Applied and Environmental Microbiology最新文献

Remote polar regions offer unique opportunities and significant challenges for antimicrobial resistance research in a near-pristine environment. While core microbiology techniques continue to have an important role in supporting environmental research, the severe cold climate presents considerable challenges to laboratory research. We explore adaptations required for core bacteriology investigations in polar regions on an unsupported remote expedition c. 600 km north of the Arctic Circle utilizing the National Collection of Type Culture bacterial strains. Methods of culture, microscopy, biochemical and phenotypic testing, vortex, and centrifuge techniques are explored. Across -21.5 to -41.0°C, culture was satisfactorily enabled using a solar-powered USB incubator and an electricity-free water-bath option utilizing white gas for a variety of standard culture media. Microscopy and biochemical tests supported organism identification. Phenotypic testing for carbapenemase-producing genes using lateral flow devices showed good performance without modification (Carba-5, 20/20 carbapenemase-producing organism tests, 100% sensitivity; 100/100 negative targets, 100% specificity). The modified centrifuge was enabled with a 3D printed disk and Dremel drill and microbial DNA extraction (ZymoBIOMICS) kits were able to extract DNA of suitable quality for analysis. With suitable adaptations, conducting core microbiology techniques (with potential relevance for more advanced techniques) is possible in the remote extreme cold environment.

Importance: Antimicrobial resistance (AMR) represents one of the key global public health threats currently facing humanity. The recent UN High-Level Meeting on AMR highlighted the need for greater knowledge generation on its environmental aspects while also considering the potential adverse effects of climate change. The polar regions of the world offer a unique opportunity for AMR research in a near-pristine environment while also holding the potential for novel resistance mechanisms and/or antimicrobial peptide discovery within melting permafrost or glacial ice. Despite considerable technological advances in microbiology, operating in severe cold environments continues to present significant operational challenges. Our report here offers a basis for adaptations to enable both environmental and clinical antimicrobial resistance, microbiome, and discovery research for operating in the harshest of remote environments.

Polyunsaturated fatty acids (PUFAs) play a crucial role in aiding bacteria to adapt to extreme and stressful environments. While there is a well-established understanding of their production, accrual, and transfer within marine ecosystems, knowledge about terrestrial environments remains limited. Investigation of the intestinal microbiome of earthworms has illuminated the presence of PUFAs presumably of microbial origin, which contrasts with the surrounding soil. To comprehensively study this phenomenon, a multi-faceted approach was employed, combining fatty acid analysis with amplicon sequencing of the PfaA-KS domain of the anaerobic fatty acid synthase gene (pfa), as well as the 16S rRNA and 18S rRNA genes. This methodology was applied to scrutinize the gut microbiome of Eisenia fetida, its compost-based dietary source, and the resultant castings. This study unveiled a distinct gut soil ecosystem from input compost and output castings in fatty acid profile as well as type and abundance of organisms. 16S sequencing provided insights into the microbial composition, showing increased relative abundance of certain Pseudomonadota, including Shewanellaceae, and Planctomycetota, including Gemmataceae within the gut microbiome compared to input bulk soil compost, while Actinomycetota and Bacillota were relatively enriched compared to the casted feces. Sequencing of the PfaA-KS domain revealed amplicon sequence variants (ASVs) belonging primarily to Shewanella. Intriguingly, the 20C PUFAs were identified only in gut soil samples, though PfaA-KS sequence abundance was highest in output castings, indicating a unique metabolism occurring only in the gut. Overall, the results indicate that Shewanella can explain PUFA enrichment in the gut environment because of the pfa gene presence detected via PfaA-KS sequence data.IMPORTANCEPrior research has demonstrated that earthworm microbiomes can potentially harbor polyunsaturated fatty acids (PUFAs) that are not found within their residing soil environment. Moreover, distinct indicator species have been pinpointed for various microbial genera in earthworm microbiomes. Nevertheless, none of these studies have integrated metataxonomic and fatty acid analyses to explore the origin of PUFA synthesis in any earthworm species, with the objective of identifying the specific organisms and locations responsible for this production. This study suggests that earthworms accumulate PUFAs produced from bacteria, especially Shewanella, activated through the gut ecosystem.

In Sweden, reforestation of managed forests relies predominantly on planting nursery-produced tree seedlings. However, the intense production using containerized cultivation systems (e.g., high seedling density, irrigation from above, regular fertilization) creates favorable conditions for fungal infections. Despite the harmful role of diseases in forest nurseries, the origin and dispersal factors of fungal pathogens remain largely unknown. A better understanding of the airborne spread of pathogens could improve the prediction of fungal infection, ultimately optimizing preventative methods and decreasing the use of fungicides. This study investigated the temporal dynamics of airborne fungi in forest nurseries, with a focus on fungal pathogens. Airborne fungi were monitored in four Swedish forest nurseries over two growing seasons using spore traps and high-throughput sequencing. Fungal pathogens were identified using bioinformatics and quantified with quantitative PCR. Results showed strong temporal shifts of airborne fungal diversity and community composition following the growing seasons. The airborne spread included high abundances of important fungal pathogens (e.g., Cladosporium sp., Botrytis cinerea, Alternaria sp., Sydowia polyspora, and Melampsora populnea) with individual temporal and spatial variations. In general, the deposited spore loads of nursery pathogens correlated positively with increased temperature and negatively with higher precipitation. This was expressed the strongest for Cladosporium sp., Alternaria sp., and M. populnea, which suggests a higher availability of fungal inoculum in warm and dry periods. This study highlights the influence of seasonality on the temporal dynamics of economically important fungal pathogens in Swedish forest nurseries, which should be considered in the development of a local decision support system.IMPORTANCEFungal diseases in forest nurseries have significant environmental and economic impacts on the tree seedling production. This study highlights the role of seasonality in the airborne spread of fungal pathogens in Swedish forest nurseries. By analyzing airborne fungal spores using advanced sequencing and monitoring techniques, key fungal pathogens and their dispersal patterns over two growing seasons were identified. The findings indicate that warmer, drier periods may increase the spread of fungal pathogens, emphasizing the need for targeted preventative measures. Understanding these temporal dynamics can help optimize the use of fungicides in forest nurseries, thereby promoting more sustainable and environmentally friendly management practices. This research provides valuable insights for improving disease management in forest nurseries, ultimately supporting sustainable tree seedling production.

Soil microbial communities play crucial roles in nutrient cycling and can help retain nitrogen in agricultural soils. Quantitative stable isotope probing (qSIP) is a useful method for investigating taxon-specific microbial growth and utilization of specific nutrients, such as nitrogen (N). Typically, qSIP is performed in a highly controlled lab setting, so the field relevance of lab qSIP studies remains unknown. We conducted and compared tandem lab and field qSIP to quantify the assimilation of ¹⁵N by maize-associated soil prokaryotic communities at two agricultural sites. Here, we show that field qSIP with ¹⁵N can be used to measure taxon-specific microbial N assimilation. Relative ¹⁵N assimilation rates were generally lower in the field, and the magnitude of this difference varied by site. Rates differed by method (lab vs field) for 19% of the top N assimilating genera. The field and lab measures were more comparable when relative assimilation rates were weighted by relative abundance to estimate the proportion of N assimilated by each genus with only ~10% of taxa differing by method. Of those that differed, the taxa consistently higher in the lab were inclined to have opportunistic lifestyle strategies, whereas those higher in the field had niches reliant on plant roots or in-tact soil structure (biofilms, mycelia). This study demonstrates that ¹⁵N-qSIP can be successfully performed using field-incubated soils to identify microbial allies in N retention and highlights the strengths and limitations of field and lab qSIP approaches.

Importance: Soil microbes are responsible for critical biogeochemical processes in natural and agricultural ecosystems. Despite their importance, the functional traits of most soil organisms remain woefully under-characterized, limiting our ability to understand how microbial populations influence the transformation of elements such as nitrogen (N) in soil. Quantitative stable isotope probing (qSIP) is a powerful tool to measure the traits of individual taxa. This method has rarely been applied in the field or with ¹⁵N to measure nitrogen assimilation. In this study, we measured genus-specific microbial nitrogen assimilation in two agricultural soils and compared field and lab ¹⁵N qSIP methods. Our results identify taxa important for nitrogen assimilation in agricultural soils, shed light on the field relevance of lab qSIP studies, and provide guidance for the future application of qSIP to measure microbial traits in the field.

Plant growth-promoting rhizobacterium Azospirillum brasilense Sp7 utilizes fructose efficiently via a fructose phosphotransferase system (Fru-PTS). Its genome encodes two putative Fru-PTS, each consisting of FruB (EIIA), FruK (Pfk), and FruA (EIIBC) proteins. We compared the proteomes of A. brasilense Sp7 grown with malate or fructose as sole carbon source, and noticed upregulation of the constituent proteins of Fru-PTS1 only on fructose. Inactivation of fruA gene of both the Fru-PTS showed that Fru-PTS1 is the main PTS involved in fructose utilization. Overexpression of fruA1 in A. brasilense Sp7 enhanced its growth on fructose showing improved consumption of fructose. This suggested that fructose utilization in A. brasilense Sp7 is limited due to the limitation of EIIBC component. A FruR-type regulator, encoded divergently to the Fru-PTS1 operon, was required for chemotaxis toward fructose. Although not an absolute necessity for the growth of fructose, FruR was required for the optimal growth of fructose. The fruB1 promoter was activated by fructose and repressed by malate, but FruR does not seem to regulate its expression. A 27-nucleotide stem-loop structure located between the -125 and -99 promoter proximal region of fruB1 was involved in fructose inducibility and malate repression. Fructose also upregulated several proteins involved in the biogenesis of a Type 6 secretion system. Here, we have shown that A. brasilense Sp7 was able to inhibit the growth of Escherichia coli and Agrobacterium tumefaciens in the presence of fructose, and that an intact T6SS was required for contact-dependent growth inhibition of the two Gram-negative bacteria.IMPORTANCEAzospirillum brasilense, a plant growth-promoting rhizobacterium, has limited ability to utilize carbohydrates and sugars. Although it is known to utilize fructose via a fructose phosphotransferase system (fructose-PTS), the genes involved in fructose utilization and the role of fructose in its biology were not well characterized. This study has shown that out of the two units of fructose-PTS encoded in its genome, fructose-PTS1 plays the major role in fructose utilization. Overexpression of the membrane component (EIIBC) improved the growth of A. brasilense on fructose. The ability of fructose to induce proteins of the Type 6 Secretion System (T6SS) enables A. brasilense to cause contact-dependent inhibition of the growth of Escherichia coli as well as A. tumefaciens. This is the first report on the fructose inducibility of T6SS in A. brasilense, which may provide a handle to control the growth of undesirable bacteria using T6SS of A. brasilense in a mixed culture.

The bacterial pathogen Vibrio coralliilyticus causes disease in coral species worldwide. The mechanisms of V. coralliilyticus coral colonization, coral microbiome interactions, and virulence factor production are understudied. In other model Vibrio species, virulence factors like biofilm formation, toxin secretion, and protease production are controlled through a density-dependent communication system called quorum sensing (QS). Comparative genomics indicated that V. coralliilyticus genomes share high sequence identity for most of the QS signaling and regulatory components identified in other Vibrio species. Here, we identify an active QS signaling pathway in two V. coralliilyticus strains with distinct infection etiologies: type strain BAA-450 and coral isolate OCN008. In V. coralliilyticus, the inter-species AI-2 autoinducer signaling pathway in both strains controls expression of the master QS transcription factor and LuxR/HapR homolog VcpR to regulate >300 genes, including protease production, biofilm formation, and two conserved type VI secretion systems (T6SSs). Activation of T6SS1 by QS results in the secretion of effectors and enables interbacterial competition and killing of prey bacteria. We conclude that the QS system in V. coralliilyticus is functional and controls the expression of genes involved in relevant bacterial behaviors typically associated with host infection.IMPORTANCEVibrio coralliilyticus infects many marine organisms, including multiple species of corals, and is a primary causative agent of tissue loss diseases and bacterial-induced bleaching. Here, we investigated a common cell-cell communication mechanism called quorum sensing, which is known to be intimately connected to virulence in other Vibrio species. Our genetic and chemical studies of V. coralliilyticus quorum sensing uncovered an active pathway that directly regulates the following key virulence factors: proteases, biofilms, and secretion systems. These findings connect bacterial signaling in communities to the infection of corals, which may lead to novel treatments and earlier diagnoses of coral diseases in reefs.

Thermoanaerobacterium thermosaccharolyticum is an anaerobic and thermophilic bacterium that has been genetically engineered for ethanol production at very high yields. However, the underlying reactions responsible for electron flow, redox equilibrium, and how they relate to ethanol production in this microbe are not fully elucidated. Therefore, we performed a series of genetic manipulations to investigate the contribution of hydrogenase genes to high ethanol yield, generating evidence for the importance of hydrogen-reacting enzymes in ethanol production. Our results indicate that a high ethanol yield, >85% of the theoretical maximum, only occurs when the hfsD, hydAB, and nfnAB genes are all present together, while the hfsB gene is absent. We propose that the products of these three gene clusters facilitate an NADPH-generating reaction via hydrogen cycling, with a stoichiometry comparable with a canonical ferredoxin:NADP⁺ oxidoreductase (FNOR; EC 1.18.1.2) reaction. The hypothesized mechanism provides a balance of nicotinamide cofactors and facilitates ferredoxin recycling, leading to progress in optimizing the energy conversion of biomass-derived sugars to ethanol.

Importance: Our study elucidates the crucial role of electron flow and redox balancing mechanisms in improving ethanol yields from renewable biomass. We delve into the mechanism of electron transfer, highlighting the potential of key genes to be leveraged for enhanced ethanol production in anaerobic microbial species. We suggest by genetic investigation the existence of a novel Ferredoxin:NADP+ Oxidoreductase (FNOR) reaction, comprising HfsD, HydAB, and NfnAB enzymes, as a promising avenue for achieving balanced stoichiometry and efficient ethanol synthesis. Our findings not only advance the understanding of microbial metabolism but also offer practical insights for developing strategies to improve bioenergy production and sustainability.

Leptospirosis is a zoonotic disease caused by Leptospira bacteria, affecting humans and a broad range of wild and domestic animals in diverse epidemiological settings (rural, urban, and wild). The disease's pathogenesis and epidemiology are complex networks not fully elucidated. Epidemiology reflects the One Health integrated approach of environment-animal-human interaction, causing severe illness in humans and animals, with consequent public health burdens. Saprophytic and pathogenic leptospires have been shown to form biofilms in vivo, in vitro, and in environmental samples. Biofilms are characterized by a polymeric matrix that confers protection against hostile environments (both inside and outside of the host), favoring bacterial survival and dissemination. Despite its significance, the role of this bacterial growth mode in leptospiral survival, transmission, and decreased antibiotic susceptibility remains poorly understood and underexplored. Even so, the literature indicates that biofilms might be correlated with lower antimicrobial susceptibility and chronicity in leptospirosis. In this minireview, we discuss the aspects of biofilm formation by Leptospira and their significance for epidemiology and therapeutic management. Understanding the current scenario provides insight into the future prospects for biofilm diagnosis, prevention, and treatment of leptospirosis.

Rhizobia are soil bacteria capable of establishing symbiosis within legume root nodules, where they reduce atmospheric N₂ into ammonia and supply it to the plant for growth. Australian soils often lack rhizobia compatible with introduced agricultural legumes, so inoculation with exotic strains has become a common practice for over 50 years. While extensive research has assessed the N₂-fixing capabilities of these inoculants, their genomics, taxonomy, and core and accessory gene phylogeny are poorly characterized. Furthermore, in some cases, inoculant strains have been developed from isolations made in Australia. It is unknown whether these strains represent naturalized exotic organisms, native rhizobia with a capacity to nodulate introduced legumes, or recombinant strains arising from horizontal transfer between introduced and native bacteria. Here, we describe the complete, closed genome sequences of 42 Australian commercial rhizobia. These strains span the genera, Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium, and Sinorhizobium, and only 23 strains were identified to species level. Within inoculant strain genomes, replicon structure and location of symbiosis genes were consistent with those of model strains for each genus, except for Rhizobium sp. SRDI969, where the symbiosis genes are chromosomally encoded. Genomic analysis of the strains isolated from Australia showed they were related to exotic strains, suggesting that they may have colonized Australian soils following undocumented introductions. These genome sequences provide the basis for accurate strain identification to manage inoculation and identify the prevalence and impact of horizontal gene transfer (HGT) on legume productivity.

Importance: Inoculation of cultivated legumes with exotic rhizobia is integral to Australian agriculture in soils lacking compatible rhizobia. The Australian inoculant program supplies phenotypically characterized high-performing strains for farmers but in most cases, little is known about the genomes of these rhizobia. Horizontal gene transfer (HGT) of symbiosis genes from inoculant strains to native non-symbiotic rhizobia frequently occurs in Australian soils and can impact the long-term stability and efficacy of legume inoculation. Here, we present the analysis of reference-quality genomes for 42 Australian commercial rhizobial inoculants. We verify and classify the genetics, genome architecture, and taxonomy of these organisms. Importantly, these genome sequences will facilitate the accurate strain identification and monitoring of inoculants in soils and plant nodules, as well as enable detection of horizontal gene transfer to native rhizobia, thus ensuring the efficacy and integrity of Australia's legume inoculation program.

Tuber japonicum, a white-colored truffle that is endemic to Japan, is promising for culinary purposes due to its unique aroma. We were able to cultivate T. japonicum in plantations of inoculated Quercus serrata seedlings for the first time. Ascocarps were found after 43 months at one site and after 61 months at another. We developed simple sequence repeat markers for multilocus genotyping of glebal tissue and ascospores and confirmed that the harvested ascocarps were derived from inocula. All paternal individuals matched the multilocus genotypes of neighboring maternal individuals, indicating frequent hermaphroditism and the absence of externally introduced individuals. Our findings provide important clues to understanding the reproductive biology of T. japonicum during the early period after the truffle plantation establishment.

Importance: Truffles are highly prized as a delicacy, but only a select few species have been successfully cultivated. In our study, we succeeded for the first time in cultivating Tuber japonicum. Two out of four plantations produced ascocarps shortly after planting, with one of them yielding a comparable weight to other cultivated truffle species. This promising productivity suggests that the fungus has potential when cultivated. Our analysis of the ascocarps' maternal and paternal genotypes, using simple sequence repeat markers, revealed hermaphroditic behavior in the fungus at our planting site. Our findings provide crucial insights into the truffle mating events.