Microbial Physiology最新文献

Background: For many years, scientists have accepted Darwin's conclusion that "Survival of the Fittest" involves successful competition with other organisms for life-endowing molecules and conditions.

Summary: Newly discovered "partial" organisms with minimal genomes that require symbiotic or parasitic relationships for growth and reproduction suggest that cooperation in addition to competition was and still is a primary driving force for survival. These two phenomena are not mutually exclusive, and both can confer a competitive advantage for survival. In fact, cooperation may have been more important in the early evolution for life on Earth before autonomous organisms developed, becoming large genome organisms.

Key messages: This suggestion has tremendous consequences with respect to our conception of the early evolution of life on Earth as well as the appearance of intercellular interactions, multicellularity and the nature of interactions between humans and their societies (e.g., Social Darwinism).

Introduction: The low yield of bioflocculants has been a bottleneck problem that limits their industrial applications. Understanding the metabolic mechanism of bacteria that produce bioflocculants could provide valuable insights and strategies to directly regulate their yield in future.

Methods: To investigate the change of metabolites in the process of bioflocculant production by a biomass-degrading bacterium, Pseudomonas boreopolis GO2, an untargeted metabolome analysis was performed.

Results: The results showed that metabolites significantly differed during the fermentation process when corn stover was used as the sole carbon source. The differential metabolites were divided into four co-expression modules based on the weighted gene co-expression network analysis. Among them, a module (yellow module) was closely related to the flocculating efficiency, and the metabolites in this module were mainly involved in carbohydrate, lipid, and amino acid metabolism. The top 30 metabolites with the highest degree in the yellow module were identified as hub metabolites for bioflocculant production. Finally, 10 hub metabolites were selected to perform the additional experiments, and the addition of L-rhamnose, tyramine, tryptophan, and glutaric acid alone all could significantly improve the flocculating efficiency of GO2 strain.

Conclusion: These results indicated that the hub metabolites were key for bioflocculant production in GO2 strain, and could help guide the improvement of high-efficiency and low-cost bioflocculant production.

Background: Dinoflagellates are a monophyletic group within the taxon Alveolata, which comprises unicellular eukaryotes. Dinoflagellates have long been studied for their organismic and morphologic diversity as well as striking cellular features. They have a main size range of 10-100 µm, a complex "cell covering", exceptionally large genomes (∼1-250 Gbp with a mean of 50,000 protein-encoding genes) spread over a variable number of highly condensed chromosomes, and perform a closed mitosis with extranuclear spindles (dinomitosis). Photosynthetic, marine, and free-living Prorocentrum cordatum is a ubiquitously occurring, bloom-forming dinoflagellate, and an emerging model system, particularly with respect to systems biology.

Summary: Focused ion beam/scanning electron microscopy (FIB/SEM) analysis of P. cordatum recently revealed (i) a flattened nucleus with unusual structural features and a total of 62 tightly packed chromosomes, (ii) a single, barrel-shaped chloroplast devoid of grana and harboring multiple starch granules, (iii) a single, highly reticular mitochondrion, and (iv) multiple phosphate and lipid storage bodies. Comprehensive proteomics of subcellular fractions suggested (i) major basic nuclear proteins to participate in chromosome condensation, (ii) composition of nuclear pores to differ from standard knowledge, (iii) photosystems I and II, chloroplast complex I, and chlorophyll a-b binding light-harvesting complex to form a large megacomplex (>1.5 MDa), and (iv) an extraordinary richness in pigment-binding proteins. Systems biology-level investigation of heat stress response demonstrated a concerted down-regulation of CO2-concentrating mechanisms, CO2-fixation, central metabolism, and monomer biosynthesis, which agrees with reduced growth yields.

Key messages: FIB/SEM analysis revealed new insights into the remarkable subcellular architecture of P. cordatum, complemented by proteogenomic unraveling of novel nuclear structures and a photosynthetic megacomplex. These recent findings are put in the wider context of current understanding of dinoflagellates.

The denitrifying betaproteobacterium Aromatoleum aromaticum EbN1T is a facultative anaerobic degradation specialist and belongs to the environmental bacteria studied best on the proteogenomic level. This review summarizes the current state of knowledge about the anaerobic and aerobic degradation (to CO2) of 47 organic growth substrates (23 aromatic, 21 aliphatic, and 3 amino acids) as well as the modes of respiratory energy conservation (denitrification vs. O2-respiration). The constructed catabolic network is comprised of 256 genes, which occupy ∼7.5% of the coding regions of the genome. In total, 219 encoded proteins have been identified by differential proteomics, yielding a proteome coverage of ∼74% of the network. Its degradation section is composed of 31 peripheral and 4 central pathways, with several peripheral modules (e.g., for 4-ethylphenol, 2-phenylethylamine, indoleacetate, and phenylpropanoids) discovered only after the complete genome [Arch Microbiol. 2005 Jan;183(1):27-36] and a first proteomic survey [Proteomics. 2007 Jun;7(13):2222-39] of A. aromaticum EbN1T were reported. The activation of recalcitrant aromatic compounds involves a suite of biochemically intriguing reactions ranging from C-H-bond activation (e.g., ethylbenzene dehydrogenase) via carboxylation (e.g., acetophenone carboxylase) to oxidative deamination (e.g., benzylamine), reductive dearomatization (benzoyl-CoA), and epoxide-forming oxygenases (e.g., phenylacetyl-CoA). The peripheral reaction sequences are substrate-specifically induced, mediated by specific transcriptional regulators with in vivo response thresholds in the nanomolar range. While lipophilic substrates (e.g., phenolics) enter the cells via passive diffusion, polar ones require active uptake that is driven by specific transporters. Next to the protein repertoire for canonical complexes I-III, denitrification, and O2-respiration (low- and high-affinity oxidases), the genome encodes an Ndh-II, a tetrathionate reductase, two ETF:quinone oxidoreductases, and two Rnf-type complexes, broadening the electron transfer flexibility of the strain. Taken together, the detailed catabolic network presented here forms a solid basis for future systems biology-level studies with A. aromaticum EbN1T.

Introduction: Aflatoxin B1 (AFB1) is a potent hepatocarcinogenic mycotoxin found in animal feed and human food components. AFB1 contamination poses severe food safety and economic consequences.

Methods: In this study, we used a coumarin-selective medium to isolate bacterial strains that can remove AFB1. Among the isolated bacterial strains, strain c4a exhibited the highest AFB1 removal activity. This strain was subjected to biochemical and phylogenetic characterization. The AFB1 removal activity of the extracellular supernatant of this strain was optimized for growth medium, reaction temperature, pH, and metal ions. The degradation products were analyzed using UPLC-ESI MS/MS.

Results: Strain c4a was found to be most closely related to Chryseobacterium timonianum. The extracellular supernatant of C. timonianum c4a grown in a modified nutrient broth (with gelatin peptone and beef extract in a 4:1 ratio) demonstrated the highest AFB1 removal activity when incubated with 1 ppm AFB1 at 60°C, pH 8, and Mn2+ or Mg2+ supplementation for 72 h. Surprisingly, the autoclaved extracellular supernatant also retained AFB1 removal activity. UPLC-ESI MS/MS analysis suggested that AFB1 was transformed into a metabolite (m/z value 285.08) by water molecule addition on furan ring double bond.

Conclusion: The AFB1 removal activity of C. timonianum c4a was extracellular, constitutive, and highly thermostable, structurally transforming AFB1 into a much less toxic product. Herein, we present the first evidence of thermostable AFB1 removal activity of a strain belonging to C. timonianum.

Introduction: Lower respiratory tract infections, including COVID-19, have a substantial global impact, making the development of diagnostic tests crucial. The study aimed to develop a new, accurate, fast, and cost-effective PCR-based detection method for SARS-CoV-2, applicable in limited settings and capable of detecting all current variants and potential future pathogens.

Methods: The study was conducted between 2020 and 2022 at the molecular biology department of Mures County Clinical Hospital (MCCH), Romania. Initially, pharyngeal and nasal secretions were collected and processed using the real-time qRT-PCR method for routine COVID-19 diagnosis. Ninety-two samples were randomly selected to develop the assay, including samples from different infection periods and negative controls. Complementary DNA was prepared from the selected samples, and the presence and integrity of the extracted RNA were evaluated by amplifying the GAPDH housekeeping gene. Primers for three specific viral genes (N, ORF1ab, and S) were designed, and their efficiency was evaluated using endpoint PCR and sequencing. Finally, the method was optimized and implemented as a one-step triplex PCR assay for routine diagnostic use.

Results: The molecular laboratory at the MCCH analyzed a total of 41,818 samples between June 2020 and December 2022. Among these samples, 26.15% tested positive for SARS-CoV-2, while 70.9% were negative and 2.95% were inconclusive or invalid. Three peaks of positive tests were observed in November 2020, April 2021, and February 2022. The study selected 92 preserved RNA samples for triplex PCR assay development, validating the primers' specificity and confirming the quality of the nucleic acids. The comparative analysis showed the efficiency and accuracy of the endpoint reverse transcription triplex PCR method (RT-PCR), indicating its potential as a cost-effective alternative to real-time reverse transcription PCR (qRT-PCR) in low-income countries with limited infrastructure for COVID-19 testing.

Conclusion: This method has the potential to facilitate large-scale diagnosis of SARS-CoV-2 infections, allowing for rapid and appropriate therapeutic management and ongoing monitoring of patients. Additionally, the method can be easily adapted for the detection of other pathogens.

Introduction: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated.

Methods: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system.

Results: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels.

Conclusion: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Introduction: The global poultry industry produces millions of tons of waste feathers every year, which can be bio-degraded to make feed, fertilizer, and daily chemicals. However, feather bio-degradation is a complex process that is not yet fully understood. This results in low degradation efficiency and difficulty in industrial applications. Omics-driven system biology research offers an effective solution to quickly and comprehensively understand the molecularmechanisms involved in a metabolic pathway.

Methods: In the early stage of this process, feathers are hydrolyzed into water-soluble keratin monomers. In this study, we used high-throughput RNA-seq technology to analyze the genes involved in the internalization and degradation of keratin monomers in Stenotrophomonas maltophilia DHHJ strain cells. Moreover, we used Co-IP with LC-MS/MS technology to search for proteins that interact with recombinant keratin monomers.

Results: We discovered TonB transports and molecular chaperones associating with the keratin monomer, which may play a crucial role in the transmembrane transport of keratin. Meanwhile, multiple proteases belonging to distinct families were identified as binding partners of keratin monomers, among which ATPases associated with diverse cellular activity (AAA+) family proteases are overrepresented. Four genes, including JJL50_15620, JJL50_17955 (TonB-dependent receptors), JJL50_03260 (ABC transporter ATP-binding protein), and JJL50_20035 (ABC transporter substrate-binding protein), were selected as representatives for determining their expressions under different culture conditions using qRT-PCR, and they were found to be upregulated in response to keratin degradation consistent with the data from RNA-seq and Co-IP.

Conclusion: This study highlights the complexity of keratin biodegradation in S. maltophilia DHHJ, in which multiple pathways are involved such as protein folding, protein transport, and several protease systems. Our findings provide new insights into the mechanism of feather degradation.

We propose that intermittent fasting (time-restricted eating), in agreement with the conclusions of other biologists, as revealed in recent publications, promotes the achievement of numerous health benefits including the extension of human and animal lifespans. Background: There is evidence, obtained both with animal model systems and with humans, that intermittent fasting has health benefits. These benefits include extended longevity, weight loss, and counteracting various disease conditions. Such procedures positively influence the benefits of human tissue-specific microbiomes and minimize the consequences of organellar apoptosis. Key Messages: In this review, we attempt to summarize the predominant evidence, published in the scientific literature, relevant to the conclusions that in general, and in many specific instances, intermittent fasting has long-term benefits to animals, including humans, with respect to overall and specific organismal health and longevity.