A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, ΔesaR, and ΔesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

Análise in vitro da expressão de genes de resposta a estresse oxidativo e osmótico da bactéria fastidiosa Leifsonia xyli subsp. xyli

Atualmente, o Brasil é o maior produtor de cana-de-açúcar (Saccharum ssp.), no qual o estado de São Paulo é responsável por mais de 50% da produção. Esta cultura é hospedeira de diversos patógenos que podem limitar sua produção, dentre os quais se destaca a bactéria Leifsonia xyli subsp. xyli (Lxx), agente causal do raquitismo da soqueira (ratoon stunting disease - RSD). Pouco se sabe sobre a fisiologia deste organismo e quais as estratégias utilizadas por este para colonizar seu hospedeiro. No entanto, sabemos que para infectar e colonizar seus hospedeiros, é necessário que bactérias parasíticas superem estresses de diversas naturezas impostas durante estes processos, como os estresses oxidativo e o osmótico. Neste contexto, os objetivos deste trabalho foram identificar in silico e analisar a expressão in vitro, por qPCR, de genes relacionados a estes dois estresses. Uma análise da sequência do genoma de Lxx identificou 35 genes, sendo 8 relacionados ao estresse oxidativo, 9 relacionados ao estresse osmótico e 11 relacionados a estresse gerais, incluindo um cluster de 6 genes envolvidos na síntese de carotenoides. A expressão destes foi avaliada 60 minutos após exposição a 30mM de H2O2 ou 7% (p/v) de polietilenoglicol 6000 (PEG 6000). Sete genes foram avaliados como normalizadores das reações de qPCR. A quantificação do grau de peroxidação lipídica indicou que ambos os tratamentos resultaram em sensível peroxidação, muito embora o efeito do tratamento com PEG 6000 tenha sido maior do que o tratamento com H2O2. A exposição ao H2O2 aumentou a expressão dos genes katA (catalase), sodA (superóxido dismutase), msrA (Sulfóxido de metionina redutase) e msrB (Sulfóxido de metionina redutase) bem como de todos os genes responsáveis pela síntese de carotenoides. Por outro lado, todos os genes relacionados ao estresse osmótico foram menos expressos na presença deste composto. Já quando a bactéria foi exposta a PEG 6000, o oposto ocorreu, ou seja, os genes relacionados ao estresse osmótico, que são otsA (Trealose-6-fosfato sintase), otsB (Trealose fosfatase), treY (Malto-oligosil trealose sintase), treZ (Malto-oligosil trealose trealoidrolase), treS (Trealose sintase), proX (Proteína de ligamento em substrato, tipo ABC glicina betaína transportadora), proW (Proteína permease, tipo ABC glicina betaína transportadora), proZ (Proteína permease, tipo ABC glicina betaína transportadora) e Naggn (Amidotransferase), além dos genes do cluster carotenoide, foram mais expressos, ao passo que alguns dos genes ligados à resposta ao estresse oxidativo foram menos expressos. Verificou-se também, através de PCR convencional utilizando primers para amplificar as regiões entre os genes carotenoides, que estes são expressos como um RNA policistrônico, constituindo assim um operon. Estes resultados validam predições anteriores baseadas na análise in silico da sequência do genoma de Lxx, confirmando que Lxx possui mecanismos responsivos aos estresses osmótico e oxidativo aos quais é submetida durante o processo de infecção de seu hospedeiro.

Potential to increase yield in lucerne (Medicago sativa subsp sativa) through introgression of Medicago sativa subsp falcata into Australian adapted material

The effect of interspecific heterosis in crosses between Medicago sativa subsp. sativa and M. sativa subsp. falcata was assessed. Three sativa and 3 falcata plants were crossed in a diallel design. Progeny dry matter yield and natural plant height were assessed in a replicated field experiment at Gatton, Queensland. Yield data were analysed using the method of residual maximum likelihood (REML) and Griffing's model 1. There were significant differences between the reciprocal, general combining ability (GCA), and specific combining ability (SCA) effects. As expected, S-1 populations were lower yielding than their respective intraspecific cross and falcata x falcata crosses were significantly lower yielding than sativa x sativa crosses. Some of the interspecific crosses indicated substantial SCA effects, yielding at least as well as the best sativa x sativa crosses. We have demonstrated the potential usefulness of unselected M. sativa subsp. falcata as a heterotic group in the improvement of yield in northern Australian adapted lucerne material, and discuss how it could be incorporated into future breeding to overcome the yield stagnation currently being experienced in Australian programs.

Genome analysis and characterisation of the exopolysaccharide produced by Bifidobacterium longum subsp. longum 35624™

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

Sensitive and specific detection of viable Mycobacterium avium subsp. paratuberculosis in raw milk by the Peptide-mediated magnetic separation (PMS)-phage assay

This study describes further validation of a previously described Peptide-mediated magnetic separation (PMS)-Phage assay, and its application to test raw cows’ milk for presence of viable Mycobacterium avium subsp. paratuberculosis (MAP). The inclusivity and exclusivity of the PMS-phage assay were initially assessed, before the 50% limit of detection (LOD50) was determined and compared with those of PMS-qPCR (targeting both IS900 and f57) and PMS-culture. These methods were then applied in parallel to test 146 individual milk samples and 22 bulk tank milk samples from Johne’s affected herds. Viable MAP were detected by the PMS-phage assay in 31 (21.2%) of 146 individual milk samples (mean plaque count of 228.1 PFU/50 ml, range 6-948 PFU/50 ml), and 13 (59.1%) of 22 bulk tank milks (mean plaque count of 136.83 PFU/50 ml, range 18-695 PFU/50 ml). In contrast, only 7 (9.1%) of 77 individual milks and 10 (45.4%) of 22 bulk tank milks tested PMS-qPCR positive, and 17 (11.6%) of 146 individual milks and 11 (50%) of 22 bulk tank milks tested PMS-culture positive. The mean 50% limits of detection (LOD50) of the PMS-phage, PMS-IS900 qPCR and PMS-f57 qPCR assays, determined by testing MAP-spiked milk, were 0.93, 135.63 and 297.35 MAP CFU/50 ml milk, respectively. Collectively, these results demonstrate that, in our laboratory, the PMS-phage assay is a sensitive and specific method to quickly detect the presence of viable MAP cells in milk. However, due to its complicated, multi-step nature, the method would not be a suitable MAP screening method for the dairy industry.

Single gene resistance to Fusarium oxysporum f. sp. cubense Race 4 in the wild banana Musa acuminata subsp. malaccensis

Fusarium wilt of banana, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. A particularly virulent strain of the pathogen, tropical race 4 (TR4), presents an emerging threat to banana producing regions throughout the world. No commercially acceptable banana cultivar is resistant to TR4 and, as with all strains of the Fusarium wilt pathogen, there is no effective chemical control. Genetic resistance to TR4 has been observed in the diploid wild banana Musa acuminata subsp. malaccensis, which has consequently received attention as a potential source of Fusarium resistance genes. The aim of this research was to determine the pattern of inheritance of the resistance trait by screening plants for resistance to Foc subtropical race 4 (SR4) and TR4. Our results showed that the F1 progeny of self-fertilized malaccensis plants challenged in pot trials against SR4 (VCGs 0120, 0129, 01211) and TR4 (VCG 01213/16) segregated for resistance according to a Mendelian ratio of 3:1 which is consistent with a single dominant gene hypothesis.

Genetic variation in growth and wood-quality traits of Corymbia citriodora subsp. variegata across three sites in south-east Queensland, Australia

Ten growth or wood-quality traits were assessed in three nearby Corymbia citriodora subsp. variegata (CCV) open-pollinated family-within-provenance trials (18 provenances represented by a total of 374 families) to provide information for the development of a breeding program targeting both pulp and solid-wood products. Growth traits (diameter at breast high over bark [DBH], height and conical volume) were assessed at 3 and 7 years of age. Wood-quality traits (density [DEN], Kraft pulp yield [KPY], modulus of elasticity [MoE] and microfibril angle [MfA]) were predicted using near-infrared spectroscopy on wood samples collected from these trials when aged between 10 and 12 years. The high average KPY, DEN and MoE, and low average MfA observed indicates CCV is very suitable for both pulp and timber products. All traits were under moderate to strong genetic control. In across- trials analyses, high (>0.4) heritability estimates were observed for height, DEN, MoE and MfA, while moderate heritability estimates (0.24 to 0.34) were observed for DBH, volume and KPY. Most traits showed very low levels of genotype × site interaction. Estimated age–age genetic correlations for growth traits were strong at both the family (0.97) and provenance (0.99) levels. Relationships among traits (additive genetic correlation estimates) were favourable, with strong and positive estimates between growth traits (0.84 to 0.98), moderate and positive values between growth and wood-quality traits (0.32 to 0.68), moderate and positive between KPY and MoE (0.64), and high and positive between DEN and MoE (0.82). However, negative (but favourable) correlations were detected between MfA and all other evaluated traits (−0.31 to −0.96). The genetic correlation between the same trait expressed on two different sites, at family level, ranged from 0.24 to 0.42 for growth traits, and from 0.29 to 0.53 for wood traits. Therefore simultaneous genetic improvement of growth and wood property traits in CCV for the target environment in south-east Queensland should be possible, given the moderate to high estimates of heritability and favourable correlations amongst all traits studied, unless genotype × site interactions are greater than was evident. © 2016 NISC (Pty) Ltd

Análisis del potencial epizoótico de cepas de Mycobacterium avium subsp. paratuberculosis aisladas de diversos hospedadores empleando modelos de infección in vitro

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Tetrazolium test for seed viability and germinability of Melocactus ernestii Vaupel subsp. ernestii and Melocactus zehntneri (Briton & Rose) Luetzelb. (Cactaceae)

Despite its ornamental value, some Melocactus species (Cactaceae) are threatened by several factors and only sexual propagation is possible. Thus, artificial seed banks are an appropriate method for their ex situ conservation. Suitable methods to germinate and to monitor viability are necessary for the seeds of these species. This work explored the use of tetrazolium test for monitoring seed viability of two Melocactus species. There was a correlation between the percentage of stained embryos, considering different cover area or tone stain, and the germination percentage. Unviable embryos did not stain. The embryos curved shape can explain the partial stain on the most of cases.

Antimicrobial activity of the essential oils of Dittrichia viscosa subsp viscosa on Helicobacter pylori

Dittrichia viscosa subsp. viscosa (Compositae) is found on edges, wood clearings and in waste places of the Iberian Peninsula. Aerial parts of D. viscosa were collected at flowering phase in September-October 2001 around Lisbon, Portugal and the essential oils isolated by hydro-distillation for 4 h using a Clevenger-type apparatus. The oils were analyzed by gas chromatography and gas chromatography-mass spectrometry. Preliminary examination of the essential oils allowed the identification of 32 components. Only four components reached percentages over 5%: fokienol (11.8%), T-muurorol (7.9%), (E)-nerolidol (5.5%) and delta-cadinene (5.0%). The essential oils were tested against Helicobacterpylori and Listeria monocytogenes. Essential oils did not have antimicrobial activity against L. monocytogenes. The essential oil at 0.88 to 22.22 mu g.ml(-1) did not inhibit the growth of H. pylori, affected the growth slightly at 44.40 mu g.ml(-1), and completely inhibited the growth at 88.80 to 133.20 mu g.ml(-1) Results show that use of D. viscosa essential oil in the treatment of gastric disorders caused by H. pylori can be effective.

Antimicrobial activity of the essential oils of Dittrichia viscosa subsp viscosa on Helicobacter pylori

Dittrichia viscosa subsp. viscosa (Compositae) is found on edges, wood clearings and in waste places of the Iberian Peninsula. Aerial parts of D. viscosa were collected at flowering phase in September-October 2001 around Lisbon, Portugal and the essential oils isolated by hydro-distillation for 4 h using a Clevenger-type apparatus. The oils were analyzed by gas chromatography and gas chromatography-mass spectrometry. Preliminary examination of the essential oils allowed the identification of 32 components. Only four components reached percentages over 5%: fokienol (11.8%), T-muurorol (7.9%), (E)-nerolidol (5.5%) and delta-cadinene (5.0%). The essential oils were tested against Helicobacterpylori and Listeria monocytogenes. Essential oils did not have antimicrobial activity against L. monocytogenes. The essential oil at 0.88 to 22.22 mu g.ml(-1) did not inhibit the growth of H. pylori, affected the growth slightly at 44.40 mu g.ml(-1), and completely inhibited the growth at 88.80 to 133.20 mu g.ml(-1) Results show that use of D. viscosa essential oil in the treatment of gastric disorders caused by H. pylori can be effective.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Discovery of stable and variable differences in the Mycobacterium avium subsp. paratuberculosis type I, II, and III genomes by pan-genome microarray analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

Estudo da infeção de corvina (Argyrosomus regius Asso, 1801) por Photobacterium damselae subsp. piscicida

As práticas intensivas que são utilizadas em aquacultura implicam que exista um contacto extremo entre indivíduos promovendo o stress, que atuando conjuntamente com diversos outros fatores, como é o caso de bactérias, são passíveis de induzir nos peixes um estado de doença. Photobacterium damselae subsp. piscicida (Phdp) é uma bactéria gram-negativa reconhecida por causar surtos graves de doença em diversas espécies de peixes. É considerada mundialmente como uma das maiores enfermidades para as práticas aquícolas, plausível de causar danos irreversíveis nestas populações. Neste trabalho pretendeu-se elaborar um protocolo de infeção com Phdp em Argyrosomus regius estabelecendo num primeiro ensaio a dose que causa mortalidade a 50% de uma população (LD50), estudando posteriormente num segundo ensaio, a infeção, o destino que a bactéria teria no peixe através de análise PCR, as consequências da infeção a nível hematológico e nos parâmetros imunitários, utilizando um modelo de coabitação, expondo indivíduos saudáveis a indivíduos doentes. Foi comprovada a virulência da estirpe AQP 17.1 de Phdp sobre indivíduos da espécie A. regius com um peso médio de 28,3±10,9g, situando-se o LD50 em 2,29×105 UFC ml-1, apresentando os peixes alguns sintomas típicos da doença crónica. O modelo de coabitação utilizado no segundo ensaio, permitiu-nos confirmar que Phdp infetou por coabitação indivíduos da espécie A. regius com peso médio de 23,4±8,6g. Os resultados sugerem que as brânquias podem ter sido um dos locais de entrada da bactéria no corpo do peixe, disseminando-se após 24 horas para o rim e intestino anterior. Phdp induziu em A. regius uma resposta imune inata que se avaliou ao longo do tempo quando comparados os indivíduos coabitantes do controlo (vetores injetados com tampão salino de Hanks) com os indivíduos coabitantes infetados (vetores injetados com suspensão de Phdp a uma concentração igual ao LD50). Esta resposta inflamatória ficou visível não só no aumento do número de leucócitos no sangue, mas também no aumento de atividade dos parâmetros humorais imunes. Os resultados sugerem que a maior atividade da resposta imunitária se deu após as 24 horas de coabitação, momento que determina também a invasão do patógeno em órgãos como o intestino anterior e o rim. Os resultados obtidos neste trabalho sugerem que Phdp pode induzir um estado de infeção em A. regius através de um modelo de coabitação, invadindo num curto espaço de tempo os órgãos do hospedeiro, desencadeando a atividade do sistema imune inato como resposta à infeção.