A new isoform of anti-lipopolysaccharide factor identified from the blue swimmer crab, Portunus pelagicus: Molecular characteristics and phylogeny

Anti-lipopolysaccharide factors are small proteins that bind and neutralize lipopolysaccharide and exhibit potent antimicrobial activities. This study presents the molecular characterization and phylogenetic analysis of the first ALF isoform (Pp-ALF1; JQ745295) identified from the hemocytes of Portunus pelagicus. The full length cDNA of Pp-ALF1 consisted of 880 base pairs encoding 293 amino acids with an ORF of 123 amino acids and contains a putative signal peptide of 24 amino acids. Pp-ALF1 possessed a predicted molecular weight (MW) of 13.86 kDa and theoretical isoelectric point (pI) of 8.49. Two highly conserved cysteine residues and putative LPS binding domain were observed in Pp-ALF1. Peptide model of Pp-ALF1 consisted of two α-helices crowded against a four-strand β-sheet. Comparison of amino acid sequences and neighbor joining tree showed that Pp-ALF1 has a maximum similarity (46%) to ALF present in Portunus trituberculatus followed by 39% similarity to ALF of Eriocheir sinensis and 38% similarity to ALFs of Scylla paramamosain and Scylla serrata. Pp-ALF1 is found to be a new isoform of ALF family and its characteristic similarity with other known ALFs signifies its role in protection against invading pathogens.

Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica

Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture

Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A

Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties andmolecular structure ofHarriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates

A Novel Isoform of the Hepatic Antimicrobial Peptide, Hepcidin (Hepc-CB1), from a Deep-Sea Fish, the Spinyjaw Greeneye Chlorophthalmus bicornis (Norman, 1939): Molecular Characterisation and Phylogeny

Hepcidin is cysteine-rich short peptide of innate immune system of fishes, equipped to perform prevention and proliferation of invading pathogens like bacteria and viruses by limiting iron availability and activating intracellular cascades. Hepcidins are diverse in teleost fishes, due to the varied aquatic environments including exposure to pathogens, oxygenation and iron concentration. In the present study, we report a 87-amino acid (aa) preprohepcidin (Hepc-CB1) with a signal peptide of 24 aa, a prodomain of 39 aa and a bioactive mature peptide of 24 aa from the gill mRNA transcripts of the deep-sea fish spinyjaw greeneye, Chlorophthalmus bicornis. Molecular characterisation and phylogenetic analysis categorised the peptide to HAMP2-like group with a mature peptide of 2.53 kDa; a net positive charge (?3) and capacity to form b-hairpin-like structure configured by 8 conserved cysteines. The present work provides new insight into the mass gene duplication events and adaptive evolution of hepcidin isoforms with respect to environmental influences and positive Darwinian selection. This work reports a novel hepcidin isoform under the group HAMP2 from a nonacanthopterygian deep-sea fish, C. bicornis

Actinomycete Isolates from Arabian Sea and Bay of Bengal: Biochemical, Molecular and Functional Characterization

The thesis is comprised of seven chapters. Chapter 1 gives a general introduction to marine actinomycetes; Chapter 2 gives an account on the morphological, biochemical and physiological characterization of marine actinomycetes. Comprehensive description of molecular identification and phylogenetic analysis of actinomycetes is dealt with in Chapter 3. The antimicrobial property with special reference to antivibrio activity is described in Chapter 4. Chapter 5 explores the melanin production ability of marine actinomycetes, characterization of melanin and evaluation of its bioactivity. Chapter 6 illustrates the study on chitinolytic Streptomyces as antifungal and insecticidal agents. Summary and Conclusion of the study is presented in Chapter 7, followed by References and Appendices.The present study provides an insight into the various actinomycetes occurring in the sediments of Arabian Sea and Bay of Bengal. Streptomyces was found to be the dominant group followed by Nocardiopsis. Eventhough generic level identification is possible by traditional phenotypic methods, species level identification necessitate a polyphasic approach including both phenotypic and genotypic characterization. Antibiotic production coupled with biogranulation property helped in the effective utilization of the actinomycetes for the control of vibrios. Melanin from Streptomyces bikiniensis was proved to be a promising antioxidant and photoprotectant. Marine actinomycetes were found to be a good source of hydrolytic enzymes and the chitinolytic isolates could be explored as biocontrol agents in terms of antifungal and insecticidal property. The present study explored the potential of marine actinomycetes especially Streptomycetes as a promising source of bioactive molecules for application in aquaculture and pharmaceutical industry.

Molecular genetic profiling of functional genes of pearl oyster, Pinctada fucata

There are a number of genes involved in the regulation of functional process in marine bivalves. In the case of pearl oyster, some of these genes have major role in the immune/defence function and biomineralization process involved in the pearl formation in them. As secondary filter feeders, pearl oysters are exposed to various kinds of stressors like bacteria, viruses, pesticides, industrial wastes, toxic metals and petroleum derivatives, making susceptible to diseases. Environmental changes and ambient stress also affect non-specific immunity, making the organisms vulnerable to infections. These stressors can trigger various cellular responses in the animals in their efforts to counteract the ill effects of the stress on them. These include the expression of defence related genes which encode factors such as antioxidant genes, pattern recognition receptor proteins etc. One of the strategies to combat these problems is to get insight into the disease resistance genes, and use them for disease control and health management. Similarly, although it is known that formation of pearl in molluscs is mediated by specialized proteins which are in turn regulated by specific genes encoding them, there is a paucity of sufficient information on these genes.In view of the above facts, studies on the defence related and pearl forming genes of the pearl oyster assumes importance from the point of view of both sustainable fishery management and aquaculture. At present, there is total lack of sufficient knowledge on the functional genes and their expressions in the Indian pearl oyster Pinctada fucata. Hence this work was taken up to identify and characterize the defence related and pearl forming genes, and study their expression through molecular means, in the Indian pearl oyster Pinctada fucata which are economically important for aquaculture at the southeast coast of India. The present study has successfully carried out the molecular identification, characterization and expression analysis of defence related antioxidant enzyme genes and pattern recognition proteins genes which play vital role in the defence against biotic and abiotic stressors. Antioxidant enzyme genes viz., Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidise (GPX) and glutathione-S-transferase (GST) were studied. Concerted approaches using the various molecular tools like polymerase chain reaction (PCR), random amplification of cDNA ends (RACE), molecular cloning and sequencing have resulted in the identification and characterization of full length sequences (924 bp) of the Cu/Zn SOD, most important antioxidant enzyme gene. BLAST search in NCBI confirmed the identity of the gene as Cu/Zn SOD. The presence of the characteristic amino acid sequences such as copper/zinc binding residues, family signature sequences and signal peptides were found out. Multiple sequence alignment comparison and phylogenetic analysis of the nucleotide and amino acid sequences using bioinformatics tools like BioEdit,MEGA etc revealed that the sequences were found to contain regions of diversity as well as homogeneity. Close evolutionary relationship between P. fucata and other aquatic invertebrates was revealed from the phylogenetic tree constructed using SOD amino acid sequence of P. fucata and other invertebrates as well as vertebrates

Characterization and Pathogenicity of Vibrio cholerae and Vibrio vulnificus from Marine environments

The genus Vibrioof the family Vibrionaceae are Gram negative, oxidasepositive, rod- or curved- rodshaped facultative anaerobes, widespread in marine and estuarine environments. Vibrio species are opportunistic human pathogens responsible for diarrhoeal disease, gastroenteritis, septicaemia and wound infections and are also pathogens of aquatic organisms, causing infections to crustaceans, bivalves and fishes. In the present study, marine environmental samples like seafood and water and sediment samples from aquafarms and mangroves were screened for the presence of Vibrio species. Of the134 isolates obtained from the various samples, 45 were segregated to the genus Vibrio on the basis of phenotypic characterization.like Gram staining, oxidase test, MoF test and salinity tolerance. Partial 16S rDNA sequence analysis was utilized for species level identification of the isolates and the strains were identified as V. cholerae(N=21), V. vulnificus(N=18), V. parahaemolyticus(N=3), V. alginolyticus (N=2) and V. azureus (N=1). The genetic relatedness and variations among the 45 Vibrio isolates were elucidated based on 16S rDNA sequences. Phenotypic characterization of the isolates was based on their response to 12 biochemical tests namely Voges-Proskauers’s (VP test), arginine dihydrolase , tolerance to 3% NaCl test, ONPG test that detects β-galactosidase activity, and tests for utilization of citrate, ornithine, mannitol, arabinose, sucrose, glucose, salicin and cellobiose. The isolates exhibited diverse biochemical patterns, some specific for the species and others indicative of their environmental source.Antibiogram for the isolates was determined subsequent to testing their susceptibility to 12 antibiotics by the disc diffusion method. Varying degrees of resistance to gentamycin (2.22%), ampicillin(62.22%), nalidixic acid (4.44%), vancomycin (86.66), cefixime (17.77%), rifampicin (20%), tetracycline (42.22%) and chloramphenicol (2.22%) was exhibited. All the isolates were susceptible to streptomycin, co-trimoxazole, trimethoprim and azithromycin. Isolates from all the three marine environments exhibited multiple antibiotic resistance, with high MAR index value. The molecular typing methods such as ERIC PCR and BOX PCR revealed intraspecies relatedness and genetic heterogeneity within the environmental isolatesof V. cholerae and V. vulnificus. The 21 strains of V. choleraewere serogroupedas non O1/ non O139 by screening for the presence O1rfb and O139 rfb marker genes by PCR. The virulence/virulence associated genes namely ctxA, ctxB, ace, VPI, hlyA, ompU, rtxA, toxR, zot, nagst, tcpA, nin and nanwere screened in V. cholerae and V. vulnificusstrains.The V. vulnificusstrains were also screened for three species specific genes viz., cps, vvhand viu. In V. cholerae strains, the virulence associated genes like VPI, hlyA, rtxA, ompU and toxR were confirmed by PCR. All the isolates, except for strain BTOS6, harbored at least one or a combination of the tested genes and V. choleraestrain BTPR5 isolated from prawn hosted the highest number of virulence associated genes. Among the V. vulnificusstrains, only 3 virulence genes, VPI, toxR and cps, were confirmed out of the 16 tested and only 7 of the isolates had these genes in one or more combinations. Strain BTPS6 from aquafarm and strain BTVE4 from mangrove samples yielded positive amplification for the three genes. The toxRgene from 9 strains of V. choleraeand 3 strains of V. vulnificus were cloned and sequenced for phylogenetic analysis based on nucleotide and the amino acid sequences. Multiple sequence alignment of the nucleotide sequences and amino acid sequences of the environmental strains of V. choleraerevealed that the toxRgene in the environmental strains are 100% homologous to themselves and to the V. choleraetoxR gene sequence available in the Genbank database. The 3 strains of V. vulnificus displayed high nucleotide and amino acid sequence similarity among themselves and to the sequences of V. cholerae and V. harveyi obtained from the GenBank database, but exhibited only 72% homology to the sequences of its close relative V. vulnificus. Structure prediction of the ToxR protein of Vibrio cholerae strain BTMA5 was by PHYRE2 software. The deduced amino acid sequence showed maximum resemblance with the structure of DNA-binding domain of response regulator2 from Escherichia coli k-12 Template based homology modelling in PHYRE2 successfully modelled the predicted protein and its secondary structure based on protein data bank (PDB) template c3zq7A. The pathogenicity studies were performed using the nematode Caenorhabditiselegansas a model system. The assessment of pathogenicity of environmental strain of V. choleraewas conducted with E. coli strain OP50 as the food source in control plates, environmental V. cholerae strain BTOS6, negative for all tested virulence genes, to check for the suitability of Vibrio sp. as a food source for the nematode;V. cholerae Co 366 ElTor, a clinical pathogenic strain and V. cholerae strain BTPR5 from seafood (Prawn) and positive for the tested virulence genes like VPI, hlyA, ompU,rtxA and toxR. It was found that V. cholerae strain BTOS6 could serve as a food source in place of E. coli strain OP50 but behavioral aberrations like sluggish movement and lawn avoidance and morphological abnormalities like pharyngeal and intestinal distensions and bagging were exhibited by the worms fed on V. cholerae Co 366 ElTor strain and environmental BTPR5 indicating their pathogenicity to the nematode. Assessment of pathogenicity of the environmental strains of V. vulnificus was performed with V. vulnificus strain BTPS6 which tested positive for 3 virulence genes, namely, cps, toxRand VPI, and V. vulnificus strain BTMM7 that did not possess any of the tested virulence genes. A reduction was observed in the life span of worms fed on environmental strain of V. vulnificusBTMM7 rather than on the ordinary laboratory food source, E. coli OP50. Behavioral abnormalities like sluggish movement, lawn avoidance and bagging were also observed in the worms fed with strain BTPS6, but the pharynx and the intestine were intact. The presence of multi drug resistant environmental Vibrio strainsthat constitute a major reservoir of diverse virulence genes are to be dealt with caution as they play a decisive role in pathogenicity and horizontal gene transfer in the marine environments.

Characterization of the molecular clockwork in the cockroach Rhyparobia maderae

Der Wechsel von Tag und Nacht erzeugt einen regelmäßigen Rhythmus von verschiedenen Umweltreizen, allen voran Licht und Temperatur. Fast jedes bis zum heutigen Tage untersuchte Lebewesen besitzt einen endogenen Mechanismus zur Zeitwahrnehmung, und diese "innere Uhr" befähigt Lebewesen dazu, sich vorausschauend an rhythmische Umwelt-Änderungen anzupassen. Circadiane Rhythmen bestehen auch ohne jegliche äußere Reize und basieren auf einem molekularen Rückkopplungs-Mechanismus, der Rhythmen in Genexpression und Proteinkonzentration von etwa 24 Stunden erzeugt. Obwohl sich die grundsätzlichen Mechanismen und Komponenten dieses molekularen Uhrwerks in allen Insekten ähneln, zeigte sich jedoch immer mehr, dass es im Detail doch wesentliche Unterschiede zwischen verschiedenen Insektengruppen gibt. Während das molekulare Uhrwerk der Fruchtfliege Drosophila melanogaster inzwischen sehr gut untersucht ist, fehlen bei den meisten Insektengruppen immernoch eingehende Untersuchungen. Fast nichts ist über die molekulare Basis von circadianen Rhythmen bei der Schabe Rhyparobia maderae bekannt, obwohl diese Art bereits seit Langem als Modellorganismus in der Chronobiologie dient. Um mit der Forschung am molekularen, circadianen System von R. maderae zu beginnen, wurde die Struktur und das Expressionsprofil der core feedback loop Gene per, tim1 und cry2 analysiert. Mittels degenerierten Primern und RACE konnte das vollständige offene Leseraster (OLR) von rmPer und rmCry2, und ein Teil des rmTim1 OLR kloniert werden. Eine phylogenetische Analyse gruppierte rmPER und rmCRY2 gemeinsam mit den Orthologa hemimetaboler Insekten. Viele bei D. melanogaster funktionell charakterisierte Domänen sind bei diesen Proteinen konserviert, was auf eine ähnliche Funktion in der inneren Uhr von R. maderae hinweist. Mittels quantitativer PCR konnte gezeigt werden, dass die mRNA von rmPer, rmTim1 und rmCry2 in verschiedenen Lichtregimen in der gleichen Phasenlage Tageszeit-abhängig schwankt. Die Phasenlage stellte sich bei unterschiedlichen Photoperioden jeweils relativ zum Beginn der Skotophase ein, mit Maxima in der ersten Hälfte der Nacht. Auch im Dauerdunkel zeigen sich Rhythmen in der rmTim1 und rmCry2 Expression. Die Amplitude der rmPer Expressionsrhythmen war jedoch so gering, dass keine signifikanten Unterschiede zwischen den einzelnen Zeitgeberzeiten (ZT) festgestellt werden konnten. Mittels Laufrad-Assays wurde untersucht wie Kurz- und Langtag Lichtregime die Verhaltensrhythmen beeinflussen. Es konnten nur Unterschiede in der Periodenlänge unter freilaufenden Bedingungen festgestellt werden, wenn höhere Lichtintensitäten (1000lx) zur Synchronisation (entrainment) genutzt wurden. Die Periode des freilaufenden Rhythmus war bei Tieren aus dem Kurztag länger. Die photoperiodische Plastizität zeigte sich also auch auf Verhaltensebene, obwohl höhere Lichtintensitäten notwendig waren um einen Effekt zu beobachten. Basierend auf den Sequenzen der zuvor klonierten OLR wurden gegen rmPER, rmTIM1 und rmCRY2 gerichtete Antikörper hergestellt. Die Antikörper gegen rmPER und rmTIM1 erkannten in western blots sehr wahrscheinlich spezifisch das jeweilige Protein. Zeitreihen von Gehirngewebe-Homogenisaten zeigten keinen offensichtlichen circadianen Rhythmus in der Proteinkonzentration, wahrscheinlich auf Grund einer Oszillation mit niedriger Amplitude. In Immunhistochemischen Färbungen konnte nur mit dem gegen rmPER gerichteten Antikörper aus Kaninchen ein Signal beobachtet werden. Beinahe jede Zelle des Zentralnervensystems war rmPER-immunreaktiv im Zellkern. Es konnten keine Unterschiede zwischen den untersuchten ZTs festgestellt werden, ähnlich wie bei den western blot Zeitreihen. In dieser Studie konnten erstmals molekulare Daten der circadianen Uhr von R. maderae erfasst und dargestellt werden. Die Uhrgene per, tim1 und cry2 werden in dieser Schabenart exprimiert und ihre Domänenstruktur sowie das circadiane Expressionsmuster ähneln dem hypothetischen ursprünglichen Insektenuhrwerk, welches der circadianen Uhr von Vertebraten nahesteht. Das molekulare Uhrwerk von R. maderae kann sich an unterschiedliche Photoperioden anpassen, und diese Anpassungen manifestieren sich im Expressionsprofil der untersuchten Uhrgene ebenso wie im Verhalten.

"Identification and Diversity of Killer Cell Ig-Like Receptors in Aotus vociferans, a New World Monkey "

Previous BAC clone analysis of the Platyrrhini owl monkey KIRs have shown an unusual genetic structure in some loci. Therefore, cDNAs encoding KIR molecules from eleven Aotus vociferans monkeys were characterized here; tenputative KIR loci were found, some of which encoded atypical proteins such as KIR4DL and transcripts predicted to encode a D0+D1 configuration (AOTVOKIR2DL1*01v1) which appear to be unique in the Aotus genus. Furthermore, alternative splicing was found as a likely mechanism for producing activator receptors in A. vociferans species. KIR proteins from New World monkeys may be split into three new lineages according to domain by domain phylogenetic analysis. Although the A. vociferans KIR family displayed a high divergence among paralogous genes, individual loci were limited in their genetic polymorphism. Selection analysis showed that both constrained and rapid evolution may operate within the AvKIR family. The frequent alternative splicing (as a likely mechanism generating activator receptors), the presence of KIR4DL and KIR2DL1 (D0+D1) molecules and other data reported here suggest that the KIR family in Aotus has had a rapid evolution, independent from its Catarrhini counterparts.from its Catarrhini counterparts.

Genomic architecture of adaptive color pattern divergence and convergence in Heliconius butterflies

Identifying the genetic changes driving adaptive variation in natural populations is key to understanding the origins of biodiversity. The mosaic of mimetic wing patterns in Heliconius butterflies makes an excellent system for exploring adaptive variation using next-generation sequencing. In this study, we use a combination of techniques to annotate the genomic interval modulating red color pattern variation, identify a narrow region responsible for adaptive divergence and convergence in Heliconius wing color patterns, and explore the evolutionary history of these adaptive alleles. We use whole genome resequencing from four hybrid zones between divergent color pattern races of Heliconius erato and two hybrid zones of the co-mimic Heliconius melpomene to examine genetic variation across 2.2 Mb of a partial reference sequence. In the intergenic region near optix, the gene previously shown to be responsible for the complex red pattern variation in Heliconius, population genetic analyses identify a shared 65-kb region of divergence that includes several sites perfectly associated with phenotype within each species. This region likely contains multiple cis-regulatory elements that control discrete expression domains of optix. The parallel signatures of genetic differentiation in H. erato and H. melpomene support a shared genetic architecture between the two distantly related co-mimics; however, phylogenetic analysis suggests mimetic patterns in each species evolved independently. Using a combination of next-generation sequencing analyses, we have refined our understanding of the genetic architecture of wing pattern variation in Heliconius and gained important insights into the evolution of novel adaptive phenotypes in natural populations.

Genotypic comparison of Pantoea agglomerans plant and clinical strains

Pantoea agglomerans strains are among the most promising biocontrol agents for a variety of bacterial and fungal plant diseases, particularly fire blight of apple and pear. However, commercial registration of P. agglomerans biocontrol products is hampered because this species is currently listed as a biosafety level 2 (BL2) organism due to clinical reports as an opportunistic human pathogen. This study compares plant-origin and clinical strains in a search for discriminating genotypic/phenotypic markers using multi-locus phylogenetic analysis and fluorescent amplified fragment length polymorphisms (fAFLP) fingerprinting. Results: Majority of the clinical isolates from culture collections were found to be improperly designated as P. agglomerans after sequence analysis. The frequent taxonomic rearrangements underwent by the Enterobacter agglomerans/Erwinia herbicola complex may be a major problem in assessing clinical associations within P. agglomerans. In the P. agglomerans sensu stricto (in the stricter sense) group, there was no discrete clustering of clinical/biocontrol strains and no marker was identified that was uniquely associated to clinical strains. A putative biocontrol-specific fAFLP marker was identified only in biocontrol strains. The partial ORF located in this band corresponded to an ABC transporter that was found in all P. agglomerans strains. Conclusion: Taxonomic mischaracterization was identified as a major problem with P. agglomerans, and current techniques removed a majority of clinical strains from this species. Although clear discrimination between P. agglomerans plant and clinical strains was not obtained with phylogenetic analysis, a single marker characteristic of biocontrol strains was identified which may be of use in strain biosafety determinations. In addition, the lack of Koch's postulate fulfilment, rare retention of clinical strains for subsequent confirmation, and the polymicrobial nature of P. agglomerans clinical reports should be considered in biosafety assessment of beneficial strains in this species

Anàlisi polifàsica de soques de "Pseudomonas fluorescens" potencials agents de biocontrol de malalties de fruiters

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases. L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP. Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn. S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes. Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies. El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempelts La caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri. Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP. Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades.

Chronology for the Aegean Late Bronze Age 1700-1400 BC

Radiocarbon (carbon-14) data from the Aegean Bronze Age 1700-1400 B.C. show that the Santorini (Thera) eruption must have occurred in the late 17th century B.C. By using carbon-14 dates from the surrounding region, cultural phases, and Bayesian statistical analysis, we established a chronology for the initial Aegean Late Bronze Age cultural phases (Late Minoan IA, IB, and II). This chronology contrasts with conventional archaeological dates and cultural synthesis: stretching out the Late Minoan IA, IB, and II phases by similar to 100 years and requiring reassessment of standard interpretations of associations between the Egyptian and Near Eastern historical dates and phases and those in the Aegean and Cyprus in the mid-second millennium B.C.

Characterization and origin of infection of Rhizoctonia solani associated with Brassica oleracea crops in the UK

An extensive study was conducted to determine where in the production chain Rhizoctonia solani became associated with UK module-raised Brassica oleracea plants. In total, 2600 plants from 52 crops were sampled directly from propagators and repeat sampled from the field. Additional soil, compost and water samples were collected from propagation nurseries and screened using conventional agar isolation methods. No isolates of R. solani were recovered from any samples collected from propagation nurseries. Furthermore, nucleic acid preparations from samples of soil and compost from propagation nurseries gave negative results when tested for R. solani using real-time PCR. Conversely, R. solani was recovered from 116 of 1300 stem bases collected from field crops. All the data collected suggested R. solani became associated with B. oleracea in the field rather than during propagation. Parsimony and Bayesian phylogenetic studies of ribosomal DNA suggested the majority of further classified isolates belonged to anastomosis groups 2-1 (48/57) and AG-4HGII (8/57), groups known to be pathogenic on Brassica spp. in other countries. Many R. solani isolates were recovered from symptomless plant material and the possibilities for such an association are discussed.

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.