An in vivo assay for conjugation-mediated recombination yields novel results for Streptomyces plasmid pIJ101

Efficient transmission of circular plasmids in Streptomyces spp. proceeds by an uncharacterized mechanism that requires a cis-acting locus of transfer (clt) and often only a single plasmid-encoded protein. For circular plasmids from other bacteria, site- and strand-specific nicking takes place at the cis-acting oriT locus via the plasmid-encoded relaxase protein prior to single-strand transfer. Using an assay originally designed to demonstrate that conjugative transfer of plasmids containing tandem oriT loci results in the formation of a single composite oriT locus, we show here that an analogous construct involving the pIJ101 clt locus apparently does not undergo such a conjugation-mediated event during plasmid transfer. Our results, which imply that streptomycete plasmids are transferred by a functionally distinct mechanism compared to oriT-containing plasmids, are complementary to other recent evidences that support a novel double-stranded model for streptomycete circular plasmid transfer.     

Direct repeat sequences are essential for function of the cis-acting locus of transfer (clt) of Streptomyces phaeochromogenes plasmid pJV1

The functionality of direct and inverted repeat sequences inside the cis acting locus of transfer (clt) of the Streptomyces plasmid pJV1 was determined by testing the effect of different deletions on plasmid transfer. The results show that the single most important element for pJV1 clt function is a series of evenly spaced 9 bp long direct repeats which match the consensus CCGCACA(C/G)(C/G), since their deletion caused a dramatic reduction in plasmid transfer. The presence of these repeats in the absence of any other clt sequences allowed plasmid transfer to occur at a frequency that was at least two orders of magnitude higher than that obtained in the complete absence of clt. A database search revealed regions with a similar organization, and in the same position, in Streptomyces plasmids pSN22 and pSLS, which have transfer proteins homologous to those of pJV1.     

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.     

Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2)

The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.     

海南典型有害植物的入侵扩散机理研究进展

有害植物成灾会对农林业生态系统、经济和环境造成巨大影响。本文对海南典型入侵植物薇甘菊、假臭草及土著灾变植物金钟藤的成灾扩散机理研究做一综述,以期为海南有害植物的研究和管理提供依据。自然及人为干扰造成的薇甘菊匍匐茎克隆片段有较高的再生能力,其再生能力和生长与储存在节间和叶子中的资源呈正相关。假臭草随土壤养分条件的改善比本地种获利更多,说明农田施肥等干扰因素可能促进假臭草相对于本地种的种群优势。金钟藤已扩散至海南五指山有旅游活动的自然林;调查和移栽试验发现,其可能是先在受干扰严重的林缘和旅游小径边定植,之后通过匍匐和攀爬生长扩散至附近的自然林。这些结果强调了人类活动、高养分条件及物种自身性状等在有害植物成灾中的作用。未来仍需系统、深入地研究有害植物成灾扩散的机理及其控制对策,为减弱有害植物的扩散和危害提供参考,并为海南国际旅游岛的建设提供安全保证。     

Isolation of a solventogenic Clostridium sp. strain: Fermentation of glycerol to n-butanol,analysis of the bcs operon region and its potential regulatory elements

A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474^T. GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v).     

Effect of mono- and polygyne social forms on transmission and spread of a microsporidium in fire ant populations

Thelohania solenopsae is a pathogen of the red imported fire ant, Solenopsis invicta, which debilitates queens and eventually causes the demise of colonies. Reductions of infected field populations signify its potential usefulness as a biological control agent. Thelohania solenopsae can be transmitted by introducing infected brood into a colony. The social forms of the fire ant, that is, monogyny (single queen per colony) or polygyny (multiple queens per colony), are associated with different behaviors, such as territoriality, that affect the degree of intercolony brood transfer. T. solenopsae was found exclusively in polygyne colonies in Florida. Non-synchronous infections of queens and transovarial transmission favor the persistence and probability of detecting infections in polygynous colonies. However, queens or alates with the monogyne genotype can be infected, and infections in monogyne field colonies have been reported from Louisiana and Argentina. Limited independent colony-founding capability and shorter dispersal of alate queens with the polygyne genotype relative to monogyne alates may facilitate the maintenance of infections in local polygynous populations. Demise of infected monogyne colonies can be twice as fast as in polygyne colonies and favors the pathogen's persistence in polygyne fire ant populations. The social form of the fire ant reflects different physiological and behavioral aspects of the queen and colony that will impact T. solenopsae spread and ultimate usefulness for biological control.     

optix functions as a link between the retinal determination network and the dpp pathway to control morphogenetic furrow progression in Drosophila

optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein–protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play an important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development.     

Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardiaduodenalis and identification of potentially zoonotic subgenotypes

Human giardiasis, caused by the intestinal flagellate Giardia duodenalis, is considered a zoonotic infection, although the role of animals in the transmission to humans is still unclear. Molecular characterisation of cysts of human and animal origin represents an objective means to validate or reject this hypothesis. In the present work, cysts were collected in Italy from humans (n=37) and animals (dogs, one cat and calves, n=46), and were characterised by PCR amplification and sequencing of the beta-giardin gene. As expected, only Assemblages A and B were identified among human isolates. The host-specific Assemblages C and D were found in the majority of dog isolates; however, 6 dog isolates were typed as Assemblage A. The cat-specific Assemblage F has been identified in the single feline isolate available. Among calf isolates, most were typed as Assemblages A (n=12) and B (n=5), whereas the host-specific Assemblage E was rarely found (n=3). Sequence heterogeneity in the beta-giardin gene allowed a number of subgenotypes to be identified within Assemblage A (8 subgenotypes), B (6 subgenotypes), D (2 subgenotypes), and E (3 subgenotypes). Five of these subgenotypes, namely A1, A2, A3, A4 and B3, were found to be associated with infections of humans, of dogs and of calves; these data, therefore, supported the role of these animals as a source of infection for humans.     

Intensity of transmission and spread of gene mutations linked to chloroquine and sulphadoxine-pyrimethamine resistance in falciparum malaria

The number of malaria parasite clones per infection-multiplicity of parasite clones-is affected by the transmission intensity, multiplicity increases with increasing transmission. This affects the frequency of parasites' sexual recombination and, if several mutations in different genes are involved, can break down drug resistant genotypes. Therefore, the effects of malaria transmission intensity on the spread of drug resistance could vary depending on the number of genes involved. Here we show that, compared to low transmission, intermediate-high transmission is associated with a 20-100-fold lower risk for the mutations linked to chloroquine resistance and a 6-17 times higher risk for those linked to sulphadoxine-pyrimethamine resistance. This is consistent with the hypothesis of a multigenic basis for chloroquine resistance and a monogenic basis for that of sulphadoxine-pyrimethamine. Reducing transmission intensity could slow the spread of resistance. However, a reduction below a critical threshold (e.g. when parasite prevalence in children 2-9 years old is around 60-80%) could, paradoxically, accelerate the spread of resistance to chloroquine and possibly to other drug combinations whose basis is multigenic. Our findings have important implications for malaria control because increasing drug resistance has a substantial impact on mortality.     

Pentalenolactone biosynthesis: Molecular cloning and assignment of biochemical function to PtlF, a short-chain dehydrogenase from Streptomyces avermitilis, and identification of a new biosynthetic intermediate

Pentalenolactone (1) is an antibiotic that has been isolated from many species of Streptomyces. The putative dehydrogenase encoded by the ptlF gene (SAV2993) found within the Streptomyces avermitilis pentalenolactone gene cluster was cloned and overexpressed in Escherichia coli. PtlF, which belongs to the short-chain dehydrogenase/oxidoreductase superfamily, was shown to catalyze the oxidation of 1-deoxy-11beta-hydroxypentalenic acid (9) to 1-deoxy-11-oxopentalenic acid (10), a new intermediate of the pentalenolactone biosynthetic pathway. The methyl ester of 10 was characterized by NMR, GC-MS and high resolution mass spectrometry. PtlF exhibited a 150-fold preference for beta-NAD(+) over beta-NADP(+). PtlF had a pH optimum of 8.0 in the physiological pH range, while a significant activity enhancement was observed from pH 9.0 to 11.3. At pH 8.0, PtlF had a k(cat) of 0.65+/-0.03 s(-1), with a K(m) for 9 of 6.5+/-1.5 microM and K(m) for NAD(+) of 25+/-3 microM.     

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.     

Effect of Gregarina sp. parasitism on the susceptibility of Blattella germanica to some control agents

Gregarines are enteric parasites of invertebrates but little is known about the negative effects of this parasitism on host species. The present study evaluates the influence of the parasitism of Gregarina sp. on the survival of Blattella germanica and methods for elimination of gregarine infection in laboratory rearing systems. Insects were dissected and the infection was detected in 80% of a sample of 50 adults. Diseased cockroaches had swollen abdomens, slower movement at high incidences of the protozoan, and short antennas. Dead cockroaches showed darkened body and putrid smell, indicating septicaemia. Infected insects were more susceptible than healthy cockroaches when treated with Metarhizium anisopliae and triflumuron.     

Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein

Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes, a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. We report on the Sco-like protein from Streptomyces lividans (Sco^Sl) and present a series of experiments that firmly establish a role for Sco^Sl as a copper metallochaperone as opposed to a role as a thiol-disulphide reductase that has been assigned to other bacterial Sco proteins. Under low copper concentrations, a Δsco mutant in S. lividans displays two phenotypes; the development switch between vegetative mycelium and aerial hyphae stalls and cytochrome c oxidase (CcO) activity is significantly decreased. At elevated copper levels, the development and CcO activity in the Δsco mutant are restored to wild-type levels and are thus independent of Sco^Sl. A CcO knockout reveals that morphological development is independent of CcO activity leading us to suggest that Sco^Sl has at least two targets in S. lividans. We establish that one Sco^Sl target is the dinuclear Cu_A domain of CcO and it is the cupric form of Sco^Sl that is functionally active. The mechanism of cupric ion capture by Sco^Sl has been investigated, and an important role for a conserved His residue is identified.