Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 ΔfhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.     

Characterization of siderophores from Ustilago maydis

Two siderophores, ferrichrome and ferrichrome A, were found in cultures of Ustilago maydis (DC) Corda. Both siderophores were found intracellularly and extracellularly. Their authenticity was confirmed by thin layer chromatography, HPLC, UV-visible spectrometry, paper electrophoresis, amino acid analysis, NMR and fast atom bombardment mass spectroscopy. Regulation of siderophore production by iron was examined. Repression of biosynthesis of extracellular siderophores occurred at 10^–5 M iron. Ferrichrome was found intracellularly at all iron concentrations employed; in general, ferrichrome A was not found to be cell-associated.     

Characterization of siderophore-mediated iron transport inGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [⁵⁹Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [⁵⁹Fe]ferrioxamine B uptake by ferrichrome was non-competitive (K _i 2.4 M), suggesting that the two siderophores do not share a common transport system. Uptake of [⁵⁹Fe]ferrichrome, [⁵⁹Fe]rhodotorulic acid and [⁵⁹Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [¹⁴C]ferrioxamine B and⁶⁷Ga-desferrioxamine B were 30% and 60% respectively, as compared to [⁵⁹Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [⁵⁹Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.     

Iron nutrition and physiological responses to iron stress in Nitrosomonas europaea

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 μM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH₃- and NH₂OH-dependent O₂ consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.     

Isolation and characterization of actinomycetes antagonistic to pathogenic Vibrio spp. from nearshore marine sediments

Summary A total of 94 actinomycete strains were isolated from the marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others were Micromonospora spp. Fifty-one percent of the actinomycete strains showed activity against the pathogenic Vibrio spp. strains. Thirty-eight percent of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar plates. Seven strains of Streptomyces were found to produce siderophores and to inhibit the growth of Vibrio spp. in vitro. Two of them belonged to the Cinerogriseus group, the most frequently isolated group of Streptomyces. The results showed that streptomycetes could be a promising source for biocontrol agents in aquaculture.     

Phylogenetic relationships of someMicrosporum andArthroderma species inferred from mitochondrial DNA analysis

Thirty-eight strains of 12Microsporum and 10Arthroderma (Nannizzia) species were investigated by analysis of mitochondrial DNA with 6 restriction enzymes, and classified into 13 genetic groups. The phylogenetic tree of the 13 groups thus established was constructed. On the tree,M. audouinii, M. langeronii, M. rivalieri, M. distortum, M. equinum, M. ferrugineum andA. otae comprise one genetic group and are suggested to be the same species.A. gypseum, A. fulvum, M. duboisii, M. ripariae, A. incurvatum, A. persicolor andA. obtusum are clustered on one of five boughs of the tree indicating their close relation.A. racemosum andA. cajetani are also closely related.     

Characterization of ferrioxamine E as the principal siderophore ofErwinia herbicola (Enterobacter agglomerans)

Summary Several strains of the enterobacterial groupErwinia herbicola (Enterobacter agglomerans) were screened for siderophore production. After 3 days of growth in a low-iron medium, all strains studied produced hydroxamate siderophores. The retention values of the main siderophore during thin-layer chromatography on silica gel plates and on HPLC reversed-phase columns were identical with those of an authentic sample of ferrioxamine E (norcardamine). Gas-chromatographic analysis of the HI hydrolyzate yielded succinic acid and 1,5-diaminopentane in equimolar amounts; fast-atom-bombardment (FAB) mass spectroscopy showed a molecular mass of 653 Da. Iron from⁵⁵Fe-labelled ferrioxamine E was well taken up by iron-starved cells ofE. herbicola (K _m=0.1 M,V _max=8 pmol mg^–1 min^–1). However, besides ferrioxamine E (100%), several exogenous siderophores such as enterobactin (94.5%), ferric citrate (78.5%), coprogen (63.5%) and ferrichrome (17.5%) served as siderophores, suggesting the presence of multiple siderophore receptors in the outer membrane ofE. herbicola.     

Ferrioxamine transport mutants and the identification of the ferrioxamine receptor protein (FoxA) inErwinia herbicola (Enterobacter agglomerans)

Summary Iron deprivation ofErwinia herbicola (Enterobacter agglomerans) induces the biosynthesis of six high-M _r outer-membrane proteins and large amounts of ferrioxamine E. Mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine and selection with ferrimycin A yielded mutants ofE. herbicola K4 (wild type), defective in the expression of a 76-kDa outer-membrane protein, as determined by SDS/polyacrylamide gel electrophoresis. While in bioassays wild-type cells showed growth promotion in the presence of ferrioxamines (B, D₁, D₂, E, G), enterobactin, citrate, ferrichrome and coprogen, these mutants failed to respond to ferrioxamines. Moreover, experiments with⁵⁵Fe-labelled siderophores confirmed that iron transport mediated by ferrioxamine E and B in the mutants was completely inhibited, whereas iron transport by other hydroxamate siderophores, such as ferrichrome and coprogen was unaffected. The results are evidence that the 76-kDa protein in the outer membrane represents the receptor protein (FoxA) for ferrioxamines inE. herbicola.     

Synthetic siderophores as biological probes

Summary Synthetic molecules that mimic the properties of the natural siderophores promise to become powerful tools in the exploration of microbial iron(III)-uptake phenomena. Such molecules can serve as probes to (i) establish the essential structural requirements for biological action, (ii) trace alternative reaction pathways and (iii) compare receptors of different biological origins. In this article a series of synthetic ferrichrome analogs will be described. The strategy adopted for the design and synthesis of these compounds will be outlined and their properties in vitro and in vivo examined. The growth promotion activity of these compounds inArthrobacter flavescens is used to map the ferrichrome receptor surface. Their activities towardsZea mays allow us to trace the plants' reductive iron(III) uptake routes. Potential applications of modified ferrichrome analogs for the isolation of ferrichrome receptors, the generation of fluorescent probes and ultimately new families of antibiotic or antifungal agents, will also be indicated.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Buzz-pollination in three nectariferousBoraginaceae and possible evolution of buzz-pollinated flowers

Buzz-pollination was observed in three nectariferousBoraginaceae spp.:Onosma gigantea Lam.,Trichodesma africana (L.)R. Br. andT. boissieri Post. An evolutionary pathway from usual nectariferous flowers to typical buzz-pollinated flowers is suggested.     

Pollination ecology ofOxalis violacea (Oxalidaceae) following a controlled grass fire

Vernal grass fires may encourage profuse flowering in clonal, colonies ofOxalis violacea. Long-styled colonies appear to be more floriferous than short-styled colonies and set a greater number of capsules. Individual flowers of both morphs live one or two days, change position on their respective pedicels and advertise nectar concealed at the base of the floral throat. AlthoughDiptera, Hymenoptera, andLepidoptera forage for nectar, bees (Andrenidae,Anthophoridae, Halictidae, andMegachilidae) probably make the only effective pollen transfers between the two morphs. Both male and female bees may transport pollen of both morphs and short-tongued bees (e.g.,Augochlorella spp.,Dialictus spp.) may be more common but as effective as pollinators as long-tongued bees (e.g.,Calliopsis andreniformis andHoplitis spp.). The conversion rate of flowers into capsules is only 13–17%. The spreading style in the short-styled morph is interpreted as an adaptation restricting insect-mediated, self-pollination but encouraging bee-stigma contact during nectar foraging.     

Allozymic variation in four Indian species of genusMus: A comparative analysis

The present study focusses on allozyme variation in the commensal house mouseMus musculus, the pygmy field miceM. booduga andM. terricolor, and the spiny mouseM. platythrix. Genetic heterozygosity was estimated using a set of 24 polymorphic biochemical genetic markers. The extent of variability present inM. booduga, M. terricolor andM. platythrix has been compared with that in theM. musculus complex. Levels of allozyme variation at species level indicate thatM. musculus has the maximum heterogeneity, followed byM. booduga andM. terricolor, whileM. platythrix shows comparatively homogeneous genetic make-up. Gene frequency data have been used to trace phylogenetic relationships among these four species.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and Iberian red deer (<Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis>) in central Spain

Emerging tick-borne diseases of humans and animals have occurred frequently during the past 30 years. These disease outbreaks appear to result from changes in the distribution of tick and vertebrate hosts, and the introduction of humans and domestic animals into tick–pathogen–wildlife cycles. Use of molecular technologies now available for identification of pathogens in ticks can provide valuable information that allows for risk analysis of emerging tick-borne diseases. In this study, the prevalence of selected pathogens in ticks collected in six locations in central Spain from the major wild ungulate species, European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus), was determined by PCR. Tick species collected included Ixodes ricinus, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma m. marginatum. Pathogens identified in ticks included piroplasmids, Anaplasma spp., Ehrlichia spp. and Rickettsia spp. Piroplasmids were identified in all tick species except I. ricinus. Ehrlichia spp. were detected in all tick species and collection locations, while Rickettsia spp., which proved to be R. slovaca and a recently identified Rickettsia sp. DnS28, were identified only in D. marginatus. A. marginale and A. phagocytophilum were detected in D. marginatus, R. bursa and Hy. m. marginatum. Concurrent infections of these pathogens were frequently observed in ticks. Notably, A. phagocytophilum, which is infective for a broad host range that includes humans and domestic and wild animals, was identified in ticks from all collection locations. The variety of ticks and tick-borne pathogens demonstrated in this study suggests a risk in central Spain for the emergence of tick-borne diseases in humans and domestic animals.     

Investigations of the host feeding preferences ofSitona weevils found commonly on white clover (Trifolium repens) in the UK

A series of laboratory and field studies were done to evaluate a range of leguminous plant species for their feeding potential by adult weevils of the genusSitona Germar. (Coleoptera: Curculionidae). Three species ofSitona, S. lineatus L.,S. flavescens Marsh. andS. hispidulus F. all of which are found commonly on white clover (Trifolium repens L.) in the UK were offered a range of 11 legume species,T. repens (white clover, cv. Olwen),T. pratense L. (red clover, cv. Marcom),T. fragiferum L. (strawberry clover, cv. Palestine),T. hybridum L. (hybrid clover, cv. Tetra),T. incarnatum L (crimson clover),T. dubium Sibth. (lesser yellow trefoil),Lotus corniculatus L. (birdsfoot-trefoil, cv. Leo),L. uliginosus Schkuhr. (large birdsfoot-trefoil),Melilotus alba Desr. (white melilot),Medicago sativa L. (lucerne, cv. Europe) andM. lupulina L. (black medick) in two laboratory experiments. The weevils were offered a choice of these legumes in one experiment whilst in the other they did not have a choice of food material. These legumes were also sown in the field and a number of measurements of damage, together with counts ofSitona spp., were made. In the laboratoryS. lineatus andS. hispidulus favoured some of the legumes to a greater or lesser extent than white clover.S. flavescens was more restricted in its feeding than the other two weevil species. In the field studyS. lineatus invaded the experimental area quickly and tended to favourMedicago spp. andMelilotus spp. Later in the yearS. flavescens dominated the sitona fauna on the experiment, with the exception of aggregations ofS. lineatus onM. sativa andM. alba. In a separate screen of 5 varieties of white clover (cvs Donna, Menna, Kersey, Olwen and Grasslands Huia), cv. Olwen appeared to be the most susceptible to sitona attack.     

Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria – Micrococcus luteus and Bacillus silvestris.Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and α-keto/α-hydroxy acids, but did not produce catecholates.This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.     

Hyaloscyphaceae in Japan (5): SomeLachnum-like members

Five members of the family Hyaloscyphaceae with multiseptate hairs and lanceolate paraphyses (Lachnum-like members in broad sense) are described:Albotricha fagicola andDasyscyphella longistipitata spp. nov.;Trichopeziza discolor, T. sulphurea, andTrichopezizella barbata, new to Japan. (4): Mycoscience42: 597–609, 2001.     

Ferrichrome in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> – an iron transport and iron storage compound

Schizosaccharomyces pombe has been assumed not to produce siderophores. Nevertheless, the genomic sequence of this fission yeast revealed the presence of siderophore biosynthetic genes for hydroxamates. Applying a bioassay based on an Aspergillus nidulans strain deficient in siderophore biosynthesis, and using reversed-phase HPLC and mass spectrometry analysis, we demonstrate that S. pombe excretes and accumulates intracellularly the hydroxamate-type siderophore ferrichrome. Under iron-limiting conditions, the cellular ferrichrome pool was present in the desferri-form, while under iron-rich conditions, in the ferri-form. In contrast to S. pombe, hydroxamate-type siderophores could not be detected in two other yeast species, Saccharomyces cerevisiae and Candida albicans.     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.