A novel calcium-concentrating compartment drives biofilm formation and persistent infections

AbstractBacterial biofilms produce a robust internal mineral layer, composed of calcite, which strengthens the colony and protects the residing bacteria from antibiotics. In this work, we provide evidence that the assembly of a functional mineralized macro-structure begins with mineral precipitation within a defined cellular compartment in a differentiated subpopulation of cells. Transcriptomic analysis of a model organism, Bacillus subtilis, revealed that calcium was essential for activation of the biofilm state, and highlighted the role of cellular metal homeostasis and carbon metabolism in biomineralization. The molecular mechanisms promoting calcite formation were conserved in pathogenic Pseudomonas aeruginosa biofilms, resulting in formation of calcite crystals tightly associated with bacterial cells in sputum samples collected from cystic fibrosis patients. Biomineralization inhibitors targeting calcium uptake and carbonate accumulation significantly reduced the damage inflicted by P. aeruginosa biofilms to lung tissues. Therefore, better understanding of the conserved molecular mechanisms promoting biofilm calcification can path the way to the development of novel classes of antibiotics to combat otherwise untreatable biofilm infections.

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GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri

Scientific Reports ◽

10.1038/s41598-021-95113-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Simone Cristina Picchi ◽

Mariana de Souza e Silva ◽

Luiz Leonardo Saldanha ◽

Henrique Ferreira ◽

Marco Aurélio Takita ◽

...

Keyword(s):

Amino Acids ◽

Cell Proliferation ◽

Plant Pathogen ◽

Model Organism ◽

Citrus Canker ◽

Bacterial Cells ◽

Xanthomonas Citri ◽

Canker Disease ◽

Potential Use

AbstractN-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC–MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.

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The role of FLOWERING LOCUS C in vernalization of Brassica: the importance of vernalization research in the face of climate change

Crop and Pasture Science ◽

10.1071/cp16468 ◽

2018 ◽

Vol 69 (1) ◽

pp. 30 ◽

Cited By ~ 18

Author(s):

Daniel J. Shea ◽

Etsuko Itabashi ◽

Satoko Takada ◽

Eigo Fukai ◽

Tomohiro Kakizaki ◽

...

Keyword(s):

Molecular Mechanisms ◽

Model Organism ◽

Climatic Changes ◽

Environmental Cues ◽

Plant Responses ◽

Agricultural Crops ◽

Regulatory Pathways ◽

Evolutionarily Conserved ◽

The Face

As climatic changes occur over the coming decades, our scientific understanding of plant responses to environmental cues will become an increasingly important consideration in the breeding of agricultural crops. This review provides a summary of the literature regarding vernalization research in Brassicaceae, covering both the historical origins of vernalization research and current understanding of the molecular mechanisms behind the regulatory pathways involved in vernalization and subsequent inflorescence. We discuss the evolutionarily conserved biology between the model organism Arabidopsis thaliana and the Brassica genus of crop cultivars and contrast the differences between the genera to illustrate the importance of Brassica-specific research into vernalization.

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The dgc2 gene encoding di-guanylate cyclase suppresses both motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana.

10.1101/2022.01.10.475764 ◽

2022 ◽

Author(s):

Kazuma Toida ◽

Wakana Kushida ◽

Hiroki Yamamoto ◽

Kyoka Yamamoto ◽

Kazuma Uesaka ◽

...

Keyword(s):

Mutant Strain ◽

Biofilm Formation ◽

Catalytic Domain ◽

Genetic Model ◽

Model Organism ◽

Gliding Motility ◽

Spontaneous Mutant ◽

Filamentous Cyanobacterium ◽

Wild Type

Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is the filamentous cyanobacterial species, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen-fixation. Although a widely used type strain (wild type) of this species has not been reported to show any motile activity, we isolated a spontaneous mutant strain which shows active motility (gliding activity) to give rise to complicated colony patters, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations on the genome in the mutant strain. We confirmed that inactivation of a candidate gene, dgc2 (LBDG_02920), in the wild type background was sufficient to give rise to motility and the morphological colony patterns. This gene encodes a protein, containing the GGDEF motif, which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the mutant strain lacking dgc2 significantly facilitated biofilm formation, suggesting a role of DGC for suppressing both gliding motility and biofilm formation. Thus, L. boryana provides an excellent genetic model to study dynamic colony pattern formation, and novel insight on a role of c-di-GMP for biofilm formation.

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Human Cytomegalovirus miR-UL70-3p Downregulates the H2O2-Induced Apoptosis by Targeting the Modulator of Apoptosis-1 (MOAP1)

International Journal of Molecular Sciences ◽

10.3390/ijms23010018 ◽

2021 ◽

Vol 23 (1) ◽

pp. 18

Author(s):

Abhishek Pandeya ◽

Raj Kumar Khalko ◽

Anup Mishra ◽

Nishant Singh ◽

Sukhveer Singh ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Molecular Mechanisms ◽

Ectopic Expression ◽

Antiviral Responses ◽

Persistent Infections ◽

Innate Immune Mechanism ◽

Infected Cells ◽

Hcmv Replication ◽

Induced Apoptosis

Human Cytomegalovirus (HCMV) is a prototypic beta herpesvirus, causing persistent infections in humans. There are medications that are used to treat the symptoms; however, there is no cure yet. Thus, understanding the molecular mechanisms of HCMV replication and its persistence may reveal new prevention strategies. HCMV evasive strategies on the antiviral responses of the human host largely rely on its significant portion of genome. Numerous studies have highlighted the importance of miRNA-mediated regulation of apoptosis, which is an innate immune mechanism that eradicates virus-infected cells. In this study, we explore the antiapoptotic role of hcmv-miR-UL70-3p in HEK293T cells. We establish that hcmv-miR-UL70-3p targets the proapoptotic gene Modulator of Apoptosis-1 (MOAP1) through interaction with its 3’UTR region of mRNA. The ectopic expression of hcmv-miR-UL70-3p mimic significantly downregulates the H2O2-induced apoptosis through the translational repression of MOAP1. Silencing of MOAP1 through siRNA also inhibits the H2O2-induced apoptosis, which further supports the hcmv-miR-UL70-3p mediated antiapoptotic effect by regulating MOAP1 expression. These results uncover a role for hcmv-miR-UL70-3p and its target MOAP1 in regulating apoptosis.

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Selective Autophagy inDrosophila

International Journal of Cell Biology ◽

10.1155/2012/146767 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 14

Author(s):

Ioannis P. Nezis

Keyword(s):

Cellular Response ◽

Molecular Mechanisms ◽

Current Knowledge ◽

Model Organism ◽

Major Pathway ◽

Selective Autophagy ◽

Cytoplasmic Material ◽

Evolutionarily Conserved ◽

High Conservation

Autophagy is an evolutionarily conserved process of cellular self-eating and is a major pathway for degradation of cytoplasmic material by the lysosomal machinery. Autophagy functions as a cellular response in nutrient starvation, but it is also associated with the removal of protein aggregates and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Although it was initially believed that autophagy occurs randomly in the cell, during the last years, there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective. Given the important role of autophagy and selective autophagy in several disease-related processes such as neurodegeneration, infections, and tumorigenesis, it is important to understand the molecular mechanisms of selective autophagy, especially at the organismal level.Drosophilais an excellent genetically modifiable model organism exhibiting high conservation in the autophagic machinery. However, the regulation and mechanisms of selective autophagy inDrosophilahave been largely unexplored. In this paper, I will present an overview of the current knowledge about selective autophagy inDrosophila.

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Mechanism of Attenuation of Uranyl Toxicity by Glutathione in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.00538-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3563-3571 ◽

Cited By ~ 6

Author(s):

Muhammad H. Obeid ◽

Jana Oertel ◽

Marc Solioz ◽

Karim Fahmy

Keyword(s):

Heavy Metals ◽

Lactococcus Lactis ◽

Metal Toxicity ◽

Molecular Mechanisms ◽

Model Organism ◽

Cell Number ◽

Metal Resistance ◽

Titration Calorimetry ◽

Content Type

ABSTRACTBoth prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grownLactococcus lactiscontaining an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 μM. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions.IMPORTANCEUnderstanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.

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Quantitative mapping of mRNA 3’ ends in Pseudomonas aeruginosa reveals putative riboregulators and a pervasive role for transcription termination in response to azithromycin

10.1101/2020.06.16.155515 ◽

2020 ◽

Author(s):

Salini Konikkat ◽

Michelle R. Scribner ◽

Rory Eutsey ◽

N. Luisa Hiller ◽

Vaughn S. Cooper ◽

...

Keyword(s):

Cystic Fibrosis ◽

Stress Responses ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Transcription Termination ◽

Transcriptional Response ◽

Chronic Infections ◽

Biofilm Growth ◽

Persistent Infections ◽

Upregulated Genes

ABSTRACTP. aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including cystic fibrosis patients. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and QS. The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using an improved genome-wide approach to quantitate RNA 3’-ends (3pMap). We also identified hundreds of P. aeruginosa genes subject to premature transcription termination in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3’-ends indicating premature transcription termination or pausing. Reciprocally, AZM reduced premature transcription termination in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced premature transcription termination downregulates expression of central transcriptional regulators, which in turn both impairs QS and biofilm formation, and stress responses, while upregulating genes associated with persistence.

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Molecular Regulatory Network Involved in Biofilm Structure Development by Acidithiobacillus thiooxidans Includes Pel Exopolysaccharide Machinery

Solid State Phenomena ◽

10.4028/www.scientific.net/ssp.262.330 ◽

2017 ◽

Vol 262 ◽

pp. 330-333 ◽

Cited By ~ 3

Author(s):

Mauricio Diaz ◽

Nicolas Guiliani

Keyword(s):

Gene Cluster ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Industrial Process ◽

Genomic Analysis ◽

Mutant Strains ◽

Relevant Role ◽

Biofilm Structure

The Acidithiobacillus genus plays a relevant role in bioleaching. The molecular understanding of biofilm formation has been pointed out to design biological strategies to improve the efficiency of this industrial process and to prevent environmental damages caused by acid mine/rock drainages. In Acidithiobacillus spp., the molecular mechanisms involved in biofilm formation are currently emerging. The second messenger cyclic diguanylate (c-di-GMP) appears as a key player for biofilm formation by Acidithiobacillus sp. Here, results obtained from genomic analysis to characterize c-di-GMP pathway in At. thiooxidans are reported. Intracellular levels of c-di-GMP have been previously measured and data indicated that they are higher in adhered cells than planktonic ones. During the course of characterization of c-di-GMP effectors, a complete pel-like gene cluster has been identified in At. thiooxidans. By using total RNA obtained from planktonic and adhered sulfur-grown cells, transcriptomic analysis revealed that pelA belonging to the pel-like gene cluster is overexpressed in adhered cells. Moreover, genetic experiments were performed to compare wild type and null-mutant strains of At. thiooxidans for assessing the role of Pel exopolysaccharide. All together, the results obtained suggest a specific role for Pel machinery in the attachment to solid energy substrates by At. thiooxidans.

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Responses of Microorganisms to Osmotic Stress

Annual Review of Microbiology ◽

10.1146/annurev-micro-020518-115504 ◽

2019 ◽

Vol 73 (1) ◽

pp. 313-334 ◽

Cited By ~ 38

Author(s):

Erhard Bremer ◽

Reinhard Krämer

Keyword(s):

Osmotic Stress ◽

Cytoplasmic Membrane ◽

Bacterial Cells ◽

Water Fluxes ◽

Cellular Compartment ◽

Secondary Messenger ◽

Water Influx ◽

Recent Developments ◽

Cellular Integrity

The cytoplasm of bacterial cells is a highly crowded cellular compartment that possesses considerable osmotic potential. As a result, and owing to the semipermeable nature of the cytoplasmic membrane and the semielastic properties of the cell wall, osmotically driven water influx will generate turgor, a hydrostatic pressure considered critical for growth and viability. Both increases and decreases in the external osmolarity inevitably trigger water fluxes across the cytoplasmic membrane, thus impinging on the degree of cellular hydration, molecular crowding, magnitude of turgor, and cellular integrity. Here, we assess mechanisms that permit the perception of osmotic stress by bacterial cells and provide an overview of the systems that allow them to genetically and physiologically cope with this ubiquitous environmental cue. We highlight recent developments implicating the secondary messenger c-di-AMP in cellular adjustment to osmotic stress and the role of osmotic forces in the life of bacteria-assembled in biofilms.

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Role of Multicellular Aggregates in Biofilm Formation

mBio ◽

10.1128/mbio.00237-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 133

Author(s):

Kasper N. Kragh ◽

Jaime B. Hutchison ◽

Gavin Melaugh ◽

Chris Rodesney ◽

Aled E. L. Roberts ◽

...

Keyword(s):

Biofilm Formation ◽

Single Cells ◽

Subsequent Development ◽

Relative Fitness ◽

Bacterial Cells ◽

Ecological Benefit ◽

Multicellular Aggregates ◽

Biofilm Development

ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. IMPORTANCE During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.

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