Biology subjects

Willemse, J.

Publications and source records attributed to Willemse, J..

17 recordsLinked to original sources

bioRxiv2026

Ecologically inspired assembly of a Bacillales synthetic community for lignin degradation

Lignin remains a major bottleneck in biomass valorization due to its heterogeneous and recalcitrant structure. Traditionally, lignin degradation has been developed using a single-microorganism or single-enzyme approach, with efforts focused on identifying ligninolytic activities of individual bacteria or fungi. Here, we establish a framework for designing lignin-degrading synthetic microbial communities (SynComs) by integrating top-down ecological filtering with bottom-up trait-based selection. This approach enables the selection of bacteria with ligninolytic capacity while retaining community members that form strong biofilms and contribute indirectly to overall community function. Proteomic profiling uncovered lignin-driven functional reprogramming of the SynCom, highlighting strain-specific enzymatic specializations that collectively enable lignin depolymerization. Our work highlights the potential of SynComs for lignin degradation and shows how rationally designed, functionally partitioned communities can enhance lignin conversion.

microbiology↗

bioRxiv2025

GreenLeafVI: A FIJI plugin for high-throughput analysis of leaf chlorophyll content

Chlorophyll breakdown is a central process during plant senescence or stress responses and leaf chlorophyll content is therefore a strong predictor of plant health. Chlorophyll quantification can be done in several ways, most of which are time-consuming or require specialized equipment. A simple alternative to these methods is the use of image-based chlorophyll estimation, which uses the color values in RGB images to calculate colorimetric visual indexes as a measure for the leaf chlorophyll content. Image-based chlorophyll measurement is non-destructive and, apart from a digital camera, requires no specialized equipment. Here, we developed the ImageJ plugin GreenLeafVI that facilitates high-throughput image analysis for measuring leaf chlorophyll content. Our plugin offers the option to white-balance images to decrease variation between images and has an optional background removal step. We show that this method can reliably quantify leaf chlorophyll content in a variety of plant species. In addition, we show that image-based chlorophyll quantification can replicate GWAS results based on traditional chlorophyll extraction methods, showing that this method is highly accurate.

plant biology↗

bioRxiv2025

Defining the minimal structural requirements of DivIVA in filamentous Actinomycetota

The morphogenetic protein DivIVA exhibits diverse functions across bacterial phyla. In Bacillota, DivIVA is primarily involved in cell division, whereas in Actinomycetota, it plays a central role in coordinating polar growth. Due to its essential nature, gaining insight into DivIVA function is challenging. Here we report on the functionality of truncated DivIVA proteins, using a unique divIVA deletion mutant created in cell wall-deficient Kitasatospora viridifaciens L-forms. DivIVA comprises an N-terminal domain, two coiled-coil regions separated by an intercoil linker, and a C-terminal domain. Deleting either the intercoil or the C-terminal region impacted branching dynamics. We also created a minimized variant wherein both were deleted simultaneously, containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. Expression of this minimized variant resulted in severe growth defects. Cells exhibited a strong increase in hyphal width and cell wall thickness, accompanied by frequent tip bursting. Finally, we successfully introduced chimeric DivIVA from the unicellular actinobacterium Mycolicibacterium smegmatis with an N-terminal domain of Kitasatospora viridifaciens, demonstrating functional conservation within the phylum. In contrast, a chimeric DivIVA from Bacillus subtilis could not support growth, underscoring that polar growth is encoded within Actinomycetota-specific amino acid motifs encoded in the first and second coiled-coil. These findings enhance our understanding of the structure-function relationship for DivIVA and present new opportunities to study polar growth. ImpactDivIVA is essential for polar growth in Actinomycetota. In Streptomycetaceae, this membrane-binding protein localizes at growing hyphal tips and along lateral hyphal walls where new branches emerge. Due to its essentiality, the structural relationship of DivIVA between unicellular and multicellular species remains elusive. Using a Kitasatospora viridifaciens L-form divIVA deletion mutant, we expressed truncated DivIVA variants to identify essential regions. Deleting two large unstructured domains produced a minimized variant containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. This strongly impacted morphogenesis, increasing hyphal width and cell wall thickness, and leading to hyphal tip bursting. Finally, we successfully substituted DivIVA of K. viridifaciens with that from Mycolicibacterium smegmatis. Furthermore, Bacillus subtilis DivIVA could not facilitate reversion, showing that polar growth depends on amino acid motifs unique to Actinomycetota. These findings enhance our understanding of the structure-function relationship of DivIVA and offer new opportunities to study polar growth.

microbiology↗

bioRxiv2023

Finding needles in haystacks: identification of novel conserved PETase enzymes in Streptomyces

The rising use of plastic results in an appalling amount of waste which scatters into the environment affecting environmental, animal, and human health. One of these plastics is PET which is mainly used for bottles and textiles. In this research, we investigate the PET degrading ability of the IsPETase homolog ScLipA from Streptomyces coelicolor. Of 96 different Streptomyces strains screened, 18 % were able to degrade the model substrate BHET. Three different variants of lipase A, named ScLipA, S2LipA and S92LipA were identified and analyzed in detail. The lipA gene was deleted from S. coelicolor M145 using CRISPR/Cas9, resulting in reduced BHET degradation. LipA overexpression in the knock-out background significantly enhanced BHET degradation. All three enzymes were expressed in E. coli BL21 for protein purification and biochemical analysis, showing that enzymatic activity most likely resides in a dimeric form of the enzyme. The optimum pH and temperature were determined to be pH 7 and 25 {degrees}C for all three variants. Using these conditions, the activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and small indents on the surface of PET films, consistent with PET-degrading activity. The frequent occurrence of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more hydrophobic substrates such as these polluting plastics in the environment.

microbiology↗

bioRxiv2022

The plant stress hormone jasmonic acid evokes defensive responses in streptomycetes

Actinobacteria are prevalent in the rhizosphere and phyllosphere of diverse plant species where they help to enhance tolerance of plants against biotic and abiotic stresses. Here, we show that the plant hormones jasmonic acid (JA) and methyljasmonate (MeJA) alter growth, development and specialized metabolism of Streptomyces. Challenge of Streptomyces coelicolor with JA or MeJA led to strongly enhanced production of the polyketide antibiotic actinorhodin. JA is toxic to Streptomycetaceae, whereby members of the genus Streptacidiphilus are generally more sensitive than streptomycetes. As a defensive response, extensive amino acid conjugation of JA was observed; the most prevalent conjugation was with glutamine (Gln), while conjugates with Val, Tyr, Phe and Leu/Ile were identified after longer exposure to JA. Synthetic JA conjugates failed to activate antibiotic production and had strongly reduced toxicity, strongly suggesting that conjugation inactivates JA and serves to detoxify the hormone. Thus, for the first time we provide evidence that plant hormones modulate growth, development and secondary metabolism of streptomycetes, whereby amino acid conjugation serves as a defense strategy by the bacteria to circumvent plant hormone toxicity. IMPORTANCEMicroorganisms that live on or inside plants greatly influence plant health. Streptomycetes are considered to have an important role in defense against plant diseases, but the mechanisms through which they protect plants are currently not fully understood. It has been suggested that streptomycetes respond to changes in the plants physiology, among others by producing protective molecules; however, little is known of the signal transduction from plant to bacterium. We here demonstrate that the plant hormones jasmonic acid (JA) and methyljasmonate (MeJA) directly influence the life cycle of streptomycetes by modulating antibiotic synthesis and promoting faster development. Moreover, the plant hormones specifically stimulate the synthesis of the polyketide antibiotic actinorhodin in Streptomyces coelicolor. Jasmonic acid is then modified in the cell by amino acid conjugation, which reduces the bioactivity of the hormone and thus quenches the signal. To the best of our knowledge, this has not been reported previously. Collectively, these results suggest a relationship between plant physiological changes and the response of streptomycetes in multiple ways.

microbiology↗

bioRxiv2022

Taxonomic and metabolic diversity of Actinobacteria isolated from faeces of a 28,000-year-old mammoth

Ancient microbial communities of permafrost soils and frozen animal remains represent an archive that has barely been explored. This yet unexplored microbial world is a vast resource that can provide us with new evolutionary insights, metabolic pathways and novel chemistry. Here, we reveal that Actinobacteria isolated from a faecal sample from the intestinal tract of a 28,000-year-old Siberian mammoth are phylogenetically and metabolically distinct from currently known modern siblings. Ancient Micromonospora, Oerskovia, Saccharopolyspora, Sanguibacter and Streptomyces species were successfully revived and their genome sequences resolved. Surprisingly, the genomes of the ancestors show a large phylogenetic distance to strains isolated today and harbour many novel biosynthetic gene clusters that may well represent uncharacterised biosynthetic potential. Metabolic profiles of the strains display production of known molecules like antimycin, conglobatin and macrotetrolides, but the majority of the mass features could not be dereplicated. Our work provides a snapshot into Actinobacteria of the past, yielding unexplored genomic information that is not yet present in current databases.

microbiology↗

bioRxiv2021

The nucleoid-associated protein Gbn binds to GATC sequences and affects sporulation and antibiotic production in Streptomyces

Bacterial chromosome structure is organized by a diverse group of proteins collectively referred to as nucleoid-associated proteins (NAPs). Many NAPs have been well studied in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of Actinobacteria NAPs and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Deletion of gbn led to alterations in development and antibiotic production in Streptomyces coelicolor. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all such motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Transcriptomics analysis showed that Gbn binding generally leads to reduced gene expression. The DNA binding profiles were nearly identical between vegetative and aerial growth. Exceptions are SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which nearly exclusively bound during vegetative growth. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes. IMPORTANCEA large part of the chemical space of bioactive natural products is derived from Actinobacteria. Many of the biosynthetic gene clusters for these compounds are cryptic, in others words, they are expressed in nature but not in the laboratory. Understanding the global regulatory networks that control gene expression is key to the development of approaches to activate this biosynthetic potential. Chromosome structure has a major impact on the control of gene expression. In bacteria, the organization of chromosome structure is mediated by a diverse group of proteins referred to collectively as nucleoid-associated proteins (NAPs), which play an important role in the control of gene expression, nucleoid structure and DNA repair. We here present the discovery of a novel and extremely pleiotropic NAP, which we refer to as Gbn. Gbn is a sporulation-specific protein that occurs only in the Actinobacteria and binds to GATC sequences, with a subtle but broad effect on global gene expression. The discovery of Gbn is a new step towards better understanding of how gene expression and chromosome structure is governed in antibiotic-producing streptomycetes.

molecular biology↗

bioRxiv2020

Branching of sporogenic aerial hyphae in sflA and sflB mutants of Streptomyces coelicolor correlates to ectopic localization of DivIVA and FtsZ in time and space

Bacterial cytokinesis starts with the polymerization of the tubulin-like FtsZ, which forms the cell division scaffold. SepF aligns FtsZ polymers and also acts as a membrane anchor for the Z-ring. While in most bacteria cell division takes place at midcell, during sporulation of Streptomyces many septa are laid down almost simultaneously in multinucleoid aerial hyphae. The genomes of streptomycetes encode two additional SepF paralogs, SflA and SflB, which can interact with SepF. Here we show that the sporogenic aerial hyphae of sflA and sflB mutants of Streptomyces coelicolor frequently branch, a phenomenon never seen in the wild-type strain. The branching coincided with ectopic localization of DivIVA along the lateral wall of sporulating aerial hyphae. Constitutive expression of SflA and SflB largely inhibited hyphal growth, further correlating SflAB activity to that of DivIVA. SflAB localized in foci prior to and after the time of sporulation-specific cell division, while SepF co-localized with active septum synthesis. Foci of FtsZ and DivIVA frequently persisted between adjacent spores in spore chains of sflA and sflB mutants, at sites occupied by SflAB in wild-type cells. This may be caused by the persistance of SepF multimers in the absence of SflAB. Taken together, our data show that SflA and SflB play an important role in the control of growth and cell division during Streptomyces development.

microbiology↗

bioRxiv2020

A novel and conserved cell wall enzyme that can substitute for the Lipid II synthase MurG

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule Lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here we present the discovery of a novel cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster including murG surprisingly produced Lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG paralogue, which encodes a putative enzyme that shares only around 31% aa sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MurG2. Orthologues of murG2 are present in 38% of all genomes of Kitasatosporae and members of the sister genus Streptomyces. CRISPRi experiments showed that K. viridifaciens murG2 can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. Altogether, these results identify MurG2 as a bona fide Lipid II synthase, thus demonstrating plasticity in cell wall synthesis.

microbiology↗

bioRxiv2019

Longitudinal cell division is associated with single mutations in the FtsZ-recruiting SsgB in Streptomyces

In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at the mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium Streptomyces, cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here we show via site-saturated mutagenesis that various amino acid substitutions in the highly conserved SsgB protein result in the production of ectopically placed septa, that sever spores diagonally or along the long axis, perpendicular to the division plane. Ectopic septa were especially prominent when cells expressed SsgB variants with substitutions in residue E120. Biochemical analysis of SsgB variant E120G revealed that its interaction with - and polymerization of - FtsZ had been maintained. The crystal structure of S. coelicolor SsgB was resolved and the position of residue E120 suggests its requirement for maintaining the proper angle of helix 3, thus providing a likely explanation for the aberrant septa formed in SsgB E120 substitution mutants. Taken together, our work presents the first example of longitudinal division in a free living bacterium, which is explained entirely by changes in the FtsZ-recruiting protein SsgB.

developmental biology↗

bioRxiv2019

Control of septum thickness by a large protein ring

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and SepF. We have isolated SepF homologues from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 41 nm. Importantly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF determines ring diameter. Importantly, when SepF chimeras with a smaller diameter were expressed in the bacterial host Bacillus subtilis, the thickness of its septa also became smaller. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mould to shape the cell wall. Significance StatementMany bacteria form a thick cell wall and divide by forming a cross wall. How they control the thickness of their cell wall and cross wall is unknown. In this study we show that in these bacteria the cell division protein SepF forms very large protein rings with diameters that correspond to the diameter of their cross walls. Importantly, when we reduced the diameter of SepF rings in the bacterial host Bacillus subtilis the cross wall also became thinner. These results provide strong evidence that a large protein ring can function as a mould to control the thickness of the cell wall that divides these bacterial cells.

microbiology↗

bioRxiv2018

Classification of the gifted natural product producer Streptomyces roseofaciens sp. nov. by polyphasic taxonomy

A novel verticillate strain of streptomycetes, Streptomyces strain MBT76T, was isolated from the QinLing mountains, which harbours more than 40 biosynthetic gene clusters for natural products. Here we present full taxonomic classification of strain MBT76T, and show that it has chemotaxonomic, genomic and morphological properties consistent with its classification in the genus Streptomyces. Strain MBT76T is part of the cluster of Streptoverticillates, a group within the genus Streptomyces that has characteristic whorl-forming spores produced in chains along the lateral wall of the hyphae. Multi-locus sequence analysis based on five housekeeping gene alleles showed that MBT76T is closely related to Streptomyces hiroshimensis. Average Nucleotide Identification (ANI) and Genome to Genome Distance Calculation (GGDC) of the genomes of strain MBT76T and S. hiroshimensis separated them into distinct species. Strain MBT76T represents a novel species of the genus Streptomyces for which we propose the name Streptomyces roseofaciens sp. nov. The type strain is MBT76T (=NCCB 100637T =DSM 106196T). The whole genome of MBT76T has 7974 predicted open reading frames and a total genome size of 8.64 Mb. Further genomic analysis showed that verticillate streptomycetes lack the sporulation gene ssgE, and our data suggest that this is a useful genetic marker for the spore-chain morphology of the verticillates.

microbiology↗

bioRxiv2017

Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity

Many actinobacteria live in close association with eukaryotes like fungi, insects, animals and plants. Plant-associated actinobacteria display (endo)symbiotic, saprophytic or pathogenic life styles, and can make up a substantial part of the endophytic community. Here, we characterised endophytic actinobacteria isolated from root tissue of Arabidopsis thaliana (Arabidopsis) plants grown in soil from a natural ecosystem. Many of these actinobacteria belong to the family of Streptomycetaceae with Streptomyces olivochromogenes and Streptomyces clavifer as well represented species. When seeds of Arabidopsis were inoculated with spores of Streptomyces strain coa1, which shows high similarity to S. olivochromogenes, roots were colonised intercellularly and, unexpectedly, also intracellularly. Subsequent exposure of endophytic isolates to plant hormones typically found in root and shoot tissues of Arabidopsis led to altered antibiotic production against Escherichia coli and Bacillus subtilis. Taken together, our work reveals remarkable colonization patterns of endophytic streptomycetes with specific traits that may allow a competitive advantage inside root tissue.

microbiology↗

bioRxiv2017

YlmD and YlmE are required for correct sporulation-specific cell division in Streptomyces coelicolor A3(2)

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

microbiology↗

bioRxiv2017

SParticle, an algorithm for the analysis of filamentous microorganisms in submerged cultures

Streptomycetes are filamentous bacteria that produce a plethora of bioactive natural products and industrial enzymes. Their mycelial lifestyle typically results in high heterogeneity in bioreactors, with morphologies ranging from fragments and open mycelial mats to dense pellets. There is a strong correlation between morphology and production in submerged cultures, with small and open mycelia favoring enzyme production, while most antibiotics are produced mainly in pellets. Here we describe SParticle, a Streptomyces Particle analysis method that combines whole slide imaging with automated image analysis to characterize the morphology of submerged grown Streptomyces cultures. SParticle allows the analysis of over a thousand particles per hour, offering a high throughput method for the imaging and statistical analysis of mycelial morphologies. The software is available as a plugin for the open source software ImageJ and allows users to create custom filters for other microbes. Therefore, SParticle is a widely applicable tool for the analysis of filamentous microorganisms in submerged cultures.

microbiology↗

bioRxiv2016

Reversible metamorphosis in a bacterium

The cell wall is a shape-defining structure that envelopes almost all bacteria. One of its main functions is to serve as a protection barrier to environmental stresses. Bacteria can be forced in a cell wall-deficient state under highly specialized conditions, which are invariably aimed at interrupting cell wall synthesis. Therefore, the relevance of such cells has remained obscure. Here we show that many filamentous actinomycetes have a natural ability to generate a new, cell wall-deficient cell type in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the canonical mycelial mode-of-growth. Remarkably, prolonged exposure of S-cells to hyperosmotic stress yielded variants that are able to proliferate indefinitely without their cell wall. This is the first report that demonstrates the formation of wall-deficient cells as a natural adaptation strategy and their potential transition into stable wall-less forms solely caused by prolonged exposure to osmotic stress. Given that actinomycetes are potent antibiotic producers, our work also provides important insights into how biosynthetic gene clusters and resistance determinants may disseminate into the environment.

microbiology↗

bioRxiv2016

Hyphal aggregation and surface attachment of Streptomyces is governed by extracellular poly-β-1,6-N-acetylglucosamine

Streptomycetes are multicellular filamentous microorganisms, which are major producers of antibiotics, anticancer drugs and industrial enzymes. When grown in submerged cultures, the preferred enzyme producer, Streptomyces lividans, forms dense mycelial aggregates or pellets, which requires the activity of the proteins encoded by the matAB and cslA-glxA. Here we show that matAB encodes the biosynthetic genes for the extracellular polymeric substance (EPS) poly-{beta}-1,6-N-acetylglucosamine or PNAG. Heterologous expression of matAB in actinomycetes that naturally lack these genes was sufficient for PNAG production and induction of mycelial aggregation. Also, overexpression of matAB in a non-pelleting cslA mutant restored pellet formation, which could effectively be antagonized by the PNAG-specific hydrolase, dispersin B. Extracellular accumulation of PNAG allowed Streptomyces to attach to hydrophilic surfaces, unlike attachment to hydrophobic surfaces, which involves a cellulase-degradable EPS produced by CslA. Altogether, our data support a model in which pellet formation depends on hydrophilic interactions mediated by PNAG and hydrophobic interactions involving the EPS produced by CslA. These new insights may be harnessed to improve growth and industrial exploitation of these highly versatile natural product and enzyme producers.

microbiology↗