Biology subjects

Kleerebezem, M.

Publications and source records attributed to Kleerebezem, M..

5 recordsLinked to original sources

bioRxiv2021

Manganese modulates metabolic activity and redox homeostasis in translationally-blocked Lactococcus cremoris, impacting metabolic persistence, cell-culturability, and flavor formation.

Manganese (Mn) is an essential trace element that is supplemented in microbial media with varying benefits across species and growth conditions. We found that growth of Lactococcus cremoris was unaffected by manganese omission from the growth medium. The main proteome adaptation to manganese omission involved increased manganese transporter production (up to 2000-fold), while the remaining 10 significant proteome changes were between 1.4 and 4 fold. Further investigation in translationally-blocked (TB), non-growing cells showed that Mn supplementation (20 M) led to approximately 1.5X faster acidification compared to Mn-free conditions. However, this faster acidification stagnated within 24 hours, likely due to draining of intracellular NADH that coincides with substantial loss of culturability. Conversely, without manganese, non-growing cells persisted to acidify for weeks, albeit at a reduced rate, but maintaining redox balance and culturability. Strikingly, despite being unculturable, -keto acid-derived aldehydes continued to accumulate in cells incubated in the presence of manganese, whereas without manganese cells predominantly formed the corresponding alcohols. This is most likely reflecting NADH availability for the alcohol dehydrogenase-catalyzed conversion. Overall, manganese influences the lactococcal acidification rate, and flavor formation capacity in a redox dependent manner. These are important industrial traits especially during cheese ripening, where cells are in a non-growing, often unculturable state.

microbiology↗

bioRxiv2020

Early-life feeding accelerates gut microbiome maturation in piglets

Early-life microbiome perturbations have been suggested to have important effects on host development, physiology, and behaviour, which can persist throughout life. We hypothesise that early feeding (access to a pre-weaning fibrous diet) can affect gut microbiome colonisation and development in neonatal piglets. In this longitudinal study, a customised fibrous diet was provided to early-fed piglets (EF; 6 litters) starting two days after birth until weaning (28 days of age) in addition to mothers milk, whereas control piglets (CON; 4 litters) suckled sows milk only. Rectal swabs were collected at multiple timepoints until six weeks of age (i.e., 2 weeks post-weaning) to investigate intestinal microbiota composition development over time using 16S rRNA gene profiling (n = 10 piglets per treatment). We observed a dynamic intestinal microbiota colonisation pattern during the pre-weaning period in both treatment groups, which rapidly stabilised within 2 weeks post-weaning. The microbial (alpha) diversity increased with age and seemed to reach a plateau in the early post-weaning time-point (day+5). The homogenous post-weaning microbiota was represented by microbial groups including Prevotella, Roseburia, Faecalibacterium, Ruminococcus, Megasphaera, Catenibacterium and Subdoligranulum. Remarkably, early feeding of neonatal piglets resulted in accelerated maturation of the intestinal microbiota at pre-weaning time-points, characterised by increased rate of microbial diversity and expanded colonisation of typical post-weaning associated microbial groups (mentioned above) at pre-weaning stages. The acceleration in EF piglets was illustrated by the simultaneous emergence of typical post-weaning-associated microbial groups and a more rapid decline of typical early-life/pre-weaning microbial genera. In addition, the individual eating behaviour scores of the piglets quantitatively correlated with the accelerated change of their microbiome. Overall, these findings show the importance of early-life nutritional strategies to influence the gut microbiota maturation in piglets.

microbiology↗

bioRxiv2020

Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses

Maternal milk is natures first functional food. It plays a crucial role in the development of the infants gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.

immunology↗

bioRxiv2018

An open microscopy framework suited for tracking dCas9 in live bacteria

Super-resolution microscopy is frequently employed in the life sciences, but the number of freely accessible and affordable microscopy frameworks, especially for single particle tracking photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed the miCube: a versatile super-resolution capable fluorescence microscope, which combines high spatiotemporal resolution, good adaptability, low price, and easy installation. By providing all details, we hope to enable interested researchers to build an identical or derivative instrument. The capabilities of the miCube are assessed with a novel sptPALM assay relying on the heterogeneous expression of target genes. Here, we elucidate mechanistic details of catalytically inactive Cas9 (dead Cas9) in live Lactococcus lactis. We show that, lacking specific DNA target sites, the binding and unbinding of dCas9 to DNA can be described using simplified rate constants of kbound[->]free = 30-80 s-1 and kfree[->]bound = 15-40 s-1. Moreover, after providing specific DNA target sites via DNA plasmids, the plasmid-bound dCas9 population size decreases with increasing dCas9 copy number via a mono-exponential decay, indicative of simple disassociation kinetics.

biophysics↗

bioRxiv2017

Unleashing natural competence in Lactococcus lactis by induction of the competence regulator ComX

In biotechnological work horses like Streptococcus thermophilus and Bacillus subtilis natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis natural competence has not been established to date. However, in silico analysis of complete genome sequences of 43 L. lactis strains revealed complete late-competence gene-sets in 2 L. lactis subspecies cremoris strains (KW2 and KW10) and 8 L. lactis subspecies lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast, comX expression in L. lactis NZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147, abolished the comX driven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to other L. lactis strains of both subspecies.\n\nSignificance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

microbiology↗