Isolation and characterization of persisters of the pathogenic microorganism Staphylococcus aureus.

The presence of antibiotic persisters is one of the leading causes of recurrent and chronic diseases. One challenge in mechanistic research on persisters is the enrichment of pure persisters. In this work, we validated a proposed method to isolate persisters with notorious Staphylococcus aureus cultures. With this, we analyzed the proteome profile of pure persisters and revealed the distinct mechanisms associated with vancomycin and enrofloxacin induced persisters. Furthermore, morphological and metabolic characterizations were performed, indicating further differences between these two persister populations. Finally, we assessed the effect of ATP repression, protein synthesis inhibition, and reactive oxygen species (ROS) level on persister formation. In conclusion, this work provides a comprehensive understanding of S. aureus vancomycin and enrofloxacin induced persisters, facilitating a better mechanistic understanding of persisters and the development of effective strategies to combat them.

Pro-inflammatory T cells-derived cytokines enhance the maturation of the human fetal intestinal epithelial barrier.

Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.

Exploiting a subtype-specific mitochondrial vulnerability for successful treatment of colorectal peritoneal metastases.

Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.

Marked IDO2 expression and activity related to autophagy and apoptosis in brain tissue of fatal tuberculous meningitis.

In about 1% of tuberculosis (TB) patients, Mycobacterium tuberculosis (M. tuberculosis) can disseminate to the meninges, causing tuberculous meningitis (TBM) with mortality rate up to 60%. Chronic granulomatous inflammation (non-necrotizing and necrotizing) in the brain is the histological hallmark of TBM. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and the generated kynurenine metabolites exert major effector functions relevant to TB granuloma functioning. Here we have assessed immunohistochemically IDO1 expression and activity and its effector function and that of its isoform, IDO2, in post-mortem brain tissue of patients that demised with neurotuberculosis. We also related these findings to brain tissue of fatal/severe COVID-19. In this study, IDO1 and IDO2 were abundantly expressed and active in tuberculoid granulomas and were associated with the presence of M. tuberculosis as well as markers of autophagy and apoptosis. Like in fatal/severe COVID-19, IDO2 was also prominent in specific brain regions, such as the inferior olivary nucleus of medulla oblongata and cerebellum, but not associated with granulomas or with M. tuberculosis. Spatially associated apoptosis was observed in TBM, whereas in fatal COVID-19 autophagy dominated. Together, our findings highlight IDO2 as a potentially relevant effector enzyme in TBM, which may relate to the symptomology of TBM.

CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

The impact of bed rest on human skeletal muscle metabolism.

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

Platelets of COVID-19 patients display mitochondrial dysfunction, oxidative stress, and energy metabolism failure compatible with cell death.

Background: Alterations in platelet function have been implicated in the pathophysiology of COVID-19 since the beginning of the pandemic. While early reports linked hyperactivated platelets to thromboembolic events in COVID-19, subsequent investigations demonstrated hyporeactive platelets with a procoagulant phenotype. Mitochondria are important for energy metabolism and the function of platelets. Objectives: Here, we sought to map the energy metabolism of platelets in a cohort of noncritically ill COVID-19 patients and assess platelet mitochondrial function, activation status, and responsiveness to external stimuli. Methods: We enrolled hospitalized COVID-19 patients and controls between October 2020 and December 2021. Platelets function and metabolism was analyzed by flow cytometry, metabolomics, glucose fluxomics, electron and fluorescence microscopy and western blot. Results: Platelets from COVID-19 patients showed increased phosphatidylserine externalization indicating a procoagulant phenotype and hyporeactivity to ex vivo stimuli, associated with profound mitochondrial dysfunction characterized by mitochondrial depolarization, lower mitochondrial DNA-encoded transcript levels, an altered mitochondrial morphology consistent with increased mitochondrial fission, and increased pyruvate/lactate ratios in platelet supernatants. Metabolic profiling by untargeted metabolomics revealed NADH, NAD+, and ATP among the top decreased metabolites in patients' platelets, suggestive of energy metabolism failure. Consistently, platelet fluxomics analyses showed a strongly reduced utilization of 13C-glucose in all major energy pathways together with a rerouting of glucose to de novo generation of purine metabolites. Patients' platelets further showed evidence of oxidative stress, together with increased glutathione oxidation and synthesis. Addition of plasma from COVID-19 patients to normal platelets partially reproduced the phenotype of patients' platelets and disclosed a temporal relationship between mitochondrial decay and (subsequent) phosphatidylserine exposure and hyporeactivity. Conclusion: These data link energy metabolism failure in platelets from COVID-19 patients with a prothrombotic platelet phenotype with features matching cell death.

Nucleocapsid protein accumulates in renal tubular epithelium of a post-COVID-19 patient.

IMPORTANCE: Even though the coronavirus disease 2019 (COVID-19) pandemic is slowly developing into a conventional infectious disease, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection are still not well understood. One of the problems is that many COVID-19 cases develop acute kidney injuries. Still, it is heavily debated whether SARS-CoV-2 virus enters and actively replicates in kidney tissue and if SARS-CoV-2 virus particles can be detected in kidney during or post-infection. Here, we demonstrated that nucleocapsid N protein was detected in kidney tubular epithelium of patients that already recovered form COVID-19. The presence of the abundantly produced N protein without signs of viral replication could have implications for the recurrence of kidney disease and have a continuing effect on the immune system.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Spatial Transcriptional Mapping Reveals Site-Specific Pathways Underlying Human Atherosclerotic Plaque Rupture.

BACKGROUND: Atherosclerotic plaque ruptures, triggered by blood flow-associated biomechanical forces, cause most myocardial infarctions and strokes. OBJECTIVES: This study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. METHODS: Histology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. RESULTS: In human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. CONCLUSIONS: Our findings show plaque site-specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

Effect of long-term antihypertensive treatment on cerebrovascular structure and function in hypertensive rats.

Midlife hypertension is an important risk factor for cognitive impairment and dementia, including Alzheimer's disease. We investigated the effects of long-term treatment with two classes of antihypertensive drugs to determine whether diverging mechanisms of blood pressure lowering impact the brain differently. Spontaneously hypertensive rats (SHR) were either left untreated or treated with a calcium channel blocker (amlodipine) or beta blocker (atenolol) until one year of age. The normotensive Wistar Kyoto rat (WKY) was used as a reference group. Both drugs lowered blood pressure equally, while only atenolol decreased heart rate. Cerebrovascular resistance was increased in SHR, which was prevented by amlodipine but not atenolol. SHR showed a larger carotid artery diameter with impaired pulsatility, which was prevented by atenolol. Cerebral arteries demonstrated inward remodelling, stiffening and endothelial dysfunction in SHR. Both treatments similarly improved these parameters. MRI revealed that SHR have smaller brains with enlarged ventricles. In addition, neurofilament light levels were increased in cerebrospinal fluid of SHR. However, neither treatment affected these parameters. In conclusion, amlodipine and atenolol both lower blood pressure, but elicit a different hemodynamic profile. Both medications improve cerebral artery structure and function, but neither drug prevented indices of brain damage in this model of hypertension.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

The immunological effects of intradermal particle-based vaccine delivery using a novel microinjection needle studied in a human skin explant model.

For intradermal (ID) immunisation, novel needle-based delivery systems have been proposed as a better alternative to the Mantoux method. However, the penetration depth of needles in the human skin and its effect on immune cells residing in the different layers of the skin has not been analyzed. A novel and user-friendly silicon microinjection needle (Bella-muTM) has been developed, which allows for a perpendicular injection due to its short needle length (1.4-1.8 mm) and ultrashort bevel. We aimed to characterize the performance of this microinjection needle in the context of the delivery of a particle-based outer membrane vesicle (OMV) vaccine using an ex vivo human skin explant model. We compared the needles of 1.4 and 1.8 mm with the conventional Mantoux method to investigate the depth of vaccine injection and the capacity of the skin antigen-presenting cell (APC) to phagocytose the OMVs. The 1.4 mm needle deposited the antigen closer to the epidermis than the 1.8 mm needle or the Mantoux method. Consequently, activation of epidermal Langerhans cells was significantly higher as determined by dendrite shortening. We found that five different subsets of dermal APCs are able to phagocytose the OMV vaccine, irrespective of the device or injection method. ID delivery using the 1.4 mm needle of a OMV-based vaccine allowed epidermal and dermal APC targeting, with superior activation of Langerhans cells. This study indicates that the use of a microinjection needle improves the delivery of vaccines in the human skin.

Mitochondrial dysfunction in human hypertrophic cardiomyopathy is linked to cardiomyocyte architecture disruption and corrected by improving NADH-driven mitochondrial respiration.

AIMS: Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce. METHODS AND RESULTS: Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with <10. Mitochondrial dysfunction was not explained by reduced abundance or fragmentation of mitochondria, as evaluated by transmission electron microscopy. Rather, improper organization of mitochondria relative to myofibrils (expressed as a percentage of disorganized mitochondria) was strongly associated with mitochondrial dysfunction. Pre-incubation with the cardiolipin-stabilizing drug elamipretide and raising mitochondrial NAD+ levels both boosted NADH-linked respiration. CONCLUSION: Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.

A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages.

Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves the transformation of infected phagolysosomes from a site of killing into a nutrient-rich replicative niche. Here, we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis, 1-tuberculosinyladenosine (1-TbAd), caused lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing M. tuberculosis, caused intralysosomal and peribacillary lipid storage patterns that matched both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters and that 1-TbAd increased M. tuberculosis growth under conditions of restricted lipid access in macrophages. Furthermore, lipidomics identified 1-TbAd-induced lipid substrates that define Gaucher's disease, Wolman's disease, and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of M. tuberculosis-induced lysosomal failure, leading to successful testing of an agonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Hyperactivation of mTORC1 in a double hit mutant zebrafish model of tuberous sclerosis complex causes increased seizure susceptibility and neurodevelopmental abnormalities.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by pathogenic variants in TSC1 and TSC2 genes. TSC patients present with seizures and brain abnormalities such as tubers and subependymal giant cells astrocytoma (SEGA). Despite common molecular and clinical features, the severity of the disease varies greatly, even intrafamilially. The second hit hypothesis suggests that an additional, inactivating mutation in the remaining functional allele causes a more severe phenotype and therefore explains the phenotypic variability. Recently, second hit mutations have been detected frequently in mTORopathies. To investigate the pathophysiological effects of second hit mutations, several mouse models have been developed. Here, we opted for a double mutant zebrafish model that carries a LOF mutation both in the tsc2 and the depdc5 gene. To the best of our knowledge, this is the first time a second-hit model has been studied in zebrafish. Significantly, the DEP domain-containing protein 5 (DEPDC5) gene has an important role in the regulation of mTORC1, and the combination of a germline TSC2 and somatic DEPDC5 mutation has been described in a TSC patient with intractable epilepsy. Our depdc5 -/- x tsc2 -/- double mutant zebrafish line displayed greatly increased levels of mammalian target of rapamycin (mTORC1) activity, augmented seizure susceptibility, and early lethality which could be rescued by rapamycin. Histological analysis of the brain revealed ventricular dilatation in the tsc2 and double homozygotes. RNA-sequencing showed a linear relation between the number of differentially expressed genes (DEGs) and the degree of mTORC1 hyperactivity. Enrichment analysis of their transcriptomes revealed that many genes associated with neurological developmental processes were downregulated and mitochondrial genes were upregulated. In particular, the transcriptome of human SEGA lesions overlapped strongly with the double homozygous zebrafish larvae. The data highlight the clinical relevance of the depdc5 -/- x tsc2 -/- double mutant zebrafish larvae that showed a more severe phenotype compared to the single mutants. Finally, analysis of gene-drug interactions identified interesting pharmacological targets for SEGA, underscoring the value of our small zebrafish vertebrate model for future drug discovery efforts.

The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages.

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.

Transfer dynamics of multi-resistance plasmids in Escherichia coli isolated from meat.

Resistance plasmids are crucial for the transfer of antimicrobial resistance and thus form a matter of concern for veterinary and human healthcare. To study plasmid transfer, foodborne Escherichia coli isolates harboring one to five known plasmids were co-incubated with a general recipient strain. Plasmid transfer rates under standardized conditions varied by a factor of almost 106, depending on the recipient/donor strain combination. After 1 hour transconjugants never accounted for more than 3% of the total number of cells. Transconjugants were formed from 14 donors within 1 hour of co-incubation, but in the case of 3 donors 24 hours were needed. Transfer rates were also measured during longer co-incubation, between different species and during repeated back and forth transfer. Longer co-incubation resulted in the transfer of more types of resistance. Maximum growth rates of donor strains varied by a factor of 3. Donor strains often had higher growth rates than the corresponding transconjugants, which grew at the same rate as or slightly faster than the recipient. Hence, possessing one or more plasmids does not seem to burden the harboring strain metabolically. Transfer was species specific and repeated transfer of one plasmid did not result in different transfer rates over time. Transmission Electron microcopy was used to analyze the morphology of the connection between co-incubated strains. Connection by more pili between the cells resulted in better aggregate formation and corresponded with higher transfer rates.

Harnessing solar power: photoautotrophy supplements the diet of a low-light dwelling sponge.

The ability of organisms to combine autotrophy and heterotrophy gives rise to one of the most successful nutritional strategies on Earth: mixotrophy. Sponges are integral members of shallow-water ecosystems and many host photosynthetic symbionts, but studies on mixotrophic sponges have focused primarily on species residing in high-light environments. Here, we quantify the contribution of photoautotrophy to the respiratory demand and total carbon diet of the sponge Chondrilla caribensis, which hosts symbiotic cyanobacteria and lives in low-light environments. Although the sponge is net heterotrophic at 20 m water depth, photosynthetically fixed carbon potentially provides up to 52% of the holobiont's respiratory demand. When considering the total mixotrophic diet, photoautotrophy contributed an estimated 7% to total daily carbon uptake. Visualization of inorganic 13C- and 15N-incorporation using nanoscale secondary ion mass spectrometry (NanoSIMS) at the single-cell level confirmed that a portion of nutrients assimilated by the prokaryotic community was translocated to host cells. Photoautotrophy can thus provide an important supplemental source of carbon for sponges, even in low-light habitats. This trophic plasticity may represent a widespread strategy for net heterotrophic sponges hosting photosymbionts, enabling the host to buffer against periods of nutritional stress.