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.

The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals

The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.

Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces

We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.

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.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

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.

Genetic uniformity of international isolates of Leifsonia xyli subsp xyli, causal agent of ratoon stunting disease of sugarcane

An international collection of the sugarcane ratoon stunting disease pathogen, Leifsonia xyli subsp. xyli, was analysed to assess genetic diversity. DNA fingerprinting using BOX primers was performed on 105 isolates, comprising 65 Australian isolates and an additional 40 isolates from Indonesia (n = 8), Japan (n = 1), USA (n = 3), Brazil (n = 2), Mali (n = 2), Zimbabwe (n = 13), South Africa (n = 9) and Reunion (n = 2). Sixty-two of these isolates were also screened using ERIC primers. No variation was found among any of the isolates. The intergenic spacer (IGS) region of the ribosomal RNA genes from 54 isolates was screened for sequence variation using single-stranded conformational polymorphism (SSCP), but none was observed. Direct sequencing of the IGS from a subset of nine isolates, representing all of the countries sampled in this study, confirmed the results of the SSCP analysis. Likewise, no sequence variation was found in the 16S ribosomal RNA genes of the same subset. Four Colombian isolates from sugarcane, morphologically similar to L. xyli subsp. xyli, were putatively shown to be an undescribed Agrococcus species of unknown pathogenicity. The lack of genetic variation among L. xyli subsp. xyli isolates, independent of time of sampling, cultivar of isolation, or country of origin, suggests the worldwide spread of a single pathogenic clone, and further suggests that sugarcane cultivars resistant to ratoon stunting disease in one area should retain this property in other regions.

Recent advances in the molecular biology of Leifsonia xyli subsp xyli, causal organism of ratoon stunting disease

Twelve years ago our understanding of ratoon stunting disease (RSD) was confined almost exclusively to diagnosis of the disease and control via farm hygiene, with little understanding of the biology of the interaction between the causal agent (Leifsonia xyli subsp. xyli) and the host plant sugarcane (Saccharum spp. hybrids). Since then, research has focused on developing the molecular tools to dissect L. xyli subsp. xyli, so that better control strategies can be developed to prevent losses from RSD. Within this review, we give a brief overview of the progression in research on L. xyli subsp. xyli and highlight future challenges. After a brief historical background on RSD, we discuss the development of molecular tools such as transformation and transposon mutagenesis and discuss the apparent lack of genetic diversity within the L. xyli subsp. xyli world population. We go on to discuss the sequencing of the genome of L. xyli subsp. xyli, describe the key findings and suggest some future research based on known deficiencies that will capitalise on this tremendous knowledge base to which we now have access.

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