Microbiological Research

Transcriptome profiling provides insights into regulatory factors involved in Trichoderma viride-Azotobacter chroococcumbiofilm formation Publication date: October 2019 Source: Microbiological Research, Volume 227 Author(s): Kulandaivelu Velmourougane, Radha Prasanna, Puram Supriya, Balasubramanian Ramakrishnan, Shobit Thapa, Anil Kumar Saxena Abstract Azotobacter chroococcum (Az) and Trichoderma viride (Tv) represent agriculturally important and beneficial plant growth promoting options which contribute towards nutrient management and biocontrol, respectively. When Az and Tv are co-cultured, they form a biofilm, which has proved promising as an inoculant in several crops; however, the basic aspects related to regulation of biofilm formation were not investigated. Therefore, whole transcriptome sequencing (Illumina NextSeq500) and gene expression analyses were undertaken, related to biofilm formation vis a vis Tv and Az growing individually. Significant changes in the transcriptome profiles of biofilm were recorded and validated through qPCR analyses. In-depth evaluation also identified several genes (phoA, phoB, glgP, alg8, sipW, purB, pssA, fadD) specifically involved in biofilm formation in Az, Tv and Tv-Az. Genes coding for RNA-dependent RNA polymerase, ABC transporters, translation elongation factor EF-1, molecular chaperones and double homeobox 4 were either up-regulated or down-regulated during biofilm formation. To our knowledge, this is the first report on the modulation of gene expression in an agriculturally beneficial association, as a biofilm. Our results provide insights into the regulatory factors involved during biofilm formation, which can help to improve the beneficial effects and develop more effective and promising plant- microbe associations.

Comparative analysis of biocontrol agent Trichoderma asperellum ACCC30536 transcriptome during its interaction with Populus davidiana × P. alba var. pyramidalis Publication date: October 2019 Source: Microbiological Research, Volume 227 Author(s): Shida Ji, Zhihua Liu, Bin Liu, Yucheng Wang Abstract After exposure to with Populus davidiana × P. alba var. pyramidalis, the expression of genes in Trichoderma asperellum were compared in four transcriptomes. The top 20 high expression genes included six heat shock proteins and three hydrophobins, indicating that Trichoderma can rapidly adapt to environment stresses and elicit a plant defense response. The genes, involved in the interaction between Trichoderma and plant, showed an increasing expression level, for example sugar transporters, EPL1s, endoxylanases, pectin lyases, and nitrilases. Interestingly, sugar transporters also showed high expression when T. asperellum was cultured on medium lacking a carbon substrate, which would contribute to T. asperellum's survival and domination in ecological niche competition. And the genes related to mycoparasitism were expressed abundantly following T. asperellum's interaction with PdPap, indicating the PdPap induction could enhance the mycoparasitic ability of T. asperellum. Twelve chitinases and five glucanases showed higher expression in transcriptome Cs, indicating that T. asperellum secretes both types of enzyme before interacting with pathogens, allowing T. asperellum to implement mycoparasitism and obtain more energy. Many novel transcripts were obtained in each transcriptome, which may play important roles in the biocontrol process of T. asperellum. Interestingly, T. asperellum undergo constitutive alternative splicing in the biocontrol process: Seven biocontrol genes were alternative spliced via intron retention. qRT-PCR analysis proved that intron retention is negatively associated with the expression of chitinase, oligopeptide transporters, and beta-lactamase. However, the percentage of MAPK intron retention was quite low, suggesting that intron retention has little effect on the function of MAPK.

Toxoplasma gondii secretory proteins and their role in invasion and pathogenesis Publication date: October 2019 Source: Microbiological Research, Volume 227 Author(s): Yang Zhang, Bo Shiun Lai, Mario Juhas, Yun Zhang Abstract T. gondii is a major opportunistic pathogen chronically infecting nearly one third of the world's population. Due to the high infection and mortality rates in immunocompromised patients and newborns, the extent or magnitude of T. gondii pathogenesis is determined mainly by host-pathogen interactions. T. gondii utilizes specialized secretory proteins to modify host cellular factors and facilitate invasion and replication. This review provides update on the recent progress in this field of research with particular emphasis on the T. gondii secretory proteins and their role in invasion and pathogenesis.

Legacy of a Pleistocene bacterial community: Patterns in community dynamics through changing ecosystems Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Shan P. Thomas, Bhavatharini Shanmuganathan, Manoj Kumar Jaiswal, Anbarasu Kumaresan, Senthil Kumar Sadasivam Abstract Bacterial communities are resilient to the environmental changes, yet the effect of long term ecological changes on bacterial communities remain poorly explored. To study the effect of prolonged environmental changes, a 25 m long sediment core was excavated from a paleo beach ridge located on the Cauvery delta, south east coast of India. Geological evidences suggested that the site has experienced multiple marine transgressions and regressions. The three paleosols from Vettaikaraniruppu (VKI) beach ridge, VKI-2 (2.8 m bgl; 3 kybp), VKI-5 (7.2 m bgl; 6 kybp) and VKI-14 (24.5 m bgl; 146 kybp) was chosen for bacterial community analysis based on their formation period. Bacterial community structure of paleosols was reconstructed using V3 hypervariable region of bacterial 16S rDNA targeted Illumina sequencing. The VKI-5 sediment layer which formed under marine environment contained highest bacterial diversity, and the community was a mix up of terrestrial and marine bacterial population. The final community VKI-2 exhibited an approximate structural pattern witnessed in the native bacterial community VKI-14 which formed during marine regression. Furthermore, marine transgression and regression experienced in VKI resulted in the formation of distinct biogeographic patterns.

Agrobacterium-mediated transformation of Ceratocystis albifundus Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): M. Sayari, M.A. van der Nest, E.T. Steenkamp, O.O. Adegeye, S. Marincowitz, B.D. Wingfield Abstract Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

A novel contact-independent T6SS that maintains redox homeostasis via Zn2+ and Mn2+ acquisition is conserved in the Burkholderia pseudomallei complex Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): David DeShazer Abstract The Burkholderia pseudomallei complex consists of six phylogenetically related Gram-negative bacterial species that include environmental saprophytes and mammalian pathogens. These microbes possess multiple type VI secretion systems (T6SS) that provide a fitness advantage in diverse niches by translocating effector molecules into prokaryotic and eukaryotic cells in a contact-dependent manner. Several recent studies have elucidated the regulation and function of T6SS-2, a novel contact-independent member of the T6SS family. Expression of the T6SS-2 gene cluster is repressed by OxyR, Zur and TctR and is activated by GvmR and reactive oxygen species (ROS). The last two genes of the T6SS-2 gene cluster encode a zincophore (TseZ) and a manganeseophore (TseM) that are exported into the extracellular milieu in a contact-independent fashion when microbes encounter oxidative stress. TseZ and TseM bind Zn2+ and Mn2+, respectively, and deliver them to bacteria where they provide protection against the lethal effects of ROS. The TonB-dependent transporters that interact with TseZ and TseM, and actively transport Zn2+ and Mn2+ across the outer membrane, have also been identified. Finally, T6SS-2 provides a contact-independent growth advantage in nutrient limited environments and is critical for virulence in Galleria mellonella larvae, but is dispensable for virulence in rodent models of infection. Graphical abstract

Aphicidal activity of Bacillus amyloliquefaciens strains in the peach-potato aphid (Myzus persicae) Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Guadalupe López-Isasmendi, Adriana Elisabet Alvarez, Gabriela Petroselli, Rosa Erra-Balsells, Marcela Carina Audisio Abstract Myzus persicae Sulzer (Hemiptera: Aphididae), is a generalist cosmopolitan insect that infests more than 400 plant species of 40 different families and is one of the major pests infesting potato crops. It causes direct damage and also spread plant viruses. The intensive use of synthetic insecticide to control aphids has led to resistant populations. Therefore, there is a need to develop biopesticides for effective control that minimizes environmental hazards. The bacteria Bacillus amyloliquefaciens is recognized as a producer of a variety of bioactive compounds. The aim here was to evaluate the aphicidal effect of B. amyloliquefaciens strains, CBMDDrag3, PGPBacCA2, and CBMDLO3, and their metabolites on the mortality and fecundity of M. persicae. Cells suspensions, heat-killed cell suspensions, cell-free supernatants, or isolated lipopeptide fractions from B. amyloliquefaciens strains were offered to aphids through artificial diets. The isolated lipopeptide fractions composed mainly of kurstakins, surfactins, iturins, and fengycins, when were administrated through diets, had no aphicidal effect against M. persicae. However, aphids fed on diets with whole cell suspensions and its cell-free supernatant of all three bacteria strains resulted in 100% mortality of adult aphids and nymphs. Specially, B. amyloliquefaciens CBMDLO3, has an effective aphicidal effect on M. persicae, used both bacterial cells and their metabolites. Moreover, heat-killed cells of B. amyloliquefaciens CBMDLO3 also had aphicidal action, although the aphid mortality was lower than on diet with living bacteria. Therefore, these results propose that B. amyloliquefaciens, could function as a novel eco-friendly biopesticide for the control of M. persicae.

Effect of growth temperature on biosynthesis and accumulation of carotenoids in cyanobacterium Anabaena sp. PCC 7120 under diazotrophic conditions Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Kinga Kłodawska, Anna Bujas, Maria Turos-Cabal, Paweł Żbik, Pengcheng Fu, Przemysław Malec Abstract Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) β-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of β-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of β-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between β-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.

Microscopic analysis of colonization of Colletotrichum abscissum in citrus tissues Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Daiani Cristina Savi, Bruno Janoski Rossi, Gustavo Rodrigues Rossi, Lisandra Santos Ferreira-Maba, Israel Henrique Bini, Edvaldo da Silva Trindade, Eduardo Henrique Goulin, Marcos Antonio Machado, Chirlei Glienke Abstract Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.

Anti-quorum sensing and anti-biofilm activity of 5-hydroxymethylfurfural against Pseudomonas aeruginosa PAO1: Insights from in vitro, in vivo and in silico studies Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Jobina Rajkumari, Subhomoi Borkotoky, Dhanasekhar Reddy, Saswat Kumar Mohanty, Ranjith Kumavath, Ayaluru Murali, Kitlangki Suchiang, Siddhardha Busi Abstract Pseudomonas aeruginosa is one of the most common pathogens associated with nosocomial infections and a great concern to immunocompromised individuals especially in the cases of cystic fibrosis, AIDS and burn wounds. The pathogenicity of P. aeruginosa is largely directed by the quorum sensing (QS) system. Hence, QS may be considered an important therapeutic target to combat P. aeruginosa infections. The anti-quorum sensing and anti-biofilm efficacy of aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF) against P. aeruginosa PAO1 were assessed. At the sub-inhibitory concentration, 5-HMF suppressed the production of QS-controlled virulence phenotypes and biofilm formation in P. aeruginosa. It was also able to significantly enhance the survival rate of C. elegans infected with P. aeruginosa. The in silico studies revealed that 5-HMF could serve as a competitive inhibitor for the auto-inducer molecules as it exhibited a strong affinity for the regulatory proteins of the QS-circuits i.e. LasR and RhlR. In addition, a significant down-regulation in the expression of QS-related genes was observed suggesting the ability of 5-HMF in mitigating the pathogenicity of P. aeruginosa.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
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alsfakia@gmail.com