Biology subjects

Dey, S.

Publications and source records attributed to Dey, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

From Sequence to Significance: A Thorough Investigation of the Distinctive Genome Traits Uncovered in C. werkmanii strain NIB003

Citrobacter werkmanii (C. werkmanii) is an opportunistic bacterium that has received very little investigation in Bangladesh, even though it is thought to be capable of causing diseases including diarrhea and urinary tract infections. Therefore, the present study focuses on the exploration of genomic features, including antibiotic resistance genes, virulent genes, and their distinctive nature in the pathogenesis of diseases. A complete genome was sequenced for the first time in Bangladesh after the morphological, biochemical and molecular identification of this bacteria. A total of 47,65,861 nucleotide base pairs makes up the genome, which contains 4669 genes, and many of the protein-coding genes can be classified into various ontological frameworks, including COG and KEGG pathways. Notably, C. werkmanii has the capability to have interactions with Escherichia coli, Salmonella, and Klebsiella while they possess the same domains and motifs, indicating a causal role in the development of diarrhea. Furthermore, the Bangladeshi isolate of C. werkmaii has observed genetic heterogeneity when compared to other strains of C. werkmanii: C. werkmanii FDAARGOS_616, C. werkmanii LCU-V21, and C. werkmanii BF-6. This identified genetic variation of sequenced C. werkmanii could drive the functional aberration in body homeostasis of bacteria. Later, molecular dynamics studies have proven the impact of their genetic variation on the protein structure, as indicated by the SASA, RMSD, Rg, and RMSF values. Finally, the findings of C. werkmanii from the sequenced genome might provide us with a better understanding of C. werkmanii and their possible role as opportunistic bacteria in diseases including diarrhea and urinary tract infections.

genomics↗

bioRxiv2023

Population size shapes trade-off dilution and adaptation to a marginal niche unconstrained by sympatric habitual conditions

Niche expansion remains significantly understudied in sympatric scenarios where marginal and habitual niches are simultaneously available. Devoid of spatial constraints, such conditions impose selection to maintain fitness in habitual (high-productivity) niches while adapting to marginal (low-productivity) niches. Hence, habitual niche composition should constrain adaptation to marginal niches. This remains untested. Similarly, it is unknown if larger populations expand their niches better. We tested these hypotheses using experimental evolution with Escherichia coli and found that larger populations consistently adapted better to both marginal and habitual niches. Whereas the habitual niche composition (constant versus fluctuating environments; environmental fluctuations varying in both predictability and speed) significantly shaped fitness in habitual niches, surprisingly, it failed to constrain adaptation to the marginal niche. Curiously, two negatively correlated habitual niches can still each be positively correlated with the marginal niche. This allows the marginal niche to dilute trade-offs between habitual niches, thereby enabling costless niche expansion.

evolutionary biology↗

bioRxiv2023

In vivo and in silico experiment on Mitragyna speciosa offers new insight of antidiabetic potentials

Diabetes Mellitus (DM) is a serious metabolic disease with several treatments available for managing it, however, they can be expensive and have side effects. Medicinal plants have been used for many years to treat numerous diseases. Mitragyna speciosa (M. speciosa) plant has been shown to have anti-diabetic properties in preclinical studies. Hence, this study aimed to investigate the phytochemical compositions and anti-diabetic effects of M. speciose, using both in vivo and in silico approaches. In the in vivo study, experimental diabetes was induced in Swiss albino mice using the drug alloxan (150 mg/kg). Four groups of diabetic mice were taken. Two of the groups were given extracts at doses of 200 mg/kg and 400 mg/kg respectively. Diabetic mice treated with the reference drug, glibenclamide (5mg/kg) were chosen as a positive control, and mice with only vehicles were considered as a negative control. The Oral Glucose Tolerance Test (OGTT) and the acute toxicity test were performed. In the in silico study, molecular docking and dynamics were performed for the identification of the plant compounds that could effectively bind with the DPP4 receptor. Analysis of the study suggested that the lethal dose (LD50) values were greater than 2000 mg/kg, indicating that a dose below this level can be selected. The OGTT results showed that both doses of M. speciose extracts significantly reduced blood glucose levels (P<0.0001). However, neither dose exhibited a significantly higher blood glucose reduction compared to glibenclamide (5 mg/kg) (P > 0.05). Phytochemical screening and the ADMET profile analysis suggested four key compounds: Mitragynine, Corynantheidine, Corynoxine, and Speciociliatine in M. speciosa. The molecular docking analysis revealed these four compounds as potential antidiabetic agents considering their high binding affinity to the DPP4 receptor. These compounds were also found to be stable in the DPP4 binding pocket, as evidenced by the molecular dynamics simulation. Lastly, it can be demonstrated that the in silico experiments confirmed M. speciosa extracts to be capable of reducing blood glucose levels because of the presence of compounds.

pharmacology and toxicology↗

bioRxiv2023

Selection for dispersal under larval malnutrition results in a non-monotonic kernel in Drosophila melanogaster

Dispersal is a key strategy for organisms to track favorable conditions and a shifting global climate has necessitated its closer investigation. While the distribution of dispersal distances (i.e. dispersal kernel) influences several aspects of population ecology, it remains unclear how kernel features are themselves affected by environmental factors. The current study addresses this question using laboratory populations of Drosophila melanogaster selected for greater dispersal under a protein-deficient larval diet. Our results reveal that dispersal-selected populations initiate dispersal more often and travel further on average than unselected controls. Although there is a significant change in the overall kernel shape, selection has not led to a greater tendency for long-distance dispersal (LDD). This is contrary to the results from a previous study on dispersal evolution under standard nutrition. We also find that dispersal evolution under larval malnutrition leads to a non-monotonic dispersal kernel, with the highest frequencies at intermediate distances. The evolved shape of the dispersal kernel and the limited LDD phenotype therefore point to significant qualitative shifts in the evolution of the dispersal kernel due to a change in larval nutrition. Our results thus provide a starting point for further investigation of such context dependence in dispersal evolution.

evolutionary biology↗

bioRxiv2023

A resource-based mechanistic framework for castration-resistant prostate cancer (CRPC)

Cancer therapy often leads to the selective elimination of drug-sensitive cells from the tumour. This can favour the growth of cells resistant to the therapeutic agent, ultimately causing a tumour relapse. Castration-resistant prostate cancer (CRPC) is a well-characterised instance of this phenomenon. In CRPC, after systemic androgen deprivation therapy (ADT), a subset of drug-resistant cancer cells autonomously produce testosterone, thus enabling tumour regrowth. A previous theoretical study has shown that such a tumour relapse can be delayed by inhibiting the growth of drug-resistant cells using biotic competition from drug-sensitive cells. In this context, the centrality of resource dynamics to intra-tumour competition in the CRPC system indicates clear scope for the construction of theoretical models that can explicitly incorporate the underlying mechanisms of tumour ecology. In the current study, we use a modified logistic framework to model cell-cell interactions in terms of the production and consumption of resources. Our results show that steady state composition of CRPC can be understood as a composite function of the availability and utilisation efficiency of two resources-oxygen and testosterone. In particular, we show that the effect of changing resource availability or use efficiency is conditioned by their general abundance regimes. Testosterone typically functions in trace amounts and thus affects steady state behaviour of the CRPC system differently from oxygen, which is usually available at higher levels. Our data thus indicate that explicit consideration of resource dynamics can produce novel and useful mechanistic understanding of CRPC. Furthermore, such a modelling approach also incorporates variables into the systems description that can be directly measured in a clinical context. This is therefore a promising avenue of research in cancer ecology that could lead to therapeutic approaches that are more clearly rooted in the biology of CRPC. HighlightsO_LICancer growth and progression can be driven by intra-tumour interactions. C_LIO_LIProstate cancer cells compete among each other for resources like testosterone and oxygen. C_LIO_LIWe model these interactions using a modified logistic model with resource dynamics. C_LIO_LIEquilibrium behaviour can be understood through resource supply and use efficiency. C_LIO_LIExplicitly ecological models could enable better strategies for cancer control. C_LI

cancer biology↗

bioRxiv2023

KLP-7/Kinesin-13 orchestrates axon-dendrite checkpoints for polarized trafficking in neurons

Neurons are compartmentalized cells with spatiotemporal distinction of anatomy and molecular repertoire. Microtubule organization in the neuron is crucial for its polarized structure and composition. Microtubule dynamics are differentially optimized in the axons and dendrites by the interplay between the microtubule-stabilizing and destabilizing factors. It is unclear how the destabilizing factors are important for developing and maintaining neuronal polarity. We investigated the function of KLP-7, a microtubule depolymerizing motor from the Kinesin-13 family, in the compartmentalization of axons and dendrites using the PVD neurons in Caenorhabditis elegans. In the absence of KLP-7, axonal proteins such as RAB-3 and SAD-1 were mislocalized to dendrites, suggesting a disruption in axon-dendrite compartmentalization. Notably, this phenomenon was independent of other depolymerizing factors like EFA-6, highlighting the specific role of KLP-7 in this process. We observed a reduced rate of microtubule polymerization and an altered polarity of microtubules in the PVD major dendrite due to the loss of klp-7. Additionally, the deletion of klp-7 led to the formation of ectopic neurites from the cell body and the ectopic localization of UNC-44/Ankyrin-G, a protein associated with the axon initial segment (AIS), to the dendrites. Additionally, live imaging of GFP::KLP-7 revealed that KLP-7 is more dynamic in the dendrites as compared to the axon. These observations indicate that the precise dynamics of KLP-7 in neurites are crucial for maintaining distinct microtubule polymerization in the axons and dendrites, thereby influencing neuronal polarity. Our findings shed light on the pivotal role of KLP-7/Kinesin-13 in the establishment of axon-dendrite checkpoints, which in turn impact the polarized trafficking of cellular components within neurons.

cell biology↗

bioRxiv2023

Functional recovery associated with dendrite regeneration in PVD neuron of C. elegans

PVD neuron of C. elegans is a highly polarized cell with well-defined axonal, and dendritic compartments. PVD neuron operates in multiple sensory modalities controlling both nociceptive touch sensation and body posture. Although both axon and dendrites of this neuron show regeneration response following laser-assisted injury, it is rather unclear how the behavior associated with this neuron is affected by the loss of these structures. It is also unclear whether neurite regrowth would lead to functional restoration in these neurons. Upon axotomy, using a femtosecond laser, we saw that harsh touch response was specifically affected leaving the body posture unperturbed. Subsequently, recovery in the touch response is highly correlated to the axon regrowth, which was dependent on DLK-1 MAP Kinase. Dendrotomy of both major and minor primary dendrites affected the wavelength and amplitude of sinusoidal movement without any apparent effect on harsh touch response. We further correlated the recovery in posture behavior to the type of dendrite regeneration events. We found that dendrite regeneration with the fusion and reconnection between the proximal and distal branches of the injured dendrite corresponded to improved recovery of posture. Our data revealed that the axons and dendrites differentially regulate the functions of PVD neurons. It also revealed that dendrite and axon regeneration are both functionally and molecularly distinct.

neuroscience↗

bioRxiv2023

Leishmania major-induced alteration of host cellular and systemic copper homeostasis drives the fate of infection

Copper plays a key role in host-pathogen interaction. We found that during Leishmania major infection, the parasite-harboring macrophage regulates its copper homeostasis pathway in a way to facilitate copper-mediated neutralization of the pathogen. Copper-ATPase ATP7A transports copper to amastigote-harboring phagolysosomes to induce stress on parasites. Leishmania in order to evade the copper stress, utilizes a variety of manipulative measures to lower the host-induced copper stress. It induces deglycosylation and degradation of host-ATP7A and downregulation of copper importer, CTR1 by cysteine oxidation. Additionally, Leishmania induces CTR1 endocytosis that arrests copper uptake. In mouse model of infection, we report an increase in systemic bioavailable copper in infected animals. Heart acts as the major organ for diverting its copper reserves to systemic circulation to fight-off infection by downregulating its CTR1. Our study explores reciprocal mechanism of manipulation of host copper homeostasis pathway by macrophage and Leishmania to gain respective advantages in host-pathogen interaction.

cell biology↗

bioRxiv2023

An atlas of protein homo-oligomerization across domains of life

Protein structures are essential to understand cellular processes in molecular detail. While advances in AI revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. Here, we describe a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. We find that 50% of archaeal, 45% of bacterial, and 20% of eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of novel homo-oligomer types. Analyzing these datasets reveals coiled-coil regions as major enablers of quaternary structure evolution in Eukaryotes. Integrating these structures with omics data shows that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations, which we find enriched at interfaces. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes, protein networks, and disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/544317v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1507a12org.highwire.dtl.DTLVardef@7e522aorg.highwire.dtl.DTLVardef@1445410org.highwire.dtl.DTLVardef@eb09f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Regulation of shelterin proteins TERF2IP and TRF2 by H3K4me3-p65 axis drives hyperglycemia dependent endothelial senescence

BackgroundEndothelial senescence has been linked to several cardiovascular diseases. Dysregulation of proteins of the shelterin complex including TRF2 and TERF2IP causes senescence as it hampers DNA repair and cell proliferation. However, whether exposure to hyperglycemia interplays with proteins of the shelterin complex thus further dictates the senescent phenotype of endothelial cells (EC) remain to be explored. Approach and ResultsIn this study, we observed elevated levels of p21 and p53 in endothelial cells upon exposure to intermittent hyperglycemia. We also noted hyperglycemia exposure increased the levels of TERF2IP and TRF2, part of the shelterin complex. No change in the level of TRF1 and TPP1 were detected. Furthermore, a robust induction was detected in p65 level upon intermittent hyperglycemia challenge. ChIP-qPCR analysis revealed enhanced H3K4me3 enrichment in the promoter regions of p65, TERF2IP and TRF2. Inhibition of catalysis of H3K4me3 either by pharmacological inhibitor or siRNA-mediated knockdown of MLL2 attenuated increase in p65, TERF2IP and TRF2 levels including reversal of senescence markers p53 and p21. Interestingly, pharmacological inhibition of NF-{kappa}B signaling also diminished abrupt increase in TERF2IP and TRF2 levels thus further reversed intermittent hyperglycemia-induced p53 and p21 levels. More importantly, co-immunoprecipitation and co-localization analysis revealed an interaction between nuclear p65 and MLL2 in EC stimulated with hyperglycemia. Further knockdown of either TERF2IP or TRF2 impaired intermittent hyperglycemia-induced p53 and p21 expression and associated endothelial senescence. ConclusionOverall, the present study describes an interplay of epigenetics in defining NF-{kappa}B signaling and shelterin proteins expression which further govern the biochemical and functional state of endothelial senescence in hyperglycemic milieu.

biochemistry↗

bioRxiv2023

UVB modifies skin immune-stroma cross-talk and promotes effector T cell recruitment during cryptic Leishmania donovani infection

Many parasites of significant public health importance assume skin residency without causing overt pathlogy. How immune and stromal cells respond to such "cryptic" infections and how exposure to UVB alters such responses in poorly understood. We combined scRNA-seq, spatial transcriptomics and inferential network analysis to address these questions in a model of cryptic skin infection by Leishmania donovani. In infected C57BL/6 mice, p-selectin and CXCL12 interactions dominate intercellular communication between leucocytes, fibroblast and endothelial cells, but effector T cell function remains muted. Following UVB exposure, increased numbers of IFN{gamma}+ CD4+ Th1 cells and NK cells enter the skin, communicating with stromal cells via CCL5-CCR5 and LFA-1-ICAM1/2. However, spatial mapping indicated that Th1 cells and macrophages occupied distinct niches after UVB exposure, likely limiting effector function. Our data provide the first holistic view of the immune landscape during cryptic L. donovani infection and demonstrate how UVB exposure fundamentally reshapes this response. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/526940v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1238cbaorg.highwire.dtl.DTLVardef@ec350eorg.highwire.dtl.DTLVardef@18f412corg.highwire.dtl.DTLVardef@1ffd468_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Molecular and cellular mechanisms by which cardiac sympathetic denervation prevents heart failure and sudden cardiac arrest

RATIONALESudden cardiac arrest (SCA) and heart failure (HF) are leading causes of death. The underlying mechanisms are incompletely understood, limiting the design of new therapies. Whereas most autonomic modulation therapies have not shown clear benefit in HF patients, growing evidence indicates cardiac sympathetic denervation (CSD) exerts cardioprotective effects. The underlying molecular and cellular mechanisms remain unexplored. OBJECTIVEBased on the hypothesis that mitochondrial reactive oxygen species (mROS) drive the pathogenesis of HF and SCA, we investigated whether CSD prevents SCA and HF by improving mitochondrial antioxidant capacity and redox balance, to correct impaired Ca2+ handling and repolarization reserve. METHODS AND RESULTSWe interrogated CSD-specific responses in pressure-overload HF models with spontaneous SCA using in vivo echocardiographic and electrocardiographic studies and in vitro biochemical and functional studies including ratiometric measures of mROS, Ca2+ and sarcomere dynamics in left ventricular myocytes. Pressure-overloaded HF reduced mitochondrial antioxidant capacity and increased mROS, which impaired {beta}-adrenergic signaling and caused SR Ca2+ leak, reducing SR Ca2+ and increasing diastolic Ca2+, impaired myofilament contraction and further increased the sympathetic stress response. CSD improved contractile function and mitigated mROS-mediated diastolic Ca2+ overload, dispersion of repolarization, triggered activity and SCA by upregulating mitochondrial antioxidant and NADPH-producing enzymes. CONCLUSIONSOur findings support a fundamental role of sympathetic stress-induced downregulation of mROS scavenging enzymes and RyR-leak mediated diastolic Ca2+ overload in HF and SCA pathogenesis that are mitigated by CSD. This first report on the molecular and cellular mechanisms of CSD supports its evaluation in additional high-risk patient groups. BRIEF SUMMARYCardiac sympathetic denervation (CSD) confers cardioprotective effects via unclear mechanisms. In a guinea pig model that uniquely mimics human pressure-overload heart failure (HF) with spontaneous sudden cardiac arrest (SCA), we interrogated CSD-specific responses using echocardiographic, electrocardiographic and biochemical measures, and ratiometric measures of mitochondrial reactive oxygen species (mROS), Ca2+ and sarcomere dynamics. Consistent with our hypothesis, CSD rescued cardioprotection by upregulating mitochondrial antioxidant and NADPH-producing enzymes, which mitigate mROS-mediated Ca2+ derangements, repolarization lability, triggered activity, HF and SCA. Our findings provide the first molecular and cellular mechanistic basis for evaluating CSD therapy in a broader group of high-risk patients.

physiology↗

bioRxiv2023

Ryanodine receptor inhibition improves cardiac repolarization reserve and contractile function and prevents sudden arrhythmic death in failing hearts.

IntroductionSudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) are a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)- mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. ObjectiveTo test the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. MethodsWe used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. ResultsDS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient {beta}-adrenergic challenge, and improved heart rate variability and cardiac function. ConclusionInhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca2+ leak-induced increases in diastolic Ca2+ and ROS-mediated RyR2 oxidation, thereby increasing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.

physiology↗

bioRxiv2022

Plasmonic Stimulation of Gold Nanorods for the Photothermal Control of Engineered Living Materials

Engineered living materials (ELMs) use encapsulated microorganisms within polymeric matrices for biosensing, drug delivery, capturing viruses, and bioremediation. It is often desirable to control their function remotely and in real time. Suitable, genetically engineered microorganisms respond to changes of their environment. Here, we combine this local sensitivity with a nanostructured encapsulation material to sensitize the ELM for infrared light. Previously, blue light has been used to stimulate microorganisms that contain optogenetic modules responsive to those wavelengths without the need for exogenous cofactors. Here, we use plasmonic gold nanorods (AuNR) that have a strong absorption maximum at 808 nm, a wavelength where human tissue is relatively transparent. Biocompatible composites of a Pluronic-based hydrogel and AuNR are prepared without agglomeration; they react to illumination by local heating. We measure a photothermal conversion efficiency of 47 % in transient temperature measurements. Steady-state temperature profiles from local photothermal heating are quantified using infrared photothermal imaging, correlated with measurements inside the gel, and applied to stimulate thermoresponsive bacteria. Using a bilayer ELM construct with the thermoresponsive bacteria and the thermoplasmonic composite gel in two separate but connected hydrogel layers, it is shown that the bacteria can be stimulated to produce a fluorescent protein using infrared light in a spatially controlled manner.

bioengineering↗

bioRxiv2022

Why Does Synergistic Activation of WASP, but Not N-WASP, by Cdc42 and PIP2 Require Cdc42 Prenylation?

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

biophysics↗

bioRxiv2022

Bacteria evolve macroscopic multicellularity via the canalization of phenotypically plastic cell clustering

The evolutionary transition from unicellular to multicellular life was a key innovation in the history of life. Given scarce fossil evidence, experimental evolution has been an important tool to study the likely first step of this transition, namely the formation of undifferentiated cellular clusters. Although multicellularity first evolved in bacteria, the extant experimental evolution literature on this subject has primarily used eukaryotes. Moreover, it focuses on mutationally driven (and not environmentally induced) phenotypes. Here we show that both Gram-negative and Gram-positive bacteria exhibit phenotypically plastic (i.e., environmentally induced) cell clustering. Under high salinity, they grow as elongated ~ 2 cm long clusters (not as individual planktonic cells). However, under habitual salinity, the clusters disintegrate and grow planktonically. We used experimental evolution with Escherichia coli to show that such clustering can be canalized successfully: the evolved bacteria inherently grow as macroscopic multicellular clusters, even without environmental induction. Highly parallel mutations in genes linked to cell wall assembly formed the genomic basis of canalized multicellularity. While the wildtype also showed cell shape plasticity across high versus low salinity, it was either canalized or reversed after evolution. Interestingly, a single mutation could canalize multicellularity by modulating plasticity at multiple levels of organization. Taken together, we show that phenotypic plasticity can prime bacteria for evolving undifferentiated macroscopic multicellularity.

evolutionary biology↗

bioRxiv2022

Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due since their complex and thick cell walls limit our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 g) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as increase in plasmid size, different methylation patterns and limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on Gibson assembly and PCR to amplify the DNA to sufficient quantities for successful transformation in L. plantarum WCFS1. This advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

synthetic biology↗

bioRxiv2022

An Arg/Ala-Rich Helix in the N-Terminal Region of M. tuberculosis FtsQ Anchors FtsZ to Membranes

Mycobacteria tuberculosis (Mtb) inflicts a quarter of the worldwide population. Most drugs for treating tuberculosis target cell growth and division. With rising drug resistance, it becomes ever more urgent to better understand Mtb cell division. This process begins with the formation of the Z-ring via polymerization of FtsZ and anchoring of the Z-ring to the inner membrane. Here, by combining solution and solid-state NMR spectroscopy with molecular dynamics simulations and other techniques, we show that the transmembrane protein FtsQ is a membrane anchor of the Mtb Z-ring. In the otherwise disordered N-terminal cytoplasmic region of FtsQ (residues 1-99), a 29-residue, Arg/Ala-rich -helix is formed that interacts with upstream acidic residues in solution and with acidic lipids at the membrane surface. The same helix also binds to the GTPase domain of FtsZ, with enormous implications for drug binding and Z-ring formation including its curvature.

biophysics↗