Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations.

Recombinant rabies virus glycoprotein (RVGP) was expressed in cell membranes of stably transfected Drosophila S2 cells using constitutive and inducible promoters. Although with quantitative differences of RVGP expression in both systems, the cDNA transcription, as evaluated by relative RVGP mRNA levels measured by qRT-PCR, sustained the amount of RVGP producing cells and the RVGP volumetric (ΠRVGP) productivity. At the transition to the stationary cell growth phase, once the cell culture slowed down its rate of multiplication, an accumulation of RVGP mRNA and RVGP was clearly observed in both cell populations. Nevertheless, cell cultures performed under sub-optimal temperatures indicated that an envisaged increase in the RVGP production is not only dependent on cell growth rate, but essentially on optimal cell metabolic state.

Combining in the melt physical and biological properties of poly(caprolactone) and chlorhexidine to obtain antimicrobial surgical monofilaments.

Bacterial infections on a sutured wound represent a critical problem, and the preparation of suture threads possessing antimicrobial properties is valuable. In this work, poly(caprolactone) (PCL) monofilaments were compounded at the concentration of 1, 2 and 4 % (w/w), respectively, to the antiseptic chlorhexidine diacetate (CHX). The incorporation was carried out in the melt by a single-step methodology, i.e. "online" approach. Mechanical tests revealed that the incorporation of CHX does not significantly change tensile properties of PCL fibres as the thermal profile adopted to prepare the compounded fibres does not compromise the antibacterial activity of CHX. In fact, CHX confers to compounded PCL fibres' antimicrobial property even at the lowest CHX concentration as revealed by microbiological assays performed on Escherichia coli, Micrococcus luteus and Bacillus subtilis strains. The scanning electron microscope micrographs and energy-dispersive X-ray analysis of compounded threads revealed that CHX is uniformly distributed on fibre surface and that the overall amount of superficial CHX increases by increasing compounded CHX concentration. This distribution determines a biphasic CHX release kinetics characterized by an initial rapid solubilisation of superficial CHX micro-crystals, followed by a slow and gradual release of CHX incorporated in the bulk. Interestingly, the compounded threads did not show any toxic effect compromising cell viability of human fibroblasts in vitro, differently from that observed using an equal amount of pure CHX. Thus, this study originally demonstrated the effectiveness of an "online" approach to confer antimicrobial properties to an organic thermoplastic polymeric material commonly used for medical devices.

Tryptophan catabolism via kynurenine production in Streptomyces coelicolor: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway.

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.

Human herpesvirus-7 in Brazilian liver transplant recipients: a follow-up comparison between molecular and immunological assays.

Human herpesvirus-6 and -7 (HHV-6, HHV-7) remain latent after primary infection and can reactivate after transplantation. HHV-6 active infection has been related to some clinical manifestation, but the role of HHV-7 remains unclear. The clinical significance of HHV-7 DNAemia is not completely known and the immune response against HHV-7 has been poorly studied in transplantation. In this study, we investigated HHV-7 DNAemia in liver transplant recipients and evaluated the immunoglobulin (Ig) G and IgM response against HHV-7. A total of 22 adult liver transplant recipients were followed up for 90 days. HHV-7 DNAemia was detected by nested polymerase chain reaction (PCR) in DNA extracted from sera. IgG and IgM detection was performed by immunofluorescent assay using HHV-7-infected cord blood mononuclear cells. A significant virus antibody response was defined as either a positive IgM or a > or =4-fold rise in the virus IgG antibody. All patients had pre-transplant HHV-7-positive serostatus. Nine of 22 (40.9%) patients presented HHV-7 DNAemia during follow-up. All these patients had anti-HHV-7-positive IgM and/or significant increase in IgG titers with concurrent or subsequent DNAemia. In patients without DNAemia and low persistent IgG antibody titers, IgM was not detected. Correlation between nested PCR and IgM detection was statistically significant (P=0.01). Our study indicates that nested PCR in DNA extraction from serum can be useful to detect and monitor HHV-7 active infection in liver transplant recipients. IgM antibody detection also can be useful as a first immunological technique to detect active infection, especially if combined with PCR.

Isolation of Gram-positive n-alkane degraders from a hydrocarbon-contaminated Mediterranean shoreline.

AIMS: To investigate the petroleum hydrocarbon (HC)-degrading potential of indigenous micro-organisms in a sandy Mediterranean coast, accidentally contaminated with petroleum-derived HCs. METHODS AND RESULTS: Using culturable methods, a population of Gram-positive n-alkane degraders was detected in the contaminated soil. Five isolates, identified as one Nocardia, two Rhodococcus and two Gordonia strains, were able to degrade medium- and long-chain n-alkanes up to C(36) as assessed by growth assays and gas chromatography-mass spectrometry analysis. Diverging alkane hydroxylase-encoding genes (alkB) were detected by PCR, using degenerated primers, in all the strains; multiple sequences were obtained from the Nocardia strain, while only one alkB gene was detected in the Rhodococcus and Gordonia strains. The majority of the alkB sequences were related to Rhodococcus alkB2, but none was identical to it. CONCLUSIONS: Actinomycetes might have a key role in bioremediation of n-alkane-contaminated sites under dry, resource-limited conditions, such as those found in the Mediterranean shorelines. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study on the bioremediation potential in Mediterranean contaminated beaches.

[The use of genetically modified microorganisms in agriculture to reduce or eliminate pesticide exposure]. / Utilizzo di microrganismi geneticamente modificati in agricoltura al fine di ridurre o eliminare l'esposizione a pesticidi.

P. graminea is the casual agent of barley leaf stripe. An early selection method of resistant types of barley leaf stripe was realized and validated in this research. This new method, based on Microrganism Genetically Modified (MOGM) GUS2 construction, obtains results comparable with those of classical method, reduces the work time, the use of chemicals (pesticides) and productive plants as greenhouses. Moreover, the use of MOGM GUS2 is restricted in laboratory ambient, therefore the risk of environmental spread is reduced. The early selection method has allowed to estimate the reaction to P. graminea agent in 12 several barley types usually farmed in Italy. The results were compared both, with the classical method data based on artificial clone Dg2 inoculum, and with natural inoculum data obtained in field. At all times we observed a ranking likeness.

First Record of Verticillium dahliae on Potato in Malta.

Potato is an important and highly valued crop throughout the Maltese Archipelago. Much of the production is exported to Holland. In January 2005, minor wilts and chlorosis of potato plants were observed in a field at Hal-Farrug, Luqa (Malta). Verticillium dahliae Kleb (1) was isolated on potato dextrose agar (PDA) from vascular tissue excised from the base of the plants. Three different isolates were obtained, all of which were typically distinguished by verticillately shaped conidiophores and the abundant production of microsclerotia on PDA. In May 2005, colonies of these three isolates were cultured in potato dextrose broth (PDB), from which conidial suspensions of each isolate were prepared with sterile distilled water to a concentration of 107 ml-1. For each isolate, 10 7-day-old potato seedlings were inoculated via root immersion in the inoculum suspension and transplanted to 20-cm diameter plastic pots containing a soil/peat mixture (1:1 [v/v]). Seedlings treated in the same way with sterile distilled water were used as a control. All plants were kept under controlled glasshouse conditions (20 ± 3°C) and watered to field capacity as required. Minor chlorosis and wilt of the pair of lower-most leaves was noted 7 days after inoculation. During subsequent weeks, wilt began to appear in the typical half-leaf form, while chlorosis was noted on all organs of the plants, including the principal stem (3). Symptoms were absent on the control plants. Measuring the weight of the new tubers produced by each plant revealed no apparent difference between inoculated and healthy plants; nevertheless, inoculated plants resulted in more tubers with a smaller diameter in respect to those of the uninoculated plants. V. dahliae was never isolated from tubers. Little to no variation in symptom severity was noted among plants inoculated with the three individual isolates. At the end of June, V. dahliae was reisolated on PDA from all inoculated plants, in particular, from vascular tissues originating from principal and lateral stems, crowns, and roots. All attempts to isolate the pathogen from control plants were unsuccessful. Molecular detection of the pathogen by using species-specific primers and real-time Scorpion PCR (2) confirmed the results obtained by the classical isolation method. The low symptom severity observed by the growers in the field, usually mistaken for normal dieback of aged plants, might explain why V. dahliae was never reported before on potatoes in the Maltese Archipelago. References: (1) D. L. Hawksworth and P. W. Talboys. No. 256. Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute (CMI), Kew, Surrey, UK, 1970. (2) F. Nigro et al. Pages 454-461 in: Proc. Convegno Internazionale di Olivicoltura. VI Giornate Scientifiche SOI, Spoleto, 2002. (3) W. R. Stevenson et al., eds. Compendium of Potato Diseases. 2nd ed. The American Phytopathological Society, St Paul, MN, 2001.

Identification of SCP2165, a new SCP2-derived plasmid of Streptomyces coelicolor A3(2).

AIMS: Characterization of SCP2165, a plasmid identified in the Gram-positive bacterium Streptomyces coelicolor A3(2). METHODS AND RESULTS: Pulsed-field gel electrophoresis (PFGE) of mycelia of a S. coelicolor strain embedded in low melting agarose revealed the presence of a plasmid. Restriction enzyme mapping and sequence analysis of a 2.1 kb fragment revealed that this plasmid could be SCP2. SCP2 and its spontaneous derivative SCP2* are self-transmissible plasmids and have chromosome mobilizing ability (c.m.a.). SCP2* has a c. 1000-fold increased c.m.a. compared with SCP2. Interestingly the plasmid, named SCP2165, shows a c.m.a. from 5x10(-2) to 1x10(-1) which is 50-100-fold higher than that described for crosses involving SCP2*. CONCLUSIONS: SCP2165 is a SCP2 derivative plasmid with the highest c.m.a. so far described for SCP2 derivative plasmids. PFGE, under conditions we used, seems to be a fast way to identify large circular plasmids in Streptomyces strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Further knowledge of the SCP2 family may allow the construction of improved SCP2-derived cloning vectors. SCP2165 could be a potential tool for conjugational transfer of gene clusters between different Streptomyces species.

Zygotic and nucellar embryo survival following dehydration and cryopreservation of citrus intact seeds.

A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16 percent (C. sinensis) or 10 percent (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50 percent after drying to 15 percent moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93 percent germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.

Bradyrhizobium sp. nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.).

AIMS: The molecular diversity of 25 strains of rhizobia, isolated in Sicily from root nodules of the Mediterranean shrubby legume Spanish broom (Spartium junceum L.), is presented in relation to the known rhizobial reference strains. METHODS AND RESULTS: Our approach to the study of the S. junceum rhizobial diversity combined the information given by the 16S and the intergenic spacer (IGS) 16S-23S rDNA polymorphic region by obtaining them in a single polymerase chain reaction (PCR) step. The PCR fragment size of the S. junceum isolates was 2400-2500 bp and that of the reference strains varied from 2400 in Bradyrhizobium strains to 2800 in Sinorhizobium strains. Inter- and intrageneric length variability was found among the reference strains. Restriction fragment length polymorphisms (RFLP) analysis allowed us to identify eight genotypes among the S. junceum rhizobia that were clustered into two groups, both related to the Bradyrhizobium lineage. Sequencing of representative strains of the two clusters confirmed these data. The 16S-IGS PCR-RFLP approach, when applied to rhizobial reference strains, allowed very close species (i.e. Rhizobium leguminosarum/R. tropici) to be separated with any of the three enzymes used; however, cluster analysis revealed inconsistencies with the 16S-based phylogenesis of rhizobia. CONCLUSIONS: Rhizobia nodulating S. junceum in the Mediterranean region belong to the Bradyrhizobium lineage. Our results confirm the resolution power of the 16S-23S rDNA in distinguishing among rhizobia genera and species, as well as the usefulness of the PCR-RFLP method applied to the entire 16S-IGS region for a rapid tracking of the known relatives of new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present paper is, to our knowledge, the first report on rhizobia nodulating a Mediterranean wild woody legume.

First Report of Rhizopycnis vagum Associated with Tomato Roots in Italy.

Field surveys were made in several central and southern Italian tomato-growing areas for Pyrenochaeta lycopersici, the cause of corky root of tomato. In addition to P. lycopersici, a different fungus was frequently isolated from roots showing typical corky root symptoms, even after disinfestation of diseased roots with 0.1% (vol/wt) mercury chloride water solution for 1 min. The fungus was isolated from primary and secondary tomato roots in 8 of 21 fields visited. The isolates were grown on potato dextrose agar (PDA), with morphological features such as color and shape of mature conidia and pycnidia, type of conidiogenesis, presence of microsclerotia, and color of colony underside noted. Preliminary identification of the fungus was Rhizopycnis vagum Farr. To confirm the identification, the internal transcribed spacer (ITS) region of rDNA of one isolate (maintained at the ISPaVe collection at the authors' address and available on request as isolate ER 940) was amplified with two universal primers, ITS5 and ITS4. The ITS fragment was sequenced, and the nucleotide sequence compared with that of R. vagum deposited in GenBank (Accession No. AF022786). Both sequences were identical supporting the identification. R. vagum is a recently described species associated with the vine decline syndrome of melon in the United States, Guatemala, Honduras (2), and Spain (3). Eight isolates were tested for pathogenicity both on tomato (five cultivars) and melon (three cultivars) using two methods. In method 1, plantlets at the cotyledonary stage were grown on blotter in petri dishes and tested by placing a 6-mm plug of colonized PDA on the tap root (1). After 7 days, the plug was removed, and the roots were checked for symptoms. In method 2, 20-day-old seedlings were transferred to pots with infested soil (50,000 CFU/g of soil) and grown for 45 days before the roots were checked for each isolate-cultivar combination. Eight and four plants were used in tests 1 and 2, respectively. With the first method, rotten, pinkish lesions with different extensions from the inoculation point were observed on all the melon cultivars tested (Pamir, Cantalupo di Charentais, and Charme). On tomato, three of eight isolates caused root necrosis of limited extent, without pinkish discolorations at the inoculation site on cvs. Monalbo and Bonnie Best, the former showing the larger lesions. The tests on plants grown in infested soil confirmed pathogenicity on both host species, although the symptoms were of minor intensity (light, small brown lesions on secondary roots, no pinkish discoloration). The symptomatic plantlets ranged from 0 to 100% on both hosts in the petri dish tests and from 0 to 100% and 0 to 50%, respectively, for tomato and melon in the pot tests, varying according to the cultivar-isolate combination. The fungus was consistently reisolated from all symptomatic plants. To our knowledge, this is the first report of R. vagum associated with tomato roots. Although the isolates showed varying degrees of virulence with respect to host species (all being pathogenic at least on one host), the virulence of R. vagum on tomato was certainly low. Nevertheless, tomato may maintain or possibly increase inoculum for melon, which often follows tomato in Italian crop rotations. References: (1) M. Clerjeau and M. Conus. Annu. Rev. Phytopatol. 5:143, 1973. (2) D. F. Farr et al. Mycologia 90:290, 1998. (3) J. García-Jiménez et al. EPPO Bull. 30:169, 2000.

Artificial chromosomes for antibiotic-producing actinomycetes.

Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chromosomes (BAC) that can be shuttled among Escherichia coli, where they replicate autonomously, and a suitable Streptomyces host, where they integrate site-specifically into the chromosome. The existence of gene clusters and of genetically amenable host strains, such as Streptomyces coelicolor or Streptomyces lividans, makes this a sensible approach. We report here that 100 kb segments of actinomycete DNA can be cloned into these vectors and introduced into genetically accessible S. lividans, where they are stably maintained in integrated form in its chromosome.

Broad spectrum thiopeptide recognition specificity of the Streptomyces lividans TipAL protein and its role in regulating gene expression.

Microbial metabolites isolated in screening programs for their ability to activate transcription of the tipA promoter (ptipA) in Streptomyces lividans define a class of cyclic thiopeptide antibiotics having dehydroalanine side chains ("tails"). Here we show that such compounds of heterogeneous primary structure (representatives tested: thiostrepton, nosiheptide, berninamycin, promothiocin) are all recognized by TipAS and TipAL, two in-frame translation products of the tipA gene. The N-terminal helix-turn-helix DNA binding motif of TipAL is homologous to the MerR family of transcriptional activators, while the C terminus forms a novel ligand-binding domain. ptipA inducers formed irreversible complexes in vitro and in vivo (presumably covalent) with TipAS by reacting with the second of the two C-terminal cysteine residues. Promothiocin and thiostrepton derivatives in which the dehydroalanine side chains were removed lost the ability to modify TipAS. They were able to induce expression of ptipA as well as the tipA gene, although with reduced activity. Thus, TipA required the thiopeptide ring structure for recognition, while the tail served either as a dispensable part of the recognition domain and/or locked thiopeptides onto TipA proteins, thus leading to an irreversible transcriptional activation. Construction and analysis of a disruption mutant showed that tipA was autogenously regulated and conferred thiopeptide resistance. Thiostrepton induced the synthesis of other proteins, some of which did not require tipA.

Antibody response in CD4-depleted mice after immunization or during early infection with Echinococcus granulosus.

The aims of this work were to investigate the existence of T-independent antigens in Echinococcus granulosus protoscoleces and to evaluate the relative contribution of T-independent stimulation to the overall antibody response in early infection. Mice depleted of CD4(+)-cells were immunized with protoscolex somatic antigens (PSA) or infected with E. granulosus protoscoleces (PSC). Results showed that the response of CD4-depleted immunized mice had the expected characteristics of a T-independent stimulation and that such T-independent stimulation was important mainly during primary response. During infection absence of CD4(+)-cells affected mainly the secretion of all IgG subclasses with the exception of IgG3 and IgM. To carry out a preliminary isolation of PSC T-independent antigens we prepared a carbohydrate enriched fraction from protoscolex antigens, using a monoclonal antibody specific for the carbohydrate moiety Gal alpha(1,4)Gal highly expressed in PSC. This fraction was mitogenic for naive mouse splenocytes and was recognized by a high percentage of the specific antibodies secreted by CD4-depleted immunized or infected mice. In summary, these results suggest that E. granulosus protoscoleces contain immunogenic T-independent antigens. Primary antibody responses to protoscolex somatic antigens and the production of IgM and IgG3 in early infection would be mainly stimulated by a T-independent mechanism.

Soybean Disease Loss Estimates for the Top 10 Soybean Producing Countries in 1994.

Soybean disease loss estimates were compiled for the 1994 harvested crop from the 10 countries with the greatest soybean production. The objective was to document the major soybean disease problems in these countries and any recent changes in the severity of individual soybean diseases. Total yield losses caused by Heterodera glycines in these 10 countries were greater than those caused by any other disease. Next in order of importance were stem canker, brown spot, and charcoal rot. The total yield loss due to disease during 1994 in these countries was 14.99 million metric tons, valued at $3.31 billion. Methods used to estimate soybean disease losses were field surveys, plant disease diagnostic clinic samples, variety trial data, information from field workers and university extension staff, research plots, grower demonstrations, and private crop consultant reports. Yield loss estimates due to a particular disease varied by country. For example, yield losses due to rust were reported from China and Indonesia, but no losses due to this disease were reported from any of the remaining eight countries. Soybean disease control research and extension efforts are needed to provide more effective preventive and therapeutic disease management strategies and systems to producers.

Synthesis and microbiological evaluations of (N-heteroaryl) arylmethanamines and their Schiff bases.

The synthesis as well as the antimicrobial and antiviral activities of new (N-heteroaryl)arylmethanamines and their Schiff bases are reported. None of the tested compounds shown activity against Herpes simplex virus type 2 and against Gram positive and Gram negative bacteria. Weak or moderate activity on poliovirus Sabin type 1, on reverse transcriptase and against Cryptococcus neoformans was shown by some of the tested compounds. Viceversa several synthesized compounds exhibited a moderate or good activity against strains of Candida albicans, while only some of the tested compounds were found moderately active against strains of Candida sp. Instead numerous new compounds 3 or 4 were active as control against isolates of plant pathogenic fungi. The obtained results are discussed on the basis of structure-activity relationships.

The dnaK operon of Streptomyces coelicolor encodes a novel heat-shock protein which binds to the promoter region of the operon.

Transcriptional studies have demonstrated that the dnaK gene of Streptomyces coelicolor A3(2) is contained within a 4.3 kb operon. The operon is transcribed from a single (transiently) heat-inducible promoter, dnaKp, that resembles the typical vegetative (sigma 70-recognized) eubacterial consensus promoter sequence. dnaK transcription was found to be heat-inducible at all stages of development in surface-grown cultures. In addition, at the normal growth temperature of 30 degrees C, dnaK transcript levels were shown to vary at different stages of development, being more abundant in young germinating cultures and in mycelium undergoing sporogenesis. The nucleotide sequence of the dnaK operon has been completed, revealing the gene organization 5'dnaK-grpE-dnaJ orfX. orfX represents a novel heat-shock gene. Its predicted product displays high similarity to the GlnR repressor proteins of Bacillus spp. and to the MerR family of eubacterial transcriptional regulators. The S. coelicolor OrfX protein has been over-produced in Escherichia coli, and DNA-binding experiments indicate that it interacts specifically with the dnaKp region, binding to three partially related inverted repeat sequences; they are centered at -75, -49 and +4, respectively, relative to the transcription start site of the operon. These results suggest that OrfX plays a direct role in the regulation of the dnaK operon.

Developmental control of the heat-shock stress regulon in Streptomyces coelicolor.

In the differentiating eubacterium Streptomyces coelicolor, nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the level of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat- and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor.

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [35S]-methionine, separated by two-dimensional polyacrylamide gel electrophoresis, and quantified using the BioImage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coli beta-galactosidase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHI fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHI fragment.

Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2).

The dnaK homologue of Streptomyces coelicolor A3(2) strain M145 has been cloned and sequenced. Nucleotide sequence analysis of a 2.5-kb region revealed an open reading frame (ORF) encoding a predicted DnaK protein of 618 amino acids (M(r) = 66,274). The dnaK coding sequence displays extreme codon bias and shows a strong preference for CGY and GGY, for Arg and Gly codons, respectively. The predicted DnaK sequence has a high Lys:Arg ratio which is not typical of streptomycete proteins. The region immediately downstream from dnaK contains an ORF for a GrpE-like protein; the predicted start codon of grpE overlaps the last two codons of dnaK, indicating that the two genes are translationally coupled. This organisation differs from that reported for other prokaryotes.