP1, was active only during growth on solid medium, when its activation coincided with aerial hyphae formation. Transcription from sigNP1 was readily detected in several whi mutants (interrupted in morphogenesis of aerial mycelium into spores), but was absent from all bld mutants tested, suggesting that sigNP1 activity was restricted to the aerial hyphae. It also depended on sigN, thus sigN was autoregulated. Mutational and transcription studies revealed no functional significance to the location of sigN next to sigF, encoding another SigB-like sigma factor. We identified another potential SigN target, nepA, encoding a putative small secreted protein. Transcription of nepA originated from a single, aerial hyphae-specific and sigN-dependent promoter. While in vitro run-off transcription using purified SigN on the Bacillus subtilis ctc promoter confirmed that SigN is an RNA polymerase sigma factor, SigN failed to initiate transcription from sigNP1 and from the nepA promoter in vitro. Additional in vivo data indicated that further nepA upstream sequences, which are likely to bind a potential activator, are required for successful transcription. Using a nepA-egfp transcriptional fusion we located nepA transcription to a novel compartment, the 'subapical stem' of the aerial hyphae. We suggest that this newly recognized compartment defines an interface between the aerial and vegetative parts of the Streptomyces colony and might also be involved in communication between these two compartments.

Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony

The bldA gene encodes the only tRNA for the UUA codon that, although dispensable in genes important for primary vegetative growth of Streptomyces spp., is important in genes that serve a regulatory purpose in the differentiation. To investigate this role further, the spatial and temporal expression profiles of the bldA-regulated and unregulated genes within a Streptomyces colony were examined using modified genes for the green fluorescent protein ( gfp) as an expression-tag. A comparative study, based on computer-assisted quantitative analysis of the GFP fluorescence, revealed that the presence of TTA codons in gfp results in a temporal delay of translation and, consequently, changed the spatial pattern of the GFP expression within a colony, especially during early differentiation. The delay of GFP expression was undetectable at 60 h post-inoculation. These results provide the first extensive evidence that the bldA does indeed play a significant regulatory role during colony differentiation.

A Quantitative Method for Determining Soil Populations of Streptomyces and Differentiating Potential Potato Scab-Inducing Strains.

A procedure is described for estimating Streptomyces populations in soil. Soils are air-dried, 10g quantities are shaken in plastic bags containing 0.1% water agar and homogenized with a Stomacher homogenizer, serial dilutions are plated on a semi-selective culture (STR) medium and incubated for 2 weeks at 22°C, and the Streptomyces colonies are enumerated. Use of STR medium reduced the bacterial and fungal colonies recovered from soil to levels below that of the Streptomyces spp. while not affecting the number of Streptomyces colonies compared with those enumerated on yeast malt extract medium. A procedure for screening large numbers of Streptomyces strains for thaxtomin production, a phytotoxin recognized as a virulence marker in S. scabies, is also described. Strains are grown on oatmeal medium, and the thaxtomin is extracted from the medium by facilitated diffusion and detected by miniature thin layer chromatography. S. scabies and S. acidiscabies strains (approximately 130 from Ontario and 70 from other locations in North America) that produced thaxtomin did not form aerial mycelia or sporulate on STR medium within 2 weeks at 22°C. Ontario S. scabies strains that produced thaxtomin A also produced melanin on STR medium. All S. scabies strains from scab lesions that produced thaxtomin A had this colony morphology, whereas only 4 to 9% of strains from soil with this colony morphology produced thaxtomin A. Using these procedures, we determined that the population of thaxtomin-producing S. scabies in soil from a potato field in Ontario with a history of potato scab was about 20,000 CFU/g soil.

Taking a genetic scalpel to the Streptomyces colony.

1997 Fred Griffith Review Lecture(Delivered at the 138th Meeting of the Society for General Microbiology, 2 September 1977).

Ectopic expression of the Streptomyces coelicolor whiE genes for polyketide spore pigment synthesis and their interaction with the act genes for actinorhodin biosynthesis

The whiE gene cluster of Streptomyces coelicolor is normally expressed shortly before sporulation in the aerial mycelium, leading to production of the grey polyketide spore pigment. By placing the whiE genes under the control of the thiostrepton-inducible tipA promoter, they were artificially expressed on plasmids or in the chromosome during vegetative growth in a strain deleted for the act genes, which control biosynthesis of the polyketide antibiotic actinorhodin. Certain combinations of whiE-ORFI-VII led to production of mycelial pigments; these were exported into the medium when whiE-ORFI was absent, but poorly in its presence. Combined with comparative sequence data, the results allowed deductions to be made, or confirmed, about the normal roles of the eight known genes, whiE-ORFI-VIII, as follows: whiE-ORFIII, IV, V encode the three components (ketosynthase, chain length factor and acyl carrier protein) of the whiE 'minimal' polyketide synthase (PKS) needed for assembly of the carbon chain of the spore pigment precursor; whiE-ORFII, VI, VII are likely to be involved in cyclizations of the nascent carbon chain; whiE-ORFVIII controls a late step in the spore pigment biosynthetic pathway, probably a hydroxylation; and whiE-ORFI may encode a protein needed for correct targeting or retention of spore pigment at an appropriate cellular location. In other experiments, genes encoding components of the act-PKS and whiE-PKS were artificially co-expressed. Each of the three whiE minimal PKS subunit genes could complement lesions in the corresponding act-PKS genes to produce actinorhodin or related mycelial pigments, and each of the three act minimal PKS genes could complement lesions in the whiE minimal PKS genes to cause spore pigmentation. Thus the two sets of PKS subunits, which are encoded by genes that have presumably diverged from a common ancestor, are still capable of biochemical 'cross-talk', but this is normally prevented because the gene sets are expressed in different 'tissues' of the differentiated Streptomyces colony. Ectopic expression of sets of whiE-PKS genes presumed to be sufficient to assemble a carbon chain caused inhibition of early growth of the strains, perhaps by causing interference with fatty acid biosynthesis; this yielded circumstantial evidence that the whiE-PKS gene products can also interact with those of the fatty acid synthase(s) of the organism.

Processing of bacterial colony images for automatic isolation and transfer

A method for processing and analysing images of bacterial colonies growing on solid bacterial medium is presented. The procedure determines the position of single colonies, intended for further inoculum transfer by a specially designed life-science laboratory robot. The image processing and analysis of Streptomyces colonies are described. The procedure can be employed to isolate antibiotic producers for the pharmaceutical industry; a similar process can be used to automate methods of charting human genomic DNA.

Structural instability of a bifunctional plasmid pZG1 and single-stranded DNA formation in Streptomyces

Structural instability of a hybrid plasmid pZG1, consisting of Escherichia coli pBR322 and Streptomyces pIJ350 plasmids, has been studied in Streptomyces. After transformation of S. lividans 1326 and S. rimosus R6 protoplasts with pZG1, transformants harbored the intact pZG1 and various deleted plasmid forms. The pattern of deleted plasmids varied with the transformant colony age and changed upon subcultivation. The presence of intact pZG1 in at least a part of the Streptomyces colonies indicated that the plasmid was capable of replicating in the transformants and that deletion events occurred after at least one round of replication. Less instability of pZG1 in S. rimosus R6 was observed when this strain was transformed with the DNA isolated from the same strain. pZG1 and its various derivatives were found in S. lividans 1326 and S. rimosus R6 as double- and single-stranded DNA molecules. Structural instability of pZG1 could therefore be due at least in part to the presence of single-stranded DNA.

Sulphur oxidation by a Streptomyces sp. growing in a carbon-deficient medium and autoclaved soil.

Streptomyces colonies, apparently all of the same species, were isolated from a range of soils using a polysulphide medium lacking an organic carbon source. Growth on this medium, and clearing of the otherwise white, opaque overlay, suggested that the organisms were capable of growing autotrophically. However, investigation of one of these isolates showed that it was unable to fix 14CO2 and did not possess the enzyme ribulose bisphosphate carboxylase, showing that it was incapable of autotrophic growth. The isolate oxidized elemental sulphur, thiosulphate and tetrathionate to sulphate in vitro in carbon-deficient medium, and also oxidized elemental sulphur to sulphate when inoculated into autoclaved soil supplemented with sulphur. It also oxidized polysulphide when growing on Czapek Dox and plate count agars. The isolate can therefore grow heterotrophically in both carbon-rich media and in media lacking organic carbon - presumably by scavenging organic carbon from the laboratory atmosphere. The possible role of these organisms in sulphur oxidation in soils is commented upon.