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Fungi are the most frequent cause of microbial spoilage in baked products, including rye bread. As the baking process destroys fungal spores in bread, the post-processing is the main source for mould contamination. Rapid detection methods are needed to track down the origin of the contamination source. In the present research we used a combined molecular approach consisting of PCR-fingerprinting with an M13 primer and further identification of each genotype by amplification and sequencing of the Internal Transcribed Spacer region, the β-tubulin gene and the D1/D2 region of the large subunit of the 28S rDNA. Different rye breads from five bakeries were stored plastic-packed for one month and the fungal colonies with unique morphology were isolated from the bread surfaces. Based on random amplified polymorphic DNA analysis using M13 primer 50 fungal isolates were clustered into eight groups and identified as Aspergillus chevalieri, Aspergillus flavus/oryzae, Aspergillus niger, Aspergillus tubingensis, Penicillium citrinum, Penicillium corylophilum, Saccharomyces cerevisiae and Wickerhamomyces anomalus species. Sequencing of the β-tubulin gene and the ITS region showed an equal efficiency for the identification of Penicillium species, whereas only the sequence of the β-tubulin gene allowed us to identify most isolates from the genus Aspergillus including closely-related black-spored Aspergillus species. Yeasts were identified at the species level based on the sequences of the Internal Transcribed Spacer region and the D1/D2 region. [ABSTRACT FROM AUTHOR]

Different aspects of bacterial degradation of organic contaminants in soil, and how to improve the efficiency and reproducibility is discussed in this review. Although bioremediation in principle includes the use of any type of organism in improving the condition of a contaminated site, most commonly bacteria are the degraders and other organisms, such as soil animals or plant roots, play a role in dissemination of bacteria and, indirectly, plasmids between bacteria, and in providing nutrients and co-substrates for the bacteria active in the degradation process. There are a number of different procedures that have been tested more-or-less successfully in attempts to improve reliability, cost efficiency and speed of bioremediation. The methods rangefrom minimal intervention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or bacterial inocula or improvement of physico-chemical conditions, all the way to excavation followed by on site or ex situ composting in its different varieties. In the past the rule has been that more intervention (leading to higher costs) has been more reliable, but novel ideas are continuously tried out, both as a means to come up with new truly functional applications and also as a line of studies in basic soil microbial ecology. Both approaches generate valuable information needed when predicting outcome of remediation activities, evaluating environmental risks, deciding on cleaning-up approaches, etc. The emphasis ofthis review is to discuss some of the novel methods for which the value has not been clearly shown, but that in our view merit continued studies and efforts to make them work, separately or in combination. [ABSTRACT FROM AUTHOR]

In recent years, microbiomes and their potential applications for human, animal or plant health, food production and environmental management came into the spotlight of major national and international policies and strategies. This has been accompanied by substantial R&D investments in both public and private sectors, with an increasing number of products entering the market. Despite widespread agreement on the potential of microbiomes and their uses across disciplines, stakeholders and countries, there is no consensus on what defines a microbiome application. This often results in non-comprehensive communication or insufficient documentation making commercialisation and acceptance of the novel products challenging. To showcase the complexity of this issue we discuss two selected, well-established applications and propose criteria defining a microbiome application and their conditions of use for clear communication, facilitating suitable regulatory frameworks and building trust among stakeholders., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Microbiomes have highly important roles for ecosystem functioning and carry out key functions that support planetary health, including nutrient cycling, climate regulation, and water filtration. Microbiomes are also intimately associated with complex multicellular organisms such as humans, other animals, plants, and insects and perform crucial roles for the health of their hosts. Although we are starting to understand that microbiomes in different systems are interconnected, there is still a poor understanding of microbiome transfer and connectivity. In this review we show how microbiomes are connected within and transferred between different habitats and discuss the functional consequences of these connections. Microbiome transfer occurs between and within abiotic (e.g., air, soil, and water) and biotic environments, and can either be mediated through different vectors (e.g., insects or food) or direct interactions. Such transfer processes may also include the transmission of pathogens or antibiotic resistance genes. However, here, we highlight the fact that microbiome transmission can have positive effects on planetary and human health, where transmitted microorganisms potentially providing novel functions may be important for the adaptation of ecosystems., Competing Interests: The authors declare no conflict of interest.

We identified the lactic acid bacteria within rye sourdoughs and starters from four bakeries with different propagation parameters and tracked their dynamics for between 5-28 months after renewal. Evaluation of bacterial communities was performed using plating, denaturing gradient gel electrophoresis, and pyrosequencing of 16S rRNA gene amplicons. Lactobacillus amylovorus and Lactobacillus frumenti or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus panis prevailed in sourdoughs propagated at higher temperature, while ambient temperature combined with a short fermentation cycle selected for Lactobacillus sanfranciscensis, Lactobacillus pontis, and Lactobacillus zymae or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus zymae. The ratio of species in bakeries employing room-temperature propagation displayed a seasonal dependence. Introduction of different and controlled propagation parameters at one bakery (higher fermentation temperature, reduced inoculum size, and extended fermentation time) resulted in stabilization of the microbial community with an increased proportion of L. helveticus and L. pontis. Despite these new propagation parameters no new species were detected.

The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1∶10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus Lactobacillus (brevis, crustorum, plantarum, and paralimentarius) were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, Kazachstania unispora (formerly Saccharomyces unisporus) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (Kazachstania unispora, Saccharomyces cerevisiae, Candida krusei and Candida glabrata) were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping.

Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.

Comparatively little is known about directed motility of environmental bacteria to common aromatic pollutants. Here, by expressing different parts of a (methyl)phenol-degradative pathway and the use of specific mutants, we show that taxis of Pseudomonas putida towards (methyl)phenols is dictated by its ability to catabolize the aromatic compound. Thus, in contrast to previously described chemoreceptor-mediated chemotaxis mechanisms towards benzoate, naphthalene and toluene, taxis in response to (methyl)phenols is mediated by metabolism-dependent behaviour. Here we show that P. putida differentially expresses three Aer-like receptors that are all polar-localized through interactions with CheA, and that inactivation of the most abundant Aer2 protein significantly decreases taxis towards phenolics. In addition, the participation of a sensory signal transduction protein composed of a PAS, a GGDEF and an EAL domain in motility towards these compounds is demonstrated. The results are discussed in the context of the versatility of metabolism-dependent coupling and the necessity for P. putida to integrate diverse metabolic signals from its native heterogeneous soil and water environments.

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues (648)RRR(650) with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

Semliki Forest virus (SFV, genus Alphavirus) has a broad host range, high efficiency of viral protein expression, and the ability to stimulate an immune response. These properties have made SFV an attractive tool for development of expression vectors, and plasmid clones containing cDNA of the SFV genome often are used. However, instability of these plasmids resulting from cryptic expression of SFV envelope proteins in Escherichia coli represents a problem both for the development of SFV-based vectors and for SFV research. In this study, an infectious plasmid of SFV, pCMV-SFV4, was constructed; its toxic effect was eliminated by intron insertion in the capsid protein encoding region. When transfected into mammalian cells, the plasmid clone was highly infectious and produced virus with properties identical to those of wild-type SFV. The inserted intron was efficiently and properly removed from the RNA genome of SFV. Therefore, this novel and stabilized infectious SFV plasmid represents a superior tool for basic studies of SFV as well as for biotechnological applications.

Semliki Forest virus (SFV, Alphavirus) induce rapid shut down of host cell protein synthesis and apoptotic death of infected vertebrate cells. Data on alphavirus-induced apoptosis are controversial. In this study, the anti-apoptotic bcl-2 gene was placed under the control of duplicated subgenomic promoter or different internal ribosome entry sites (IRES) and expressed using a novel bicistronic SFV vector. The use of IRES containing vectors resulted in high-level Bcl-2 synthesis during the early stages of infection. Nevertheless, in infected BHK-21 cells translational shutdown was almost complete by 6h post-infection, which was similar to infection with appropriate control vectors. These results indicate that very early and high-level bcl-2 expression did not have a protective effect against SFV induced shutdown of host cell translation. No apoptotic cells were detected at those time points for any SFV vectors. Furthermore, Bcl-2 expression did not protect BHK-21 or AT3-neo cells at later time points, and infection of BHK-21 or AT3-neo cells with SFV replicon vectors or with wild-type SFV4 did not lead to release of cytochrome c from mitochondria. Taken together, our data suggest that SFV induced death in BHK-21 or AT3-neo cells is not triggered by the intrinsic pathway of apoptosis.

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1-4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1-nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1-nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex.

Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the dmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.

The purpose of this study was to evaluate the influence of introduced bacteria containing a contaminant degrading plasmid on the growth and survival of pine seedlings and mycorrhizosphere microbial flora in contaminated soil. The Pseudomonas fluorescens strain OS81, originally isolated from fungal hyphae in contaminated soil, was supplied with the TOL plasmid pWW0::Km (to generate OS81(pWW0::Km)) and inoculated in humus-soil microcosms with and without pine seedlings mycorrhized with Suillus bovinus. After 3 months of regular treatment with m-toluate (mTA) solutions, the introduced catabolic plasmid was found to be disseminated in the indigenous bacterial population of both mycorrhizosphere and soil uncolonized by the fungus. Transconjugants were represented by bacteria of the genera Pseudomonas and Burkholderia and their number correlated positively with the concentration of mTA applied. Indigenous mTA degrading bacteria with low similarity to Burkholderia species were also enriched in microcosms. They were mostly associated with mycorrhizal soil or fungal structures and virtually absent in microcosms without pines. The total number of Tol(+) bacteria was higher in mycorrhizospheric soil compared with bulk soil. Inoculation with P. fluorescens OS81(pWW0::Km) had a positive effect on the development of roots and fungus in contaminated soil. Both inoculation with the P. fluorescens OS81(pWW0::Km) and mTA contamination as well as the presence of mycorrhized pine roots and fungal hyphae had an effect on the microbial community structure of soil as measured by carbon source oxidation patterns. However, the impact of mTA on the microbial community was more prominent. The study indicates that an effect on plant and fungal development can be obtained by manipulating the mycorrhizosphere. Both introduction of the bacterium carrying the degradative plasmid and the plasmid itself are likely to have a positive effect not only on the organisms involved, but also on bioremediation of contaminated soil, a factor that was not directly monitored here.