Antonie van Leeuwenhoek

A method for preparing and storing reduced streptolysin O is described. The reliability of this method has been confirmed by the results of the titrations of different batches of reduced streptolysin O at different times after preparation.

Survival of Pseudomonas sp. SF4c and Pseudomonas sp. SF10b (two plant-growth-promoting bacteria isolated from wheat rhizosphere) was investigated in microcosms. Spontaneous rifampicin-resistant mutants derived from these strains (showing both growth rate and viability comparable to the wild-strains) were used to monitor the strains in bulk soil and wheat rhizosphere. Studies were carried out for 60 days in pots containing non-sterile fertilized or non-fertilized soil. The number of viable cells of both mutant strains declined during the first days but then became established in the wheat rhizosphere at an appropriate cell density in both kinds of soil. Survival of the strains was better in the rhizosphere than in the bulk soil. Finally, the antagonism of Pseudomonas spp. against phytopatogenic fungi was evaluated in vitro. Both strains inhibited the mycelial growth (or the resistance structures) of some of the phytopathogenic fungi tested, though variation in this antagonism was observed in different media. This inhibition could be due to the production of extracellular enzymes, hydrogen cyanide or siderophores, signifying that these microorganisms might be applied in agriculture to minimize the utilization of chemical pesticides and fertilizers.

Plants interact with a myriad of microbial cells in the rhizosphere, an environment that is considered to be important for plant development. However, the differential structuring of rhizosphere microbial communities due to plant cultivation under differential agricultural practices remains to be described for most plant species. Here we describe the rhizosphere microbiome of grapevine cultivated under conventional and organic practices, using a combination of cultivation-independent approaches. The quantification of bacterial 16S rRNA and nifH genes, by quantitative PCR (qPCR), revealed similar amounts of these genes in the rhizosphere in both vineyards. PCR-DGGE was used to detect differences in the structure of bacterial communities, including both the complete whole communities and specific fractions, such as Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and those harboring the nitrogen-fixing related gene nifH. When analyzed by a multivariate approach (redundancy analysis), the shifts observed in the bacterial communities were poorly explained by variations in the physical and chemical characteristics of the rhizosphere. These approaches were complemented by high-throughput sequencing (67,830 sequences) based on the V6 region of the 16S rRNA gene, identifying the major bacterial groups present in the rhizosphere of grapevines: Proteobacteria, Actinobacteria, Firmicutes, Bacteriodetes, Acidobacteria, Cloroflexi, Verrucomicrobia and Planctomycetes, which occur in distinct proportions in the rhizosphere from each vineyard. The differences might be related to the selection of plant metabolism upon distinct reservoirs of microbial cells found in each vineyard. The results fill a gap in the knowledge of the rhizosphere of grapevines and also show distinctions in these bacterial communities due to agricultural practices.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, for which the mortality rate is high. Host peripheral blood is a body site for the immune clearance of pathogens mediated by release of inflammatory factors. However, “out of control” inflammatory factor release can contribute to host death. To further understand the changes in the transcription level of immune-related effectors, samples of peripheral blood mononuclear cells (PBMCs) collected from piglets at different stages of infection (0, 24 and 120 h) were sequenced on an Illumina HiSeq™ 4000 platform. We found 3818 differentially expressed genes (DEGs) in the 24 h-infection group compared to the 0 h-infection group (Pb24-Vs-Pb0). DEGs mainly involved in the Gene ontology and KEGG pathways that included nucleic acid metabolism regulation, cell growth, cell differentiation, and organ morphological maintenance were not significantly enriched (P > 0.05). However, DEGs associated with protein kinase activity, receptor activation, metabolism, local adhesion and immune inflammatory responses were significantly enriched in Pb120-Vs-Pb24 (P < 0.05), as were those related to the T cell receptor signalling pathway, with most being down-regulated compared to the preceding stage (Pb24-Vs-Pb0). In PBMCs there were some changes in glucose metabolism, local adhesion and the immune inflammatory response (Pb120-Vs-Pb0). In addition, up-regulated DEGs, such as IL8, IL1β, and CCL2, and were significantly enriched in immune-inflammatory related pathways compared to the uninfected stage, although they began to decline after 24 h.

Tebuconazole is the most widely used fungicide in agriculture. Due to its long half-life, tebuconazole residues can be found in the environment media such as in soil and water bodies. Here, the metabolic pathway of tebuconazole was studied in Cunninghamella elegans (C. elegans). Approximately 98% of tebuconazole was degraded within 7 days, accompanied by the accumulation of five metabolites. The structures of the metabolites were completely or tentatively identified by gas chromatography-mass spectrometry (GC–MS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To identify representative oxidative enzymes that may be involved in the metabolic process, treatment with piperonyl butoxide (PB) and methimazole (MZ) was performed. PB had a strong inhibitory effect on the metabolic reactions, while MZ had a weak inhibitory effect. The results suggest that cytochrome P450 (CYP) and flavin-dependent monooxygenase are involved in the metabolism of tebuconazole. Based on the results, we propose a metabolic pathway for the fungal metabolism of tebuconazole. Data are of interest to gain insight into the toxicological effects of tebuconazole and for tebuconazole bioremediation.

This study aimed at measuring the nucleotide non-randomness in the region downstream of start codons in bacterial genes and to see if the non-randomness differs between biased and unbiased genes, in terms of the effective number of codons (Nc) and the codon adaptation index (CAI). In Escherichia coli, there was a marked elevation in nucleotide conservation for the genes having low Nc-values compared to the genes having high Nc-values, i.e the more biased genes showed a higher level of non-randomness. Likewise, the genes displaying high CAI-values showed stronger nucleotide conservation than the genes of low CAI-values. This elevated conservation is visible up to approximately 15-17 nucleotides downstream of the start codon, after which there is little difference. This indicates that there may be distinct selectional mechanisms acting upon the first 5-6 codons within genes in E. coli. In B. subtilis, these effects are less pronounced, if present at all. Furthermore, analyses of codons used in this region were not in support of the hypothesis that the elevation in nucleotide non-randomness is a question of selection for certain optimal codons.

Extracellular DNA can play a structural role in the microbial environment. Here evidence is presented that an environmental isolate of Acidovorax temperans utilises extracellular DNA for intercellular and cell-surface attachment and that Type IV pili and electrostatic interactions play a role in this interaction. Preliminary attempts to isolate and purify extracellular polysaccharides from A. temperans strain CB2 yielded significant amounts of DNA raising the question of whether this molecule was present as a structural component in the extracellular matrix. The role of DNA in attachment was indicated by experiments in which the addition of DNase to liquid medium inhibited the attachment of Acidovorax to glass wool. A Tn5 insertional mutant, lacking Type IV pili, was unable to initiate attachment. Addition of DNase caused rapid detachment of bound cells, but no detachment occurred when proteinase, RNase or inactivated DNase were used. Addition of MgCl2 also caused significant detachment, supporting the possible mechanistic role of electrostatic interactions in the attachment process. Although attachment was apparent in early to mid-log phase growth, surprisingly DNA was not detected in the culture supernatant until late stationary phase and coincided with an appreciable loss of cell viability. This suggests that during log-phase growth attachment is mediated by eDNA that is released in low quantities and/or is highly localised within the extracellular matrix and also that stationary phase DNA release through widespread cell lysis may be a separate and unrelated event.

A novel actinobacterium, designated strain BN506T, was isolated from soil collected from Tuz (Salt) Lake, Konya, Turkey, and was characterised to determine its taxonomic position. The isolate was found to have morphological and chemotaxonomic properties associated with members of the genus Streptomonospora. The isolate was found to grow optimally at 37 °C and in the presence of 10 % (w/v) NaCl but not in the absence of NaCl. Phylogenetic analyses based on an almost-complete 16S rRNA gene sequences indicated that isolate is closely related to members of the genus Streptomonospora and forms a distinct phyletic line in the Streptomonospora phylogenetic tree. Strain BN506T is closely related to Streptomonospora halophila YIM 91355T (98.1 % sequence similarity). Sequence similarities with other type strains of the genus Streptomyces were lower than 98.0 %. The cell wall of the novel strain was found to contain meso-diaminopimelic acid. Whole cell hydrolysates were found to contain galactose, glucose and ribose. The predominant menaquinone was identified as MK-10(H8) (57.0 %). The polar lipids detected were identified as diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. The major fatty acids were found to be anteiso-C17:0, iso-C16:0 and 10 methyl C18:0. Based on 16S rRNA gene sequence analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data, strain BN506T was identified as a member of a novel species of the genus Streptomonospora, for which the name Streptomonospora tuzyakensis sp. nov. (type strain BN506T = DSM 45930T = KCTC 29210T) is proposed.