Contrasting fertilization and phenological stages shape microbial-mediated phosphorus cycling in a maize agroecosystem.

Phosphorus (P) is essential for plants but often limited in soils, with microbes playing a key role in its cycling. P deficiency in crops can be mitigated by applying by-products like sludge and struvite to enhance yield and sustainability. Here, we evaluated the contribution of four different types of fertilizers: i) conventional NPK; ii) sludge; iii) struvite; and iv) struvite+sludge in a semiarid maize plantation to the availability of P and the responses of the soil microbiome. We investigated the effects of these treatments on the relative abundance of bacterial and archaeal genes and proteins related to organic P mineralization, inorganic P solubilization, and the P starvation response regulation through a multi-omic approach. Moreover, we explored the impact of maize phenology by collecting samples at germination and flowering stages. Our findings suggest that the phenological stage has a notable impact on the abundance of P cycle genes within bacterial and archaeal communities, particularly regarding the solubilization of inorganic P. Furthermore, significant variations were observed in the relative abundance of genes associated with different P cycles in response to various fertilizer treatments. Sludge and struvite application improved P availability, which was related to an increase in the relative abundance of Sphingomonas (Proteobacteria) and Luteitalea (Acidobacteria) respectively, and genes related to inorganic P solubilization. Furthermore, we observed a substantial taxonomic clustering of functional processes associated with the P cycle. Among the dominant bacterial populations containing P-related genes, those microbes possessing genes linked to the solubilization of inorganic P typically did not harbor genes associated with the mineralization of organic P. This phenomenon was particularly evident among members of Actinobacteria. Overall, we reveal important shifts in bacterial and archaeal communities and associated molecular processes, stressing the intricate interplay between fertilization, phenology, and P cycling in agroecosystems.

When drought meets forest management: Effects on the soil microbial community of a Holm oak forest ecosystem.

The growth and survival of plants in semiarid Mediterranean forests can be improved through the benefits conferred by thinning, a forest management practice that removes trees and reduces the competition between the remaining ones. Here, we evaluate the impacts of induced drought (the exclusion of 25% of the natural rainfall for 5 years) and thinning, and their interaction, with the objective of determining whether the thinning of Holm oak (Quercus ilex L.) modulates the resistance of the soil microbial community to drought. Sequencing of 16S rRNA and ITS amplicons revealed that drought, thinning, and their interaction influenced the composition of the bacterial community, while the fungal community was exclusively affected by thinning. Thinning consisted of the removal of the aboveground parts of the Holm oak trees, which were thereafter left in forest stand. Thinning contributed to the C and N contents, with parallel increases in microbial biomass, particularly in summer. Drought increased the amounts of total organic C and total N, likely due to the reduced enzyme activities. Indeed, the composition of the bacterial community was modulated primarily by the indirect and long-term effects of drought - the accumulation of soil organic matter - rather than by the direct effect of the lower water content imposed by the drought treatments. Thinning under drought conditions did not increase soil organic C (SOC) content. However, the resistance of the soil microbial community to drought was fostered by thinning, particularly at the functional level, as indicated by the enzyme activities related to C, N and P cycles. These responses were associated to variations in the composition of the microbial communities in thinned, drought-exposed plots, in comparison to unthinned, drought-exposed plots. In conclusion, the interaction between forest management and drought influenced the soil microbial community of a Holm oak-dominated Mediterranean ecosystem.

The extracellular metaproteome of soils under semiarid climate: A methodological comparison of extraction buffers.

We compare the protein extraction efficiencies, as well as the phylogenetic and functional information provided, of two extraction protocols in soils that differ mainly in their organic matter and clay contents, the main factors limiting protein extraction in semiarid soils. These protocols utilise extractants commonly used for the assay of extracellular enzyme activities. The first method was based on the utilisation of the modified universal buffer (MUB). The second was based on the extraction of humic substances with sodium pyrophosphate. When compared to the total amount of proteins in soil, the results indicate a very-low extraction efficiency for both protocols. Analysis in an Orbitrap Fusion mass spectrometer and further searching against an "ad hoc" metagenome evidenced that the phylogenetic and functional information retrieved from the extracellular soil metaproteome can be biased by the extraction buffer.

It's all about functionality: How can metaproteomics help us to discuss the attributes of ecological relevance in soil?

Soil metaproteomics has been recently developed in order to link the phylogeny and functionality of soil microorganisms. After the development of protein extraction and peptide analysis, the use of improved mass-spectrometric systems has allowed the description of the microbial composition and the identification of proteins in soils subjected to deforestation, restoration, contamination and different agricultural managements. In this letter, we encourage one step-forward for soil metaproteomics. We believe that proteomics can complement ecological attributes that control the dynamics of soil microbial populations. Among these attributes, we highlight the resistance, resilience and plasticity of microbial populations. Providing with functional basis to these attributes will extend our knowledge on the microbial-mediated mechanisms regulating the impacts of climate change and agricultural management in the near future.

The ecological and physiological responses of the microbial community from a semiarid soil to hydrocarbon contamination and its bioremediation using compost amendment.

The linkage between phylogenetic and functional processes may provide profound insights into the effects of hydrocarbon contamination and biodegradation processes in high-diversity environments. Here, the impacts of petroleum contamination and the bioremediation potential of compost amendment, as enhancer of the microbial activity in semiarid soils, were evaluated in a model experiment. The analysis of phospholipid fatty-acids (PLFAs) and metaproteomics allowed the study of biomass, phylogenetic and physiological responses of the microbial community in polluted semiarid soils. Petroleum pollution induced an increase of proteobacterial proteins during the contamination, while the relative abundance of Rhizobiales lowered in comparison to the non-contaminated soil. Despite only 0.55% of the metaproteome of the compost-treated soil was involved in biodegradation processes, the addition of compost promoted the removal of polycyclic aromatic hydrocarbons (PAHs) and alkanes up to 88% after 50 days. However, natural biodegradation of hydrocarbons was not significant in soils without compost. Compost-assisted bioremediation was mainly driven by Sphingomonadales and uncultured bacteria that showed an increased abundance of catabolic enzymes such as catechol 2,3-dioxygenases, cis-dihydrodiol dehydrogenase and 2-hydroxymuconic semialdehyde. For the first time, metaproteomics revealed the functional and phylogenetic relationships of petroleum contamination in soil and the microbial key players involved in the compost-assisted bioremediation.

Quantitative analysis of the intra- and inter-subject variability of the whole salivary proteome.

BACKGROUND AND OBJECTIVE: Interest in human saliva is increasing for disease-specific biomarker discovery studies. However, protein composition of whole saliva can grossly vary with physiological and environmental factors over time and it comprises human as well as bacterial proteins. MATERIAL AND METHODS: We compared intra- and inter-subject variabilities using complementary gel-based (two-dimensional difference gel electrophoresis, 2-D DIGE) and gel-free (liquid chromatography tandem mass spectrometry, LC-MS/MS) proteomics profiling of saliva. Unstimulated whole saliva of four subjects was examined at three different time-points (08.00 h, 12.00 h and 17.00 h) and variability of the saliva proteome was analyzed on two successive days by LC-MS/MS. RESULTS: In the 2-D DIGE experiment, the median coefficient of variation (CV) for intra-subject variability was significantly lower (CV of 0.39) than that for inter-subject variability (CV of 0.57; CV of technical replicates 0.17). LC-MS/MS data confirmed the significantly lower variation within subjects over time (CV of 0.37) than the inter-subject variability (CV of 0.53; CV of technical replicates 0.11), and that the inter-subject variability was not time-dependent. CONCLUSION: Both techniques revealed similar trends of variations on technical, intra- and inter-subject level but provided peptide and protein focused information and should thus be used as complementary approaches. The data presented indicate that 2-D DIGE as well as LC-MS/MS approaches are suitable for biomarker screening in saliva.

Phylogenetic and proteomic analysis of an anaerobic toluene-degrading community.

AIMS: This study intended to unravel the physiological interplay in an anaerobic microbial community that degrades toluene under sulfate-reducing conditions combining proteomic and genetic techniques. METHODS AND RESULTS: An enriched toluene-degrading community (Zz5-7) growing in batch cultures was investigated by DNA- and protein-based analyses. The affiliation and diversity of the community were analysed using 16S ribosomal RNA (rRNA) genes as a phylogenetic marker as well as bssA and dsrAB genes as functional markers. Metaproteome analysis was carried out by a global protein extraction and a subsequent protein separation by two-dimensional gel electrophoresis (2-DE). About 85% of the proteins in the spots were identified by nano-liquid chromatography coupled with electrospray mass spectrometry (nano-LC-ESI-MS/MS) analysis. DNA sequencing of bssA and the most abundant dsrAB amplicons revealed high similarities to a member of the Desulfobulbaceae, which was also predominant according to 16S rRNA gene amplicons. Metaproteome analysis provided 202 unambiguous protein identifications derived from 236 unique protein spots. The proteins involved in anaerobic toluene activation, dissimilatory sulfate reduction, hydrogen production/consumption and autotrophic carbon fixation were mainly affiliated to members of the Desulfobulbaceae and several other Deltaproteobacteria. CONCLUSION: Phylogenetic and metaproteomic analyses revealed a member of the Desulfobulbaceae as the key player of anaerobic toluene degradation in a sulfate-reducing consortium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that combines genetic and proteomic analyses to indicate the interactions in an anaerobic toluene-degrading microbial consortium.

NBDT (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene)--a novel fluorescent dye for studying mechanisms of toluene uptake into vital bacteria.

Uptake of small hydrophobic substances such as toluene into bacteria is widely assumed to occur by passive diffusion. Some toluene degrading bacteria, however, are described to contain uptake systems which may be involved in the transport of this compound. In this study, a fluorescently labeled toluene analogue dye (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene; NBDT), flow cytometry, and shot gun proteome analysis were used to follow toluene uptake into bacteria in more detail. The new dye has excitation peaks at 444 and 475 nm and an emission peak at 537 nm. The toluene-degraders P. putida mt-2 and P. putida F1 as well as P. putida KT2440 and E. coli K12 as negative controls were included. To enable quantification of NBDT uptake, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to inactivate NBDT efflux pumps. The porin inhibitor cadaverine was added to study the porin-mediated influx of toluene. Cadaverine reduced NBDT uptake by toluene-grown P. putida mt-2 and F1 by 25% and 42%, respectively, thus revealing an involvement and possibly a regulatory function of porins in the uptake of the toxic substrate toluene. Shot gun proteome measurements gave evidence for the presence of toluene transporting porins in P. putida mt-2 grown on toluene but not when grown on glucose.

Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete Phoma sp. UHH 5-1-03.

A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer ( approximately 150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography-tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.

Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene.

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO(4) and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k(cat)/K(m)) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s(-1) mM(-1), respectively, with K(m) values of 4.2, 67.8, and 104.9 microM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15 degrees C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.