New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0-6.4% decrease), glycerol (31.4-78.5% increase), and acetic acid (53.0-60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications. KEY POINTS: •Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii •Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid •Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genes.

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

Starvation induces shrinkage of the bacterial cytoplasm.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

Heat Stress and Microbial Stress Induced Defensive Phenol Accumulation in Medicinal Plant Sparganium stoloniferum.

An approach based on the heat stress and microbial stress model of the medicinal plant Sparganium stoloniferum was proposed to elucidate the regulation and mechanism of bioactive phenol accumulation. This method integrates LC-MS/MS analysis, 16S rRNA sequencing, RT-qPCR, and molecular assays to investigate the regulation of phenolic metabolite biosynthesis in S. stoloniferum rhizome (SL) under stress. Previous research has shown that the metabolites and genes involved in phenol biosynthesis correlate to the upregulation of genes involved in plant-pathogen interactions. High-temperature and the presence of Pseudomonas bacteria were observed alongside SL growth. Under conditions of heat stress or Pseudomonas bacteria stress, both the metabolites and genes involved in phenol biosynthesis were upregulated. The regulation of phenol content and phenol biosynthesis gene expression suggests that phenol-based chemical defense of SL is stimulated under stress. Furthermore, the rapid accumulation of phenolic substances relied on the consumption of amino acids. Three defensive proteins, namely Ss4CL, SsC4H, and SsF3'5'H, were identified and verified to elucidate phenol biosynthesis in SL. Overall, this study enhances our understanding of the phenol-based chemical defense of SL, indicating that bioactive phenol substances result from SL's responses to the environment and providing new insights for growing the high-phenol-content medicinal herb SL.

Decoding the PLFA profiling of microbial community structure in soils contaminated with municipal solid wastes.

This study aimed to assess the influence of municipal solid waste (MSW) disposal on soil microbial communities. Soil samples from 20 different locations of an MSW dumping site contaminated with toxic heavy metals (HMs) and a native forest (as control) were collected for phospholipid fatty acid (PLFA) profiling to predict microbial community responses towards unsegregated disposal of MSW. PLFA biomarkers specific to arbuscular mycorrhizal fungi (AMF), Gram-negative and Gram-positive bacteria, fungi, eukaryotes, actinomycetes, anaerobes, and microbial stress markers-fungi: bacteria (F/B) ratio, Gram-positive/Gram-negative (GP/GN) ratio, Gram-negative stress (GNStr) ratio and predator/prey ratio along with AMF spore density and the total HM content (Cu, Cr, Cd, Mn, Zn, and Ni) were assessed. The results showed that all of the PLFA microbial biomarkers and the F/B ratio were positively correlated, while HMs and microbial stress markers were negatively correlated. The significant correlation of AMF biomass with all microbial groups, the F/B ratio, and T. PLFA confirmed its significance as a key predictor of microbial biomass. With AMF and T. PLFA, Cd and Cr had a weak or negative connection. Among the toxic HMs, Zn and Cd had the greatest impact on microbial populations. Vegetation did not have any significant effect on soil microbial communities. This research will aid in the development of bioinoculants for the bioremediation of MSW-polluted sites and will improve our understanding of the soil microbial community's ability to resist, recover, and adapt to toxic waste contamination.

Microbial Metal Resistance within Structured Environments Is Inversely Related to Environmental Pore Size.

The physical environments in which microorganisms naturally reside rarely have homogeneous structure, and changes in their porous architecture may have effects on microbial activities that are not typically captured in conventional laboratory studies. In this study, to investigate the influence of environmental structure on microbial responses to stress, we constructed structured environments with different pore properties (determined by X-ray computed tomography). First, using glass beads in different arrangements and inoculated with the soil yeast Saitozyma podzolica, increases in the average equivalent spherical diameters (ESD) of a structure's porous architecture led to decreased survival of the yeast under a toxic metal challenge with lead nitrate. This relationship was reproduced when yeasts were introduced into additively manufactured lattice structures, comprising regular arrays with ESDs comparable to those of the bead structures. The pore ESD dependency of metal resistance was not attributable to differences in cell density in microenvironments delimited by different pore sizes, supporting the inference that pore size specifically was the important parameter in determining survival of stress. These findings highlight the importance of the physical architecture of an organism's immediate environment for its response to environmental perturbation, while offering new tools for investigating these interactions in the laboratory. IMPORTANCE Interactions between cells and their structured environments are poorly understood but have significant implications for organismal success in both natural and nonnatural settings. This work used a multidisciplinary approach to develop laboratory models with which the influence of a key parameter of environmental structure-pore size-on cell activities can be dissected. Using these new methods in tandem with additive manufacturing, we demonstrated that resistance of yeast soil isolates to stress (from a common metal pollutant) is inversely related to pore size of their environment. This has important ramifications for understanding how microorganisms respond to stress in different environments. The findings also establish new pathways for resolving the effects of physical environment on microbial activity, enabling important understanding that is not readily attainable with traditional bulk sampling and analysis approaches.

Bioreactors based on immobilized fungi: bioremediation under non-sterile conditions.

White-rot fungi are renowned for their remarkable potential to degrade a wide range of organic pollutants. They are applicable in standard bioreactors offering both the use of the continuous mode of action and easy upscaling of the biodegradation process. The recent advance in this field consisted in the use of various fungi and different types of reactors in the treatment of real wastewaters. Most degradation studies involving white-rot fungi carried out so far used controlled, aseptic conditions. However, during bioremediation of real wastewaters, the degradation capacity of the fungi would be significantly affected by autochthonous microorganisms. Consequently, for the development of sustainable bioremediation technologies, it is important to understand the mechanisms involved in the intermicrobial interactions occurring during the bioremediation process. This review summarizes recent applications of white-rot fungi to biodegradation of recalcitrant organopollutants under non-sterile conditions describing the invading microorganism(s) and the way how they affect the stability and degradation efficiency of the fungal bioreactor cultures. In addition, studies where fungal cultures were exposed to defined microbial stress are also reported documenting the effect and mechanisms of microbial interactions. Advanced OMICs techniques, specifically the genomics and metabolomics analyses, are suggested to help in identification of the invading microorganisms and in discovery of mechanisms taking part in the interspecific interactions.

Biodegradation of clotrimazole and modification of cell properties after metabolic stress and upon addition of saponins.

Azole fungicides constitute an extensive group of potential emerging pollutants which can be found in natural environment. This study focuses on the biodegradation of clotrimazole and the characterization of cell surface properties of microorganisms capable of degradation of this compound. The influence of long-term contact of bacteria with clotrimazole and the impact of the addition of Saponaria officinalis extract on cell surface modification was also checked. The biodegradation of clotrimazole did not exceed 70%. The presence of plant extract increased biodegradation of fungicide. The cells metabolic activity after one-month exposure to clotrimazole was the highest for each tested strain. Moreover, metabolic stress led to a strong modification of cell surface properties. The results are promising for determining the impact of clotrimazole on environmental microorganisms.

Electrochemical techniques reveal that total ammonium stress increases electron flow to anode respiration in mixed-species bacterial anode biofilms.

When anode-respiring bacteria (ARB) respire electrons to an anode in microbial electrochemical cells (MXCs), they harvest only a small amount of free energy. This means that ARB must have a high substrate-oxidation rate coupled with a high ratio of electrons used for respiration compared to total electrons removed by substrate utilization. It also means that they are especially susceptible to inhibition that slows anode respiration or lowers their biomass yield. Using several electrochemical techniques, we show that a relatively high total ammonium-nitrogen (TAN) concentration (2.2 g TAN/L) induced significant stress on the ARB biofilms, lowering their true yield and forcing the ARB to boost the ratio of electrons respired per electrons consumed from the substrate. In particular, a higher respiration rate, measured as current density (j), was associated with slower growth and a lower net yield, compared to an ARB biofilm grown with a lower ammonium concentration (0.2 g TAN/L). Further increases in influent TAN (to 3 and then to 4.4 g TAN/L) caused nearly complete inhibition of anode respiration. However, the ARB could recover from high-TAN inhibition after a shift of the MXC's feed to 0.2 g TAN/L. In summary, ARB biofilms were inhibited by a high TAN concentration, but could divert more electron flow toward anode respiration with modest inhibition and recover when severe inhibition was relieved. Biotechnol. Bioeng. 2017;114: 1151-1159. © 2017 Wiley Periodicals, Inc.

Simultaneous removal of calcium, phosphorus, and bisphenol A from industrial wastewater by Stutzerimonas sp. ZW5 via microbially induced calcium precipitation (MICP): Kinetics, mechanism, and stress response.

The biological treatment of complex industrial wastewater has always been a research hotspot. In this experiment, a salt-tolerant strain Stutzerimonas sp. ZW5 with aerobic denitrification and biomineralization ability was screened, and the optimum conditions of ZW5 were explored by kinetics. The removal efficiencies of nitrate (NO3--N), bisphenol A (BPA), phosphorus (PO43--P), and calcium (Ca2+) were 94.47 %, 100 %, 98.87 %, and 83.04 %, respectively. The removal mechanism of BPA was the adsorption of microbial induced calcium precipitation (MICP) and extracellular polymeric substances (EPS). Moreover, BPA could weaken the electron transfer ability and growth metabolism of microorganisms and affect the structure of biominerals. At the same time, the stress response of microorganisms would increase the secretion of EPS to promote the process of biomineralization. Through nitrogen balance experiments, it was found that the addition of BPA would lead to a decrease in the proportion of gaseous nitrogen. This experiment offers novel perspectives on the treatment of industrial effluents and microbial stress response.

Applications of Fourier Transform-Infrared spectroscopy in microbial cell biology and environmental microbiology: advances, challenges, and future perspectives.

Microorganisms play pivotal roles in shaping ecosystems and biogeochemical cycles. Their intricate interactions involve complex biochemical processes. Fourier Transform-Infrared (FT-IR) spectroscopy is a powerful tool for monitoring these interactions, revealing microorganism composition and responses to the environment. This review explores the diversity of applications of FT-IR spectroscopy within the field of microbiology, highlighting its specific utility in microbial cell biology and environmental microbiology. It emphasizes key applications such as microbial identification, process monitoring, cell wall analysis, biofilm examination, stress response assessment, and environmental interaction investigation, showcasing the crucial role of FT-IR in advancing our understanding of microbial systems. Furthermore, we address challenges including sample complexity, data interpretation nuances, and the need for integration with complementary techniques. Future prospects for FT-IR in environmental microbiology include a wide range of transformative applications and advancements. These include the development of comprehensive and standardized FT-IR libraries for precise microbial identification, the integration of advanced analytical techniques, the adoption of high-throughput and single-cell analysis, real-time environmental monitoring using portable FT-IR systems and the incorporation of FT-IR data into ecological modeling for predictive insights into microbial responses to environmental changes. These innovative avenues promise to significantly advance our understanding of microorganisms and their complex interactions within various ecosystems.

Corrigendum: Applications of Fourier Transform-Infrared spectroscopy in microbial cell biology and environmental microbiology: advances, challenges, and future perspectives.

[This corrects the article DOI: 10.3389/fmicb.2023.1304081.].

The role of magnetite (Fe3O4) particles for enhancing the performance and granulation of anammox.

In this study, two lab-scale sequencing batch reactors each with an effective volume of 2.3 L were operated as C-AMX (no carrier addition) and M-AMX (magnetite carrier added) for 147 days with synthetic wastewater at an NLR range of 0.19-0.47 kgN/m3/d. The long-term effect of magnetite on the granulation and performance of anammox bacteria in terms of nitrogen removal and other essential parameters were confirmed. In phase I (1-24 days), M-AMX took approximately 12 days to obtain a nitrogen removal rate (NRR) above 80 % of the initial input nitrogen. Although free nitrous acid inhibited the reactor at a high concentration at the onset of phase III, the NRR of M-AMX recovered about 3.7 times faster than that of C-AMX. In addition, it was confirmed that the M-AMX granules had a dense and compact structure compared to C-AMX, and the presence of the carrier promoted the development of these resilient granules. While the measured microbial stress gradually increased in C-AMX reactor, a vice versa was observed in the M-AMX reactor as granulation proceeded. Compared to other alternative iron-based carrier particles, the stable crystal structure of magnetite as a carrier created a mechanism where filamentous bacteria groups were repelled from the granulation hence the microbial stress in the M-AMX in the final phase was 61.54 % lower than that in the C-AMX. The iron rich environment created by the magnetite addition led to Ignavibacteria, (a Feammox bacteria) increasing significantly in the M-AMX bioreactor.

Molecular characterization of a heat shock protein 21 (Hsp21) from red swamp crayfish, Procambarus clarkii in response to immune stimulation.

Small heat shock proteins are a molecular chaperone and implicated in various physiological and stress processes in animals. However, the immunological functions of Hsp genes remain to elucidate in the crustaceans, particularly in red swamp crayfish, Procambarus clarkii. Here we report the cloning of heat shock protein 21 from the P. clarkii (hereafter Pc-Hsp21). The open reading frame of Pc-Hsp21 was 555 base pairs, encoding a protein of 184 amino acid residues with an alpha-crystallin family domain. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive transcript expression of Pc-Hsp21 in the tested tissue, with the highest in hepatopancreas. The transcript abundance for this gene enhanced in hepatopancreas following immune challenge with the lipopolysaccharide, peptidoglycan, and poly I:C compared to the control group. The depletion of Pc-Hsp21 by double-stranded RNA altered transcript expression profiles of several genes in hepatopancreas, genes involved in the crucial immunological pathways of P. clarkii. These results suggest that Pc-Hsp21 plays an essential biological role in the microbial stress response by modulating the expression of immune-related genes in P. clarkii.

Activation of prophenoloxidase and hyperglycemia as indicators of microbial stress in the blue swimmer crab Portunus pelagicus.

Portunus pelagicus is exposed to different kinds of microorganisms leading to high metabolic stress that affects its life. The present study evaluates the activity of Phenoloxidase (PO), which is an enzyme that is actively involved in the activation of the immune defense system and hyperglycemia in P. pelagicus challenged with Escherichia coli and Vibrio harveyi injections. The results revealed a major impact of microbial injection on PO activity and significant variations in hemolymph glucose and CHH levels. Reduction of glucose level was observed after 24 h microbial incubation (275.26 ± 28.85 and 175.23 ± 21.70 µg/ml in V. harveyi and E. coli injected crabs, respectively). An elevated level of CHH (13.54 ± 0.55 fmol/ml) was observed in V. harveyi-injected crabs, and increased PO activity was recorded in E. coli-injected crabs. The results of the present study indicate that microbial stress leads to the activation of the defense system and hyperglycemia in P. pelagicus.

The Activation of Mucosal-Associated Invariant T (MAIT) Cells Is Affected by Microbial Diversity and Riboflavin Utilization in vitro.

Recent research has demonstrated that MAIT cells are activated by individual bacterial or yeasts species that possess the riboflavin biosynthesis pathway. However, little is known about the MAIT cell activating potential of microbial communities and the contribution of individual community members. Here, we analyze the MAIT cell activating potential of a human intestinal model community (SIHUMIx) as well as intestinal microbiota after bioreactor cultivation. We determined the contribution of individual SIHUMIx community members to the MAIT cell activating potential and investigated whether microbial stress can influence their MAIT cell activating potential. The MAIT cell activating potential of SIHUMIx was directly related to the relative species abundances in the community. We therefore suggest an additive relationship between the species abundances and their MAIT cell activating potential. In diverse microbial communities, we found that a low MAIT cell activating potential was associated with high microbial diversity and a high level of riboflavin demand and vice versa. We suggest that microbial diversity might affect MAIT cell activation via riboflavin utilization within the community. Microbial acid stress significantly reduced the MAIT cell activating potential of SIHUMIx by impairing riboflavin availability through increasing the riboflavin demand. We show that MAIT cells can perceive microbial stress due to changes in riboflavin utilization and that riboflavin availability might also play a central role for the MAIT cell activating potential of diverse microbiota.

Microbial stress meeting: From systems to molecules and back.

The 4th Microbial Stress Meeting: from Systems to Moleculesand Back was held in April 2018 in Kinsale, Ireland. The meeting covered five main topics: 1. Stress at the systems and structural level; 2. Responses to osmotic and acid stress; 3. Stress responses in single cells; 4. Stress in host-pathogen interactions; and 5. Biotechnological optimisation of microorganisms through engineering and evolution, over three days. Almost 130 delegates, from 24 countries and both the industrial and academic sectors, attended the meeting, presenting 9 lectures, 28 short talks and 52 posters. The meeting showcased the diverse and rapid advancements in microbial stress research, from the single cell level to mixed populations. In this report, a summary of the highlights from the meeting is presented.

Taking control over microbial populations: Current approaches for exploiting biological noise in bioprocesses.

Phenotypic plasticity of microbial cells has attracted much attention and several research efforts have been dedicated to the description of methods aiming at characterizing phenotypic heterogeneity and its impact on microbial populations. However, different approaches have also been suggested in order to take benefit from noise in a bioprocess perspective, e.g. by increasing the robustness or productivity of a microbial population. This review is dedicated to outline these controlling methods. A common issue, that has still to be addressed, is the experimental identification and the mathematical expression of noise. Indeed, the effective interfacing of microbial physiology with external parameters that can be used for controlling physiology depends on the acquisition of reliable signals. Latest technologies, like single cell microfluidics and advanced flow cytometric approaches, enable linking physiology, noise, heterogeneity in productive microbes with environmental cues and hence allow correctly mapping and predicting biological behavior via mathematical representations. However, like in the field of electronics, signals are perpetually subjected to noise. If appropriately interpreted, this noise can give an additional insight into the behavior of the individual cells within a microbial population of interest. This review focuses on recent progress made at describing, treating and exploiting biological noise in the context of microbial populations used in various bioprocess applications.

Noisy Response to Antibiotic Stress Predicts Subsequent Single-Cell Survival in an Acidic Environment.

Antibiotics elicit drastic changes in microbial gene expression, including the induction of stress response genes. While certain stress responses are known to "cross-protect" bacteria from other stressors, it is unclear whether cellular responses to antibiotics have a similar protective role. By measuring the genome-wide transcriptional response dynamics of Escherichia coli to four antibiotics, we found that trimethoprim induces a rapid acid stress response that protects bacteria from subsequent exposure to acid. Combining microfluidics with time-lapse imaging to monitor survival and acid stress response in single cells revealed that the noisy expression of the acid resistance operon gadBC correlates with single-cell survival. Cells with higher gadBC expression following trimethoprim maintain higher intracellular pH and survive the acid stress longer. The seemingly random single-cell survival under acid stress can therefore be predicted from gadBC expression and rationalized in terms of GadB/C molecular function. Overall, we provide a roadmap for identifying the molecular mechanisms of single-cell cross-protection between antibiotics and other stressors.