Specialization in a Nitrogen-Fixing Symbiosis: Proteome Differences Between Sinorhizobium medicae Bacteria and Bacteroids.

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. In all, 3,215 proteins were identified, over half of the open reading frames predicted from the genomic sequence. The abundance of 1,361 proteins displayed strong lifestyle bias. In total, 1,131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised approximately 12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis or gluconeogenesis, and the pentose phosphate pathway; however, several amino acid degradation pathways were repressed. This suggests that bacteroids maintain a relatively independent anabolic metabolism. Tricarboxylic acid cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in S. meliloti Rm1021 were detected, and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library.

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

Saccharomyces cerevisiae genome-wide mutant screen for sensitivity to 2,4-diacetylphloroglucinol, an antibiotic produced by Pseudomonas fluorescens.

2,4-Diacetylphloroglucinol (2,4-DAPG), an antibiotic produced by Pseudomonas fluorescens, has broad-spectrum antibiotic activity, inhibiting organisms ranging from viruses, bacteria, and fungi to higher plants and mammalian cells. The biosynthesis and regulation of 2,4-DAPG in P. fluorescens are well described, but the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a deletion library of Saccharomyces cerevisiae in broth and agar medium supplemented with 2,4-DAPG. We identified 231 mutants that showed increased sensitivity to 2,4-DAPG under both conditions, including 22 multidrug resistance-related mutants. Three major physiological functions correlated with an increase in sensitivity to 2,4-DAPG: membrane function, reactive oxygen regulation, and cell homeostasis. Physiological studies with wild-type yeast validated the results of the mutant screens. The chemical-genetic fitness profile of 2,4-DAPG resembled those of menthol, sodium azide, and hydrogen peroxide determined in previous high-throughput screening studies. Collectively, these findings indicate that 2,4-DAPG acts on multiple basic cellular processes.

Isolation, characterization, and sensitivity to 2,4-diacetylphloroglucinol of isolates of Phialophora spp. from Washington wheat fields.

Dark pigmented fungi of the Gaeumannomyces-Phialophora complex were isolated from the roots of wheat grown in fields in eastern Washington State. These fungi were identified as Phialophora spp. on the basis of morphological and genetic characteristics. The isolates produced lobed hyphopodia on wheat coleoptiles, phialides, and hyaline phialospores. Sequence comparison of internal transcribed spacer regions indicated that the Phialophora isolates were clearly separated from other Gaeumannomyces spp. Primers AV1 and AV3 amplified 1.3-kb portions of an avenacinase-like gene in the Phialophora isolates. Phylogenetic trees of the avenacinase-like gene in the Phialophora spp. also clearly separated them from other Gaeumannomyces spp. The Phialophora isolates were moderately virulent on wheat and barley and produced confined black lesions on the roots of wild oat and two oat cultivars. Among isolates tested for their sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), the 90% effective dose values were 11.9 to 48.2 microg ml(-1). A representative Phialophora isolate reduced the severity of take-all on wheat caused by two different isolates of Gaeumannomyces graminis var. tritici. To our knowledge, this study provides the first report of an avenacinase-like gene in Phialophora spp. and demonstrated that the fungus is significantly less sensitive to 2,4-DAPG than G. graminis var. tritici.

Diversity, virulence, and 2,4-diacetylphloroglucinol sensitivity of Gaeumannomyces graminis var. tritici isolates from Washington state.

We determined whether isolates of the take-all pathogen Gaeumannomyces graminis var. tritici become less sensitive to 2,4-diacetylphloroglucinol (2,4-DAPG) during wheat monoculture as a result of exposure to the antibiotic over multiple growing seasons. Isolates of G. graminis var. tritici were baited from roots of native grasses collected from noncropped fields and from roots of wheat from fields with different cropping histories near Lind, Ritzville, Pullman, and Almota, WA. Isolates were characterized by using morphological traits, G. graminis variety-specific polymerase chain reaction and pathogenicity tests. The sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was determined by measuring radial growth of each isolate. The 90% effective dose value was 3.1 to 4.4 microg ml(-1) for 2,4-DAPG-sensitive isolates, 4.5 to 6.1 microg ml(-1) for moderately sensitive isolates, and 6.2 to 11.1 microg ml(-1) for less sensitive isolates. Sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was normally distributed in all fields and was not correlated with geographic origin or cropping history of the field. There was no correlation between virulence on wheat and geographical origin, or virulence and sensitivity to 2,4-DAPG. These results indicate that G. graminis var. tritici does not become less sensitive to 2,4-DAPG during extended wheat monoculture.