Multiple roles for iron in microbial physiology: Bacterial oxygen sensing by heme-based sensors.

After completing this article, readers should be able to: 1. Describe the role of iron in proteins involved in neuronal and glial energy metabolism, myelin production, and monoamine neurotransmitter metabolism. 2. Indicate the neonates who are at risk for iron deficiency. 3. Explain how iron deficiency affects the biochemistry, structure, signaling molecules, and electrophysiology of the hippocampus. 4. Describe the primary causes of iron overload in the neonate. 5. Explain why the preterm infant is at particular risk for iron deficiency. This article is designed to acquaint readers with the role of iron in early brain development. Several important principles that govern the effects of nutrients on the immature brain are discussed, followed by an explanation of the roles of iron during development. Because the developing brain is sensitive to iron overload as well as iron deficiency, both are discussed in the context of human studies and in model systems. The first guiding principle of nutrient-brain interactions during development is the concept of timing, dose, and duration. (1) The positive or negative effects of any nutrient depend on which brain regions or processes are developing at the time that nutrient status is altered and the requirements of those regions for the nutrient during that time of development. Generally, the more rapidly an area is developing, the more vulnerable it is. Dependence on specific nutrients during phases of rapid proliferation or differentiation results in the second principle, a regionalization of brain effects during periods of nutrient deficiency. In the case of iron deficiency, certain brain areas that are dependent on iron for proper development are more vulnerable than areas that are less dependent on iron. Additionally, these same brain areas are likely to be more vulnerable at certain time points than at others. To exert a maximal effect on regional brain development, the timing of iron …

Background: Listeria monocytogenes is a food-borne pathogen with strong environmental adaptability. It can survive at high temperatures and in acidic high-salt and other unfavorable stressful environments and consequently form a biofilm. Objectives: This study aimed to explore the roles of lmo2672 on the environmental adaptability, biofilm formation, and motility of L. monocytogenes. Methods: We analyzed the molecular characteristics of lmo2672 protein, constructed a lmo2672 gene deletion strain of L. monocytogenes strain (L. monocytogenes-Δlmo2672), using the homologous recombination technique and compared the environmental adaptability, biofilm formation, and motility of L. monocytogenes-Δlmo2672 with its parental strain L. monocytogenes EGD-e. The impacts of lmo2672 gene deficiency on the transcription of genes associated with flagella formation and the environmental adaptability of L. monocytogenes were also determined. Results: The results showed that the lmo2672 contains two helix-turn-helix (HTH) DNA-binding domains. Compared to L. monocytogenes EGD-e, L. monocytogenes-Δlmo2672 grew significantly slower at different temperatures (37°C and 42°C) under different osmotic pressures (5% and 8% NaCl) and in different media containing 0.3% bile salts, 5 mM H2O2, or 1% Triton X-100 (P < 0.05). Moreover, LM-Δlmo2672 showed a significant decrease in biofilm formation (P < 0.01) and motility (P < 0.05) at 48 h of culture. Furthermore, the transcriptional levels of regulatory gene prfA and flagella-related genes motA, fliP and fliE significantly decreased in LM-Δlmo2672 (P < 0.05). Conclusions: In general, these findings indicated that lmo2672 played critical regulatory roles in the environmental adaptability, biofilm formation, and motility of L. monocytogenes, thus providing new insights into the regulatory mechanism of lmo2672 in L. monocytogenes.

The oxygenation of the atmosphere - one of the most fundamental transformations in Earth's history - dramatically altered the chemical composition of the oceans and provides a compelling example of how life can reshape planetary surface environments. Furthermore, it is commonly proposed that surface oxygen levels played a key role in controlling the timing and tempo of the origin and early diversification of animals. Although oxygen levels were likely more dynamic than previously imagined, we make a case here that emerging records provide evidence for low atmospheric oxygen levels for the majority of Earth's history. Specifically, we review records and present a conceptual framework that suggest that background oxygen levels were below 1% of the present atmospheric level during the billon years leading up to the diversification of early animals. Evidence for low background oxygen levels through much of the Proterozoic bolsters the case that environmental conditions were a critical factor in controlling the structure of ecosystems through Earth's history.

Toxin-Antitoxin (TA) systems are abundant in prokaryotes and play an important role in various biological processes such as plasmid maintenance, phage inhibition, stress response, biofilm formation, and dormant persister cell generation. TA loci are abundant in pathogenic intracellular micro-organisms and help in their adaptation to the harsh host environment such as nutrient deprivation, oxidation, immune response, and antimicrobials. Several studies have reported the involvement of TA loci in establishing successful infection, intracellular survival, better colonization, adaptation to host stresses, and chronic infection. Overall, the TA loci play a crucial role in bacterial virulence and pathogenesis. Nonetheless, there are some controversies about the role of TA system in stress response, biofilm and persister formation. In this review, we describe the role of the TA systems in bacterial virulence. We discuss the important features of each type of TA system and the recent discoveries identifying key contributions of TA loci in bacterial pathogenesis.

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.

Chapter 1: Inorganic Chemistry Essentials. Introduction . Essential Chemical Elements. Metals in Biological Systems: A Survey. Inorganic Chemistry Basics. Biological Metal Ion Complexation. Thermodynamics. Kinetics. Electronic and Geometric Structures of Metals in Biological Systems. Bioorganometallic Chemistry. Electron Transfer. Introduction to Biological Metal Homeostasis, Transport, and Storage. Chapter 2: Biochemistry Fundamentals. Introduction. Proteins. Amino Acid Building Blocks. Protein Structure. Protein Sequencing and Proteomics. Protein Function, Enzymes and Enzyme Kinetics. Nucleic Acids. DNA and RNA Building Blocks. DNA and RNA Molecular Structures. Transmission of Genetic Information. Genetic Mutations and Site-Directed Mutagenesis. Genes and Cloning. Genomics and the Human Genome. The ATP-binding cassette (ABC) Transporter Superfamily. Chapter 3: Instrumental Methods. Introduction. Analytical Instrument-Based Methods. Spectroscopy. X-ray Absorption Spectroscopy (XAS) and Extended X-ray Absorption Fine Structure (EXAFS). Theoretical Aspects and Hardware. Descriptive Examples. X-ray Crystallography. Introduction. Crystallization and Crystal Habits. Theory and Hardware. Descriptive Examples. Electron Paramagnetic Resonance. Theory and Determination of g-values. Hyperfine and Superhyperfine Interactions. Descriptive Examples. Nuclear Magnetic Resonance. Theoretical Aspects. Nuclear Screening and the Chemical Shift. Spin-Spin coupling. Techniques of Spectral Integration and Spin-Spin Decoupling. Nuclear Magnetic Relaxation. The nuclear Overhauser Effect (NOE). Obtaining the NMR spectrum. Two-dimensional NMR Spectroscopy. Correlation Spectroscopy (COSY). Nuclear Overhauser Effect Spectroscopy (NOESY). Descriptive Examples. Mossbauer Spectroscopy. Theoretical Aspects. Quadrupole Splitting and the Isomer Shift. Magnetic Hyperfine Interactions. Descriptive Examples. Atomic Force Microscopy. Fast Methods. Stopped-Flow Kinetic Methods. Flash Photolysis. Time-Resolved Crystallography. Chapter 4: Introduction to Computer-Based Methods. Computer Hardware . Molecular Modeling and Molecular Mechanics. Introduction to MM. Molecular Modeling, Molecular Mechanics and Molecular Dynamics. Biomolecule Modeling. Molecular Modeling Descriptive Examples. Quantum Mechanics-Based Computational Methods. Introduction. Ab-initio Methods. Density Functional Theory. Semi-empirical Methods. Computer Software for Chemistry. Mathematical Software. World Wide Web Online Resources. Nomenclature and Visualization Resources. Online Societies, Literature, Materials, Equipment Webservers. Chapter 5: Group I and II metals in biological systems. Transport and storage of metal ions. Homeostasis involving Na+, K+, Ca2+, Mg2+, Zn2+ (as well as P, Cl, and H). Movement of molecules and ions across membranes. Proton pumps. Ion pumps. Active versus passive transport . Na+/ K+ ATPases. Magnesium dependent biomolecules. Kinases. ATP-pumps. Catalytic RNA and Magnesium. Magnesium in chlorophyll. Calcium dependent biomolecules. Calmodulins and triggering. Biochemistry of nitrogen oxides, nitroxyl, and peroxynitrite ion. Chapter 6: Iron Containing Proteins and Enzymes. Iron-containing proteins with porphyrin ligand systems. Hemoglobin and Myoglobin (first edition material somewhat condensed). Cytochrome P-450: a monooxygenase. Iron-containing cytochromes in photosynthesis. Green plant cytochromes. Cytochrome bc559. Cytochrome bf. Bacterial cytochromes. Cytochrome c2. Cytochrome bc1. Iron-containing cytochromes in the respiratory pathway. Cytochrome c. Cytochrome c oxidase. Non-Heme Iron-containing proteins. MMO-Methane Monooxygenase. Catechol dioxygenases. Iron-oxo clusters. Transferrin for metal transport . Ferritin for metal storage. Hemerythrin. Purple acid phosphatase. Iron-sulfur cluster proteins. Rubredoxin. Ferrodoxins. Aconitase. Chapter 7: Enzymes containing zinc, nickel, and enzymes with multiple metal cofactors. Zinc homeostasis, transport, and storage. Zinc Enzymes. Alkaline phosphatase. Alcohol dehydrogenase. Aspartate transcarbamoylase. Carbonic anhydrase. Carboxypeptidase A and B. Zinc Fingers. Nickel Enzymes. Urease. F430. Enzymes with multiple metal cofactors. Hydrogenases. Carbon monoxide dehydrogenases. Chapter 8 Disease, Disease Diagnosis and Treatment Utilizing Inorganic Species. Metal Transport and Metallochaperones. Disease States. Metal deficiency. Metal toxicity. Copper toxicity. FALS. Wilson and Menkes diseases. Disease Diagnosis. Technetium Imaging Agents. Gadolinium MRI Imaging Agents. Quantum Dots for In vivo Imaging. Metals in Medicine. Manganese superoxide mimetics. Vanadium-based Diabetes Drugs. Platinum-containing Anticancer Agents. Nanomedicine.

Impact of microbial physiology and microbial community structure on pharmaceutical fate driven by dissolved oxygen concentration in nitrifying bioreactors

Author response: eDNA-stimulated cell dispersion from Caulobacter crescentus biofilms upon oxygen limitation is dependent on a toxin–antitoxin system

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. In Caulobacter crescentus biofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here, we show that the toxin–antitoxin system (TAS) ParDE4 stimulates cell death in areas of a biofilm with decreased O2 availability. In conditions where O2 availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, a programmed cell death mechanism using an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability and favors colonization of a new, more hospitable environment. Editor's evaluation In this work, the authors present compelling evidence that a toxin-antitoxin system contributes to biofilm dispersal under oxygen limited conditions. This work makes important contributions to two areas of microbial physiology; functional understanding of toxin-antitoxin systems, which have remained largely elusive, and mechanistic regulation or biofilm dispersal, is a critical, but less understood aspect of biofilm physiology. https://doi.org/10.7554/eLife.80808.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Bacteria are more social than what had long been expected. While they can swim around on their own, most of them in fact settle down as part of a surface-bound community. The plaque on our teeth and the gooey deposit in our bathroom pipes are the visible results of this communal lifestyle. Inside this slimy ‘biofilm’, cells share resources and are protected from toxic substances such as antibiotics. However, being tied to one spot is not always a good thing: it may be advantageous for a cell in a biofilm to strike out on its own and resume ‘single life’ if local conditions deteriorate. Caulobacter crescentus bacteria do not always have this choice, as adult cells in this species become permanently glued into place upon joining a biofilm. When these divide, however, their daughters have a choice: swim away, or stick with the group. Previous research has shown that this decision is influenced by the health of the community. Dying cells release DNA fragments which disable the structures allowing newborn cells to adhere to the environment, and a high mortality rate in the biofilm therefore forces unattached cells to leave the colony. Berne et al. wanted to build on these results and examine how exactly cells die in the biofilm. In particular, the deaths could be sudden and random, with the bacteria succumbing to injury; or they could result from cells activating one of their built-in self-destruct programs. To investigate this question, genetically modified C. crescentus bacteria were grown in the laboratory and exposed to different environments. Combining genetic and microscopic approaches revealed that as a biofilm becomes too crowded, certain individuals self-destruct via a cell death program known as the toxin-antitoxin system. Further experiments showed that low oxygen availability was the signal that triggered self-destruction. Drops in oxygen levels can happen when the environment becomes hostile or when a colony is overpopulated. The results by Berne et al. therefore suggest that by triggering self-destruction in certain members of the community, reduced oxygen access leads to newborn cells swimming away, which in turn prevents further overcrowding and allows new, more hospitable locations to be colonized. Biofilms are of growing interest in a wide range of human industries, but they also present formidable challenges. This is particularly the case in healthcare, as they tend to infest medical devices and help disease-causing species to resist treatments. Understanding how bacteria are encouraged to join or leave their colony is necessary to better control biofilms to our advantage. Introduction Biofilms are multicellular communities attached to a surface, where complex exchanges and interactions occur between the different members. Biofilms first start with the attachment of individual bacterial cells to a surface and then grow into a more complex community when attached bacteria divide and new ones join. The biofilm lifestyle is considered beneficial for bacteria, as they usually provide protection from xenobiotic stresses and predators, and increase collective nutrient availability (Flemming et al., 2016). However, when conditions deteriorate, cells can leave the biofilm through a process referred to as dispersal, disseminate to new environments, and form new biofilms, enabling the colonization of new niches (Guilhen et al., 2017). Dispersal is triggered in response to various environment and biological cues, and understanding its regulation is important to determine how biofilms can be controlled. Many Alphaproteobacteria use a strong polar adhesin to irreversibly attach to surfaces and form biofilms (Berne et al., 2015; Berne et al., 2018b), with Caulobacter crescentus holdfast being the best characterized example. C. crescentus has a dimorphic life cycle, where each division cycle yields a sessile mother stalked cell and a motile daughter swarmer cell. Newborn motile swarmer cells bear a single flagellum and multiple pili at the new pole. After the cell cycle progresses beyond a certain point, or upon contact with a surface, the newborn cells secrete a holdfast at the same pole and differentiate into stalked cells by retracting their pili, ejecting their flagellum, and synthesizing a thin cylindrical extension of the cell envelope called the stalk, which pushes the holdfast away from the cell body. While the chemical composition of the holdfast is not entirely elucidated, it is composed of polysaccharides with four different monosaccharide constituents, as well as DNA and peptide molecules of unknown nature (Merker and Smit, 1988; Hernando-Pérez et al., 2018; Hershey et al., 2019). Holdfast is an extremely strong bioadhesin (Tsang et al., 2006; Berne et al., 2013) crucial for irreversible cell adhesion to solid surfaces (Ong et al., 1990; Bodenmiller et al., 2004), colonization of air–liquid interfaces (Fiebig, 2019), and biofilm formation (Entcheva-Dimitrov and Spormann, 2004). In some bacterial species, extracellular DNA (eDNA) plays a stabilizing role in the biofilm matrix (Okshevsky and Meyer, 2015; Campoccia et al., 2021). In contrast, we previously showed that C. crescentus eDNA produced via cell lysis negatively regulates biofilm formation and stimulates cell dispersal. eDNA binding to unattached holdfasts inhibits their adhesiveness, thereby inhibiting cell attachment to surfaces (Berne et al., 2010). In contrast, eDNA does not dislodge previously bound holdfasts. Therefore, eDNA prevents newborn swarmer cells from joining mature biofilms, but does not dissociate existing biofilms. Because inhibition by eDNA is proportional to its concentration, we proposed that eDNA serves as a rheostat-like environmental cue to trigger dispersal when conditions are detrimental and cause cell death. However, it was not known if eDNA release is the simple consequence of random cell death occurring in the biofilm as conditions worsen, or if it is the result of an active mechanism, such as programmed cell death (PCD) (Berne et al., 2010; Kirkpatrick and Viollier, 2010). In this study, we demonstrate that cell death and eDNA release in a biofilm are regulated by a PCD mechanism that responds to oxygen availability. PCD in bacteria includes all genetically encoded mechanisms that lead to cell lysis (Lewis, 2000; Bayles, 2014). Toxin-antitoxin systems (TAS) are important regulators of PCD (Rice and Bayles, 2008; Peeters and de Jonge, 2018). These systems are comprised of a stable toxin and its unstable antitoxin cognate. The antitoxin molecule usually antagonizes the toxin under ‘steady state’ growth conditions; but, in PCD-triggering conditions, the antitoxin is inactivated, leading to an excess of free toxins that target key cellular processes in response to various environmental signals (Harms et al., 2018; Wang et al., 2021). There are currently eight types of TAS described in bacteria. The classification depends on the nature of the antitoxin (RNA in types I, III, and VIII, or small protein in the other TAS types), and the toxin (small protein in all but type VIII where the toxin is a small RNA), and how the antitoxin neutralizes the toxin activity (Song and Wood, 2020a, Singh et al., 2021; Srivastava et al., 2021). TAS are widespread in bacterial and archaeal genomes, but despite their abundance, the biological relevance of most TAS is still elusive (Fraikin et al., 2020). TAS were first described as plasmid addiction modules that ensure plasmid stabilization via post-segregational killing of plasmid-free cells (Ogura and Hiraga, 1983; Gerdes et al., 1986). TAS have also been shown to promote addiction to certain chromosomally encoded elements such as integrative conjugative elements (Wozniak and Waldor, 2009) or CRISPR-cas loci (Li et al., 2021). In addition, TAS have been described as defense mechanism against phage infection where host translation is inhibited by the phage (Pecota and Wood, 1996; Fineran et al., 2009; Song and Wood, 2020b, LeRoux and Laub, 2022; Vassallo et al., 2022). In bacterial cells that lose their plasmid/chromosomal element encoding the TAS, or that are infected by phage, the amount of labile antitoxin rapidly decreases, leading to toxin activation and subsequent cell death. In addition to the well accepted role of TAS in plasmid addiction and phage exclusion, TAS have been linked to diverse physiological responses, such as biofilm formation, stress response, and persistence (Kamruzzaman et al., 2021), although this is still debated (Ronneau and Helaine, 2019; Wade and Laub, 2019; Song and Wood, 2020a, Jurėnas et al., 2022). Many TAS have been reported to be transcriptionally upregulated under environmental stress conditions (Jurėnas et al., 2022), but this increase does not necessarily trigger liberation of an active toxin (LeRoux et al., 2020). Among the 18 TAS identified in the C. crescentus genome (Ely, 2021), 13 have been studied experimentally, and belong to four different groups: (1) four paralogous RelBE (Fiebig et al., 2010) operons and one HigBA (Kirkpatrick et al., 2016) operon, belonging to the type II systems where the toxins (RelE or HigB) are known to be mRNA endonucleases; (2) four type II systems belonging to the ParDE family (Fiebig et al., 2010), where ParE toxins are usually DNA gyrase inhibitors; (3) three paralogs of HipBA, also a type II system, where the HipA toxins inhibit protein synthesis (Huang et al., 2020; Zhou et al., 2021); and (4) SocAB, the only member of the type VI TAS described so far, where the SocB toxin directly inhibits DNA replication (Aakre et al., 2013). The environmental conditions that trigger any of these TAS and their biological function are not yet fully identified. In this study, we show that the ParDE4 TAS is involved in PCD and eDNA release in C. crescentus biofilms where it stimulates cell dispersal. We show that areas of a biofilm with decreased O2 availability experience more cell death. Cell viability is improved in a ∆parDE4 mutant biofilm, especially in areas of decreased O2 availability, generating less cell lysis and less eDNA release. We also show that cell dispersal is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, PCD by an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability. Results The ParDE4 TAS is involved in biofilm inhibition and eDNA release of C. crescentus grown under static conditions We previously showed that, in C. crescentus, eDNA is a cue that can trigger biofilm inhibition and dispersion by binding to holdfasts and reducing their adhesiveness. This mechanism is a result of cell lysis and eDNA release in the biofilm (Berne et al., 2010). To investigate if this eDNA release is the product of a specific PCD mechanism, we tested if a TAS was involved in promoting cell death in the biofilm, as previously suggested (Kirkpatrick and Viollier, 2010). If such a TAS is inactivated, one should observe less cell death, less eDNA release, and more biofilm formation. We examined the four ParDE-like and four RelBE-like individual in-frame deletion mutants previously described (Fiebig et al., 2010), as well as mutants lacking the four ParDE (‘All parDE− ' mutant), the four RelBE (‘All relBE− ' mutant) and the eight ParDE/RelBE operons (‘All parDE− All relBE− ' mutant’), for their ability to form biofilms compared to C. crescentus CB15 wild-type (WT). For these static biofilm assays, we grew cells in two-ml plastic microfuge tubes sealed with AeraSeal breathable film, to allow for gas exchange, and incubated them statically at 30°C (Figure 1). We defined these growth conditions as ‘moderate aeration’. All the tested mutants grew similarly to WT under these conditions (Figure 1—figure supplement 1). Figure 1 with 2 supplements see all Download asset Open asset Role of the eight toxin–antitoxin systems (TAS) in cell death, extracellular DNA (eDNA) release, and biofilm formation. C. crescentus WT and the different TAS in-frame deletion mutants were grown for 48 hr under moderate aeration conditions at 30°C in M2G medium, as depicted on the left. (A) Percentage of dead cells in the planktonic phase; results are expressed as a percentage of the total cells (live + dead) in the sample, quantified using the BacLight Live/Dead kit. (B) Biofilm formation, quantified by crystal violet staining; results are expressed as a percentage of biofilm formed compared to WT. (C) Quantification of eDNA released in the planktonic phase, using PicoGreen. Results are given as the average of four independent experiments, each run in duplicate, and the error bars represent the standard error of the mean (SEM). Statistical comparisons are calculated using Student’s unpaired t-tests; only samples statistically different from WT are shown. **p < 0.01. We tested the ability of the TAS mutants to form biofilms after 48 hr, and quantified cell death and eDNA release under these growth conditions (Figure 1). Among single mutants, ∆parDE4 was the only strain that behaved differently compared to WT. The percentage of dead cells was lower in this mutant and it produced ~30% more biofilm than the other strains (Figure 1A, B). Furthermore, it released only about half of the amount of eDNA in the planktonic phase compared to WT (Figure 1C and Figure 1—figure supplement 1B). The All parDE− and the All parDE− All relBE− strains, where all four parDE operons and all parDE plus all relBE operons were deleted respectively, behaved like the ∆parDE4 single deletion mutant (Figure 1). These results suggest that ParDE4 plays a role in cell death and eDNA release under our experimental conditions and that the observed changes in eDNA concentration yield differences in biofilm regulation. To test if this phenotype was specific for C. crescentus cells that are able to form biofilms, we deleted the hfsDAB holdfast synthesis cluster in the ∆parDE4 background to generate a strain unable to produce holdfast, and therefore unable to adhere to surfaces and form biofilms. The double mutant ∆parDE4 ∆hfsDAB phenocopied the ∆parDE4 strain, with lower eDNA and lower proportion of dead cells (Figure 1—figure supplement 2). These results indicate that the function of ParDE4 does not require cells to be adhered to a surface and suggest that it might be responding to the differences in medium aeration as described in a later section. The ParDE4 TAS plays a role in cell death in mature biofilms of C. crescentus The parDE4 operon is composed of the parD4 antitoxin gene (CC2985/CCNA_03080) and the parE4 toxin gene (CC2984/CCNA_03079), overlapping by 21 bp (Nierman et al., 2001; Fiebig et al., 2010; Marks et al., 2010). To assess the role of ParDE4 in cell death, eDNA release, and biofilm formation over time, we monitored biofilm formation on sterile microscopy-grade clear polyvinyl chloride (PVC) strips grown under moderate aeration as depicted in Figure 1A. Over time, we also quantified eDNA release and cell death occurring in WT and ∆parDE4 (Figure 2 and Figure 2—figure supplement 1). Cell death was reduced in ∆parDE4 biofilms compared to WT, especially at longer time points when the biofilm reached maturation (Figure 2A, B). In addition, less eDNA was released in these mutant cultures (Figure 2C). We also observed an increase in attached biomass in the ∆parDE4 mutant (Figure 2D). These results support our previous findings that biofilm inhibition, eDNA release, and cell death are correlated (Berne et al., 2010). Furthermore, these results indicate that ParDE4 is involved in stimulating cell death and eDNA release, yielding a change in biofilm formation. Since eDNA stimulates dispersal from the biofilm (Berne et al., 2010), both the reduced cell death and eDNA release in the ∆parDE4 mutant might contribute to the increased biofilm formation. Figure 2 with 1 supplement see all Download asset Open asset Involvement of the ParDE4 TAS in cell death, extracellular DNA (eDNA) release, and biofilm regulation. (A) Biofilm formed on polyvinyl chloride (PVC) strips stained with the BacLight Live/Dead reagent at different incubation times. represent of the (live and signals by bars (B) Percentage of dead cells over time in the biofilm, calculated from BacLight Live/Dead stained cells using (C) eDNA release in the planktonic phase over time, quantified using Biofilm formation over time, quantified by the attached biomass with crystal C. crescentus WT and ∆parDE4 are by solid and were grown in M2G The results are given as the average of two independent experiments, each run in and the error bars represent the standard error of the mean (SEM). The antitoxin against cell death in the biofilm In TAS, cell death usually when is an in levels of toxins and produced in the cell (Harms et al., 2018). To assess the role of antitoxin we expressed it using the low plasmid (Fiebig et al., 2010; et al., and monitored biofilm formation and eDNA release when the antitoxin is When we expressed the antitoxin in WT, biofilm formation was increased and eDNA concentration was decreased (Figure However, in a mutant lacking the was of parD4 expression on biofilm formation and eDNA concentration (Figure that (1) has a against cell lysis and eDNA release, thereby biofilm formation, and (2) this depends on the of the toxin Figure with 1 supplement see all Download asset Open asset of parD4 antitoxin expression on biofilm formation and extracellular DNA (eDNA) release. The parD4 was into the low plasmid and expressed using the Biofilm formation and eDNA release for strains the parD4 antitoxin gene or the plasmid in WT (A) and were grown in M2G medium + 1 The results are given as the average of three independent experiments and the error bars represent the standard error of the mean (SEM). we wanted to determine if the of the ∆parDE4 mutant was due to of cell death and eDNA release, or also to an response to eDNA biofilm inhibition in the ∆parDE4 We tested how WT and ∆parDE4 behaved in the of eDNA by the amount of biofilm formed in the of from cultures of different We showed previously that eDNA present in medium from cultures inhibits biofilm formation (Berne et al., 2010). various of eDNA from cultures of WT or ∆parDE4 grown to phase were in biofilm assays, as previously (Berne et al., 2010). Because ∆parDE4 less eDNA than WT compared to of eDNA present in the medium of a for ∆parDE4 and WT, respectively, see Figure supplement we first the amount of eDNA present and an amount of medium to the same concentration of The amount of biofilm formed by WT and ∆parDE4 was for the same total amount of eDNA (Figure supplement that, when exposed to the same amount and of both WT and ∆parDE4 form of biofilm and that eDNA from WT or ∆parDE4 have the same biofilm Furthermore, the inhibition response is positively correlated with the amount of eDNA present in the medium in a for both strains (Figure supplement in with our previous results (Berne et al., 2010). Therefore, the increase in biofilm formation by the ∆parDE4 mutant is not due to an response to eDNA but is due to less cell death a more increase to less cell dispersal by ParDE4 promotes dispersal in the biofilm To the of biofilm formation in the WT and ∆parDE4 strains, we grew a of differently WT and ∆parDE4 at a in The of each in the biofilm was monitored over time (Figure in the of the biofilm could be observed at of biofilm maturation (Figure with formation of due to This is in with previous of C. crescentus biofilm growth in cells (Entcheva-Dimitrov and Spormann, et al., 2019). While in the of biofilm formation the of WT and ∆parDE4 was the mutant rapidly the WT at later After hr, around of the attached bacteria were ∆parDE4 (Figure Figure Download asset Open asset Biofilm formation and dispersion in of WT and ∆parDE4 were to a and grown in cells over (A) of biofilms grown in is in and the other one in (B) of each over Results are given as a percentage of total + both calculated from of of time point, independent experiments where were (C) Cell dispersal as released in the of the Results are to the of colonies for WT at hr of the were using M2G Results are expressed as an average of of samples time cells were run in independent experiments where were bars represent the standard error of the mean (SEM). To test the dispersal rate of both strains, we dispersal from the biofilm by the of cells released from the biofilm over time in the This was by samples of the cell and the of single cells released from each (Figure was more biomass of ∆parDE4 cells than WT in the biofilm were more WT cells released over time compared to that the dispersal of WT is more (Figure In these results with of previous suggest that the observed increased biofilm formation in the ∆parDE4 is due to a of increased attached biomass of reduced cell death and decreased dispersion The ParDE4 response is correlated with O2 availability Previous work the regulation of the parDE4 operon as a function of O2 availability on growing cells (Fiebig et al., 2010). by under stress O2 showed a in parD4 not statistically (Fiebig et al., 2010). In our PCD triggered by the ParDE4 TAS is more when the biofilm maturation (Figure 2). of environmental changes occur as the biofilm of O2 availability. Since C. crescentus is an O2 could be a detrimental triggering cell death and dispersal, as is the case in other species et al., In to test the of O2 on cell death, we grew cells with as compared to the ‘moderate (Figure a in eDNA release not occur in cells grown under aeration compared to the moderate aeration conditions (Figure and Figure 1—figure supplement that ParDE4 is not active under conditions. To determine if ParDE4 expression is regulated by aeration conditions, we monitored its using a under aeration growth compared to growth under moderate aeration conditions. To growth conditions as different O2 availability, we a to the of encoding the This gene is expressed when C. crescentus cells experience O2 levels et al., and its expression can be as a to O2 availability. expression was more active under moderate aeration conditions (Figure that O2 availability is limited under growth conditions. We that was two to three under aeration growth (Figure that ParDE4 expression is regulated by O2 availability. Figure Download asset Open asset expression is under aeration growth conditions. (A) of cultures grown in M2G under conditions different of and moderate (B) Quantification of extracellular DNA (eDNA) released in the planktonic phase of WT and ∆parDE4 quantified using Results are given as the average of independent experiments and the error bars represent the standard error of the mean (SEM). (C) activity of and in WT grown under and moderate aeration conditions in The results represent the average of independent cultures on three different and the error bars represent the Since the results suggested that O2 availability is important for biofilm we tested two growth conditions to an of O2 and a (Figure as by the levels in each (Figure We first that the expression of is correlated to the O2 in the cultures (Figure We then compared the of eDNA released in ∆parDE4 to WT in the different aeration conditions. We that was as eDNA released by WT compared to ∆parDE4 under conditions where O2 levels are the most reduced the levels were under aeration (Figure with the in eDNA release, we that biofilm formation by the ∆parDE4 increased

The influence of dissolved oxygen level and medium on biofilm formation by Campylobacter jejuni

Campylobacter jejuni and related species are important human pathogens, causing acute human enterocolitis, and they are the most common cause of food-borne diarrhoea in many industrialized countries. Previous infection with certain strains of C. jejuni is also linked with the development of the neurological disorder Guillain-Barre syndrome (GBS). Despite its importance as a human pathogen, relatively little is understood of the mechanisms of C. jejuni-associated disease. The recent release of the complete genome sequence of C. jejuni strain NCTC 11168, together with new strategies for random and directed mutagenesis, have allowed a better insight into some of the genetic determinants of C. jejuni virulence. In this review current knowledge on the pathogenesis of C. jejuni infection is summarized, and possible directions of future research indicated.

Campylobacter jejuni is one of the most frequent causes of bacterial gastrointestinal food-borne infection worldwide. Biofilms have been suggested to play a role in survival of these bacteria, which is fastidious in its growth requirements, in the environment. The overall objective of this study was to investigate the effect of environment, genetic and antibiotic resistance factors on biofilm formation by a range of C. jejuni strains isolated from poultry and C. jejuni ATCC 33291 strain. The influence of prior modes of growth, temperature, medium and substrate surface on biofilm formation by the C. jejuni strains was investigated. The results obtained indicated that cells grown as sessile culture and in lower nutrient media generally have a greater ability to form biofilm while growth at different temperatures affect biofilm formation in a strain dependent manner. The results also showed that C. jejuni were able to attach and form biofilms on different abiotic surfaces but none of them demonstrated strong biofilm formation. This finding suggests that environmental factors did affect biofilm formation by C. jejuni and they are more likely to persist in the environment in the form of mixed-species rather than mono-species biofilms. The effect of different media with different dissolved oxygen (DO) levels on biofilm formation by C. jejuni under different oxygen conditions was also determined in this study. The results obtained suggested that different broths used for C. jejuni biofilm generation had different DO contents under different incubation conditions and this is likely to affect the biofilm forming ability of C. jejuni, a finding which confounds previous studies that suggested that biofilm formation by C. jejuni is enhanced under aerobic conditions. The influence of different antibiotics with different modes of action on biofilm formation by C. jejuni was also investigated. The results obtained showed that the presence of certain antibiotics induced biofilm formation by some C. jejuni strains tested. These findings indicate that the presence of antibiotics in the environment as a result of their widespread use to treat or prevent diseases might lead to induction of biofilm formation by some strains which is a public health concern. Biofilm formation by selected C. jejuni strains in mono- and mixed-culture on different abiotic surfaces under flow and aerobic conditions mimicking those in the environment was also investigated using a modified Robbins Device. The results indicated that C. jejuni survival is enhanced by forming mixed-species biofilm with Pseudomonas aeruginosa as compared to mono-species C. jejuni biofilm. These findings provided an indication that the presence of other bacteria species in the environment might enhance the survival of C. jejuni under conditions which are detrimental to them. The influence of the genetic makeup of C. jejuni strains on their biofilm formation was investigated through whole genome sequencing and by constructing mutant strains through transposon mutagenesis. Some genes that might be involved in biofilm formation by C. jejuni have been identified. These genes provide a resource for future investigations in order to understand the molecular mechanisms underlying the biofilm formation by C. jejuni. This study provides a better understanding on the ability of antibiotic resistant C. jejuni strains to form biofilm in vitro under different growth conditions, especially under conditions mimicking those in the environment. In addition, further research on the genes potentially involved in biofilm formation, which were identified in this study, may contribute to knowledge of the molecular mechanisms involved in biofilm formation by C. jejuni. A clearer understanding of how C. jejuni survives in the environment (either by forming biofilm or attach to surfaces or other biofilms) could contribute to development of new strategies to prevent or eliminate C. jejuni

Decision letter: Iron status influences mitochondrial disease progression in Complex I-deficient mice

Editor's evaluation: Iron status influences mitochondrial disease progression in Complex I-deficient mice

Author response: Iron status influences mitochondrial disease progression in Complex I-deficient mice