Microbiological Research

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

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.

Regulation of antimonite oxidation and resistance by the phosphate regulator PhoB in Agrobacterium tumefaciens GW4 Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Jingxin Li, Zixu Qiao, Manman Shi, Yuxiao Zhang, Gejiao Wang Abstract Microbial oxidation of antimonite [Sb(III)] to antimonate [Sb(V)] is a detoxification process which contributes to Sb(III) resistance. Antimonite oxidase AnoA is essential for Sb(III) oxidation, however, the regulation mechanism is still unknown. Recently, we found that the expressions of phosphate transporters were induced by Sb(III) using proteomics analysis in Agrobacterium tumefaciens GW4, thus, we predicted that the phosphate regulator PhoB may regulate bacterial Sb(III) oxidation and resistance. In this study, comprehensive analyses were performed and the results showed that (1) Genomic analysis revealed two phoB (named as phoB1 and phoB2) and one phoR gene in strain GW4; (2) Reporter gene assay showed that both phoB1 and phoB2 were induced in low phosphate condition (50 μM), but only phoB2 was induced by Sb(III); (3) Genes knock-out/complementation, Sb(III) oxidation and Sb(III) resistance tests showed that deletion of phoB2 significantly inhibited the expression of anoA and decreased bacterial Sb(III) oxidation efficiency and Sb(III) resistant. In contrast, deletion of phoB1did not obviously affect anoA's expression level and Sb(III) oxidation/resistance; (4) A putative Pho motif was predicted in several A. tumefaciens strains and electrophoretic mobility shift assay (EMSA) showed that PhoB2 could bind with the promoter sequence of anoA; (5) Site-directed mutagenesis and short fragment EMSA revealed the exact DNA binding sequence for the protein-DNA interaction. These results showed that PhoB2 positively regulates Sb(III) oxidation and PhoB2 is also associated with Sb(III) resistance. Such regulation mechanism may provide a great contribution for bacterial survival in the environment with Sb and for bioremediation application.

RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens Publication date: September 2019 Source: Microbiological Research, Volume 226 Author(s): Eduardo Henrique Goulin, Diogo Manzano Galdeano, Laís Moreira Granato, Emilyn Emy Matsumura, Ronaldo José Durigan Dalio, Marcos Antonio Machado Abstract Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.

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.

Codon-usage frequency mediated SNPs selection in lasR gene of cystic fibrosis Pseudomonas aeruginosa isolates Publication date: June–August 2019 Source: Microbiological Research, Volumes 223–225 Author(s): Huifang Qiu, Yuanhao Li, Weijun Dai Abstract Pseudomonas aeruginosa is an opportunistic pathogen with high clinical relevance for hospital infections of patients. Accumulating DNA sequencing results of clinical P. aeruginosa isolates have revealed frequent mutations in lasR gene, which encodes the highest arches component of quorum-sensing system (QS). We analyzed the sequencing data of lasR gene from a large collection of cystic fibrosis (CF) P. aeruginosa isolates. Our systematical analyses revealed that single nucleotide polymorphisms (SNPs) selection in lasR gene were largely constrained by codon-usage frequency. As a whole, SNP-substituted codons encoding unconserved amino acid resulted in unfavored codons with relatively low codon-usage frequency, while those associating with conserved amino acid were not strictly regulated in such way. These SNPs substitutions gives rise to diverse functional LasR isoforms and contributes to the relative growth fitness of recombinant lasR variant strains. Our survey reveals a novel pattern of SNPs selections in lasR gene of CF isolates. Our findings could be served as a powerful resource for understanding adaptive mechanism of clinical isolates under environmental constrains and developing anti-bacteria drugs for CF patients.

Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides Publication date: June–August 2019 Source: Microbiological Research, Volumes 223–225 Author(s): Muhammad Azeem, Marina Barba-Aliaga, Anna Karin Borg-Karlson, Olle Terenius, Anders Broberg, Gunaratna Kuttuva Rajarao Abstract Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps. Graphical abstract

Beneficial effects of inoculation of growth-promoting bacteria in strawberry Publication date: June–August 2019 Source: Microbiological Research, Volumes 223–225 Author(s): Fernanda Marcondes de Andrade, Thiago de Assis Pereira, Thiago Pereira Souza, Paulo Henrique Sales Guimarães, Adalvan Daniel Martins, Rosane Freitas Schwan, Moacir Pasqual, Joyce Dória Abstract Plant growth-promoting bacteria have been highlighted by their potential for application in plant production, allowing the reduction of the use of fertilizers and pesticides, which is due to the ability to stimulate the growth of plants by nitrogen-fixation and production of phytohormones, such as indole-3-acetic acid (IAA). The objective of this study was to verify the potential of plant growth promotion of 25 wild isolates from the Agricultural Microbiology Culture Collection of the Federal University of Lavras (CCMA-UFLA) through the evaluation of the biological nitrogen-fixation capacity and the production of IAA. In addition, the growth of three selected strains inoculated on roots of strawberry seedlings in greenhouse conditions was evaluated. The experiment was conducted in a completely randomized design (CRD), with an 8 × 2 factorial schemes involving eight combinations of bacteria: alone, in pairs and threes, plus the control without inoculation. Two fertilizer levels were used (0% and 50% of nitrogen), totaling 16 treatments with eight replicates each. After 75 days, variables such as root length, root dry weight, aerial part length, aerial part dry weight, leaf number, total dry mass and ultrastructural analysis of the inoculated and uninoculated roots, were evaluated. The results showed that the strawberry crop responded positively to inoculation with the three bacteria combined Azospirillum brasilense (Ab-V5) + Burkholderia cepacia (CCMA 0056) + Enterobacter cloacae (CCMA 1285) compared to the uninoculated controls. More expressive responses in terms of plant growth were observed in relation to the combined inoculation of the three bacterial strains plus fertilizer application with 50% of nitrogen.

Alexandros Sfakianakis
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alsfakia@gmail.com