Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

italic> of the Whole nif Genes of Klebsiella oxytoca,a Nitrogen Fixer in the Rhizosphere of Rice

We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6 kb). A non nif DNA fragment was deleted from the plasmid with XhoI, and a smaller plasmid, pNOK31 (nif+, Ap^r, 31.1 kb), was reconstructed.

We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5a1 nif genes as to the EcoRI, HindIII, BamHI and XhoI sites, but differed considerably in the PstI, SalI and BglII sites.

E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280 nmol and 390 nmol/48 hr per 7 ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.     

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis

NB176 is a Bacillus thuringiensis mutant derived by λ-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ-irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.     

Molecular characterization of an adenylate cyclase gene of the cyanobacterium Spirulina platensis

A cyaA gene, encoding an adenylate cyclase, was isolated from a filamentous cyanobacterium, Spirulina platensis, by functional complementation of a cya mutant of Escherichia coli, defective in adenylate cyclase activity. The predicted gene product of cyaA contains a signal peptide-like domain, a putative sensor domain similar to the gene product of vsrA of Pseudomonas solanacearum, a putative membrane-spanning domain and an adenylate cyclase-like catalytic domain. Two other positive clones that complemented the E. coli mutant were isolated from the same cyanobacterium, suggesting that several cya genes are functioning in S. platensis.     

Mutational analysis of the MAL1 locus of Saccharomyces: identification and functional characterization of three genes

Summary Fermentation of maltose by Saccharomyces strains depends on the presence of any one of five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 or MAL6). Earlier mutational analyses of MAL2 and MAL6 containing strains have identified a single complementation group at each of these two loci. However complementation analysis between naturally occurring Mal^– Saccharomyces strains isolated from the wild demonstrated the presence of two complementation groups (designated MALp and MALg) at the MAL1, MAL3 and MAL6 loci. The available evidence suggests that the MALp gene is functionally equivalent to the complementation group identified by mutational analysis at the MAL6 locus and that this gene encodes a protein involved in the regulation of the coordinate induction of both maltase and maltose permease synthesis.In this paper we report the isolation, in a well characterized MAL1 strain, of 47 mutants unable to ferment maltose. All the mutants, with one exception, map at the MAL1 locus. These mal1 mutants, except for one, are recessive to MAL1 and fall into two major complementation groups. Evidence is presented that these two classes of mutants identify both a gene involved in the regulation of maltose fermentation (MAL1R) and a gene involved in maltose transport (MAL1T). We also report here the isolation of a temperature sensitive maltose nonfermenting mutant mapping at the MAL1 locus identifying a third gene (MAL1S) at this locus. The maltase synthesized by this mutant, when assayed in cell-free extracts, is significantly more thermolabile than the wild type enzyme. Our findings demonstrate that MAL1 is a complex locus comprising at least three genes: MAL1R, a gene involved in the coordinate regulation of the synthesis of maltase and maltose transport; MAL1T, a gene encoding a component of the maltose transport system; and MAL1S, a likely candidate for the structural gene for maltase.     

Molecular phylogeny of Macrolycus (Coleoptera: Lycidae) with description of new species from China

Macrolycus is a genus of net‐winged beetles with 69 species distributed in the eastern Palearctic and northernmost part of the Oriental region. The first molecular phylogeny of Macrolycus was produced using an rrnL + tRNA‐Leu + nad1 mtDNA fragment. The major lineages and species limits were identified with morphology and molecular data. We propose that Cerceros is a subgenus of Macrolycus to enable identification of all adult specimens in the genus without DNA sequencing. Two species groups are proposed in Macrolycus s. str. and six in Cerceros. Additionally, twelve Macrolycus species are newly described from China: M. aquilinus, M. baihualingensis, M. bicolor, M. guangxiensis, M. jianfenglingensis, M. kuatunensis, M. lizipingensis, M. parvus, M. phoeniceus, M. rhodoneurus, M. rosaceus and M. sichuanensis. Macrolycus holzschuhi is proposed to be a junior subjective synonym of M. jeanvoinei. The highest diversity of Macrolycus is found in southern China. The species from the main islands of Japan are placed in two species groups: M. excellens is a sister to remaining species of the M. murzini group and the M. flabellatus group is a monophylum of closely related species in a sister position to the M. bicolor group.     

The Evolution of RecD Outside of the RecBCD Complex

The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.     

Conservation of upstream regulators of scute on the notum of cyclorraphous Diptera

Bristles on the notum of many cyclorraphous flies are arranged into species-specific stereotyped patterns. Differences in the spatial expression of the proneural gene scute correlate with the positions of bristles in those species looked at so far. However, the examination of a number of genes encoding trans-regulatory factors, such as pannier, stripe, u-shaped, caupolican and wingless, indicates that they are expressed in conserved domains on the prospective notum. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera, and that many differences are likely to be due to changes in cis-regulatory sequences of scute. In contrast, in Anopheles gambiae, a basal species with no stereotyped bristle pattern, the expression patterns of pannier and wingless are not conserved, and expression of AgASH, the Anopheles proneural gene, does not correlate in a similar manner with the bristle pattern. We discuss the possibility that independently acting cis-regulatory sequences at the scute locus may have arisen in the lineage giving rise to cyclorraphous flies.     

Detection of a novel subspecies of Francisella noatunensis as endosymbiont of the ciliate Euplotes raikovi

Francisella are facultative intracellular bacteria causing severe disease in a broad range of animals. Two species are notable: Francisella tularensis, the causative organism of tularemia and a putative warfare agent, and Francisella noatunensis, an emerging fish pathogen causing significant losses in wild and farmed fish. Although various aspects of Francisella biology have been intensively studied, their natural reservoir in periods between massive outbreaks remains mysterious. Protists have been suspected to serve as a disguised vector of Francisella and co-culturing attempts demonstrate that some species are able to survive and multiply within protozoan cells. Here, we report the first finding of a natural occurrence of Francisella sp. as a protist endosymbiont. By molecular and morphological approaches, we identified intracellular bacteria localized in a strain of the marine ciliate Euplotes raikovi, isolated from the coast of Adriatic Sea. Phylogenetic analysis placed these endosymbionts within the genus Francisella, in close but distinct association with F. noatunensis. We suggest the establishment of a novel subspecies within F. noatunensis and propose the cytoplasmatic endosymbiont of E. raikovi as “Candidatus F. noatunensis subsp. endociliophora” subsp. nov.     

A note on the structure of system state spaces and its implications on the existence of non-repeatable experiments

The implication of state space structure on the existence of a repeatable experimentE designed to determine if a states∈L has propertyP or notP is investigated. It is shown that if a state spaceL is connected, then no experimentE is repeatable. This formalism is used to demonstrate that if a propertyP has an associated set of points inL which is dense with dense complement inL, then there exists no repeatable experimentE which can be used to test whethers has propertyP or notP. Other consequences of this formalization are discussed.     

Intensity of nitrification in soil as a function of time and soil moisture

Proceeding from three previously derived expressions for the intensity of nitrification in soil as a function of time (logΣN=K.logt+q), as a function of incubation moisture (logΣN=A.pF _i+B), as a function of initial moisture (logΣN=C.pF _v+D), it was shown that the nitrification intensity as a function of time and of moisture can be expressed by the bilinear function log ΣN=a.pF _i.logT+b.pF _i+c.logt+d; as a function of time and of initial moisture by the bilinear function logΣ=N=a.pF _v.logt+b.pF _v+c.logt+d; as a function of initial and incubation moisture by the bilinear function log ΣN=a.pF _ipF _v+b.pF _i+c.pF _v+d. The intensity of nitrification as a function of time, incubation moisture and initial moisture may be expressed by the multilinear function log ΣN=a.pF _i.pF _v.logt+b.pF _i.pF _v+c.pF _i.logt+d.pF _v.logt+e .pF _i+f.pF _v=g.logt+h. This function is valid for all the incubation moistures lying between pF _i 3.0 and 4.0 and for all initial moistures between 3.5 and 5.9 provided that the incubation temperature remains constant.     

A comparison of the carotenoids of strains of Oscillatoria and Spirulina (Cyanobacteria)

The qualitative and quantitative carotenoid composition is reported for (i) a red and a green strain of Oscillatoria limnetica and a green strain of Spirulina platensis cultivated under identical conditions and (ii) a red and a green strain of Spirulina subsalsa grown under identical conditions. No correlation between colour and carotenoid content was obtained. However, differences in carotenoid composition within the Oscillatoria and Spirulina strains were observed. Both oscillol diglycoside ex Oscillatoria limnetica and myxol glycoside ex Spirulina platensis were mixed α-glycosides with chinovose-fucose present in ca 7:2 ratio. Analytical procedures are given. The chemosystematic implications for the current taxonomy of the genera Oscillatoria and Spirulina are discussed.     

Organisation and expression of a cluster of yolk protein genes in the Australian sheep blowfly, Lucilia cuprina

The Australian sheep blowfly Lucilia cuprina is a major pest for the Australian and New Zealand sheep industries. With the long-term aim of making a strain of L. cuprina suitable for a genetic control program, we previously developed a tetracycline-repressible female lethal genetic system in Drosophila. A key part of this system is a female-specific promoter from a yolk protein (yp) gene controlling expression of the tetracycline-dependent transactivator (tTA). Here we report the sequence of a 14.2 kb genomic clone from L. cuprina that contains a cluster of three complete yp genes and one partial yp gene. The Lcyp genes are specifically expressed in females that have received a protein meal. A bioinformatic analysis of the promoter of one of the yp genes (LcypA) identified several putative binding sites for DSX, a known regulator of yp gene expression in other Diptera. A transgenic strain of L. cuprina was made that contained the LcypA promoter driving the expression of the Escherichia coli lacZ reporter gene. Transgenic females express high levels of β-galactosidase after a protein meal. Thus the LcypA promoter could be used to obtain female-specific expression of tTA in transgenic L. cuprina.     

Overexpression of fIbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brIA and premature initiation of development

Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for fIbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild-type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of fIbA using an Inducible promoter resulted in misscheduled expression of brlA in vegetative ceils and caused hyphal tips to differentiate into spore-producing structures. Sequence analysis of a nearly full-length fIbA cDNA clone showed that fibA is predicted to encode a 717-amino-acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that fIbA plays a related role in a signalling pathway for Aspergillus conidiophore development.     

A comparison of attack rates in a native and an introduced population of the parasitoid Cotesia glomerata

The attack rate of a population of the braconid parasitoid Cotesia glomerata, introduced into the USA over 100 years ago as a parasitoid of Pieris rapae, was compared with that of a native British population, which normally attacks P. brassicae, and with that of a P. rapae specialist, Cotesia rubecula. British C. glomerata attacked P. brassicae at a much higher rate than it attacked P. rapae. In comparison with British C. glomerata, C. rubecula showed a higher attack rate with P. rapae. American C. glomerata attacked P. rapae at a slightly higher rate than did British C. glomerata, but not at as high a rate as that achieved by C. rubecula. The differences in each comparison were statistically significant. The possible causes of the differences between British and American C. glomerata attacking P. rapae are discussed. They may be due to genetic or environmental effects. Egg load did not appear to be a factor limiting the number of hosts parasitized under the conditions of the experiments.     

A statistical model of the reproduction of aphids

1. The logistic function has been generally used to describe the reproductive process of a “population” of animal. However, this model can not give us any information about the reproductive process of “individuals” in the population. In this study a statistical model on the basis of the reproduction of individuals of barley aphid is presented to find the proportion of the mature individuals, the heterogeneity in reproductive ability of the aphids, etc.
2. The model is constructed as follows:
3. The probability that j insects are found on a plant at time t₀ is represented as Q(j).
4. The probability that h individuals of j have reproductive ability, say, mature individuals, in the period t₀ to t₁ is represented as B(h/j)=_jC_hw^h(1−w)^j−h, where w is the proportion of mature individuals.
5. In a population with a homogeneous reproductive ability, the probability that each parent lays i offspring in the period t₀ to t₁ is represented as P(i/m)=e^−mmⁱ/i!, where m is mean. And, in a population, m changes according to the gamma distribution. Hence the probability that a parent lays i offspring between t₀ and t₁ is represented as , where p and k are parameters of negative binomial distribution. The probability that h parents on a plant lays s offspring is represented as .
6. From the assumptions mentioned above, the probability that s offspring are to be found at time t₁ on a plant with the original j individuals at time t₀ is represented by
7. The experimental populations were demonstrated to fit well to the model.

     

Responses of genotypes from species of Trifolium, Ornithopus, Biserrula and Hedysarum to a highly virulent race of Phytophthora clandestina and new sources of resistance

Thirty‐six genotypes, including 15 cultivars and 10 breeding lines of Trifolium subterraneum, a single genotype of each of seven other species of Trifolium (viz. Trifolium dasyurum, Trifolium glanduliferum, Trifolium incarnatum, Trifolium michelanium, Trifolium purpureum, Trifolium spumosum and Trifolium vesiculosum), Biserrula pelecinus, Hedysarum coronarium, Ornithopus compressus and Ornithopus sativus were screened under controlled environmental conditions for resistance to root disease caused by the most pathogenic race of Phytophthora clandestina occurring in Australia, namely race 177. This is the first time any of these genera/species other than T.subterraneum has ever been screened for its response to P. clandestina. The root disease caused by P.clandestina is the first report of susceptibility to this pathogen for the seven other species of Trifolium and also for B.pelecinus, H.coronarium and O.sativus. Within T.subterraneum, a very high level of resistance was identified in cvs Denmark, Junee and Meteora [scores ≤1.5 (0–5 scale where 0 = no disease) across two separate screening tests] and in the breeding lines SL027 and SM023 (scores ≤1.3 across two separate screening tests). Six of the seven other species of Trifolium (viz. T.dasyurum, T.glanduliferum, T.incarnatum, T.michelanium, T.purpureum and T.spumosum) showed a high level of resistance (scores ≤0.8 across two separate screening tests), while T.vesiculosum showed a disease score of ≤1.2 across both screening tests. O.compressus showed no disease in either test, and O.sativus showed a disease score of ≤0.7 across both screening tests. H.coronarium was susceptible with a disease score of ≤2.8 across two separate screening tests, while B.pelecinus was highly susceptible with disease scores of 3.5 and 4.6 in these tests. The high levels of resistance identified against P.clandestina are useful sources of resistance that can be exploited commercially, either directly to minimise damage from this disease or as parents in breeding programs to develop cultivars within the genera/species tested with improved resistance to this highly pathogenic race of P.clandestina.