Biology subjects

Mangayil, R.

Publications and source records attributed to Mangayil, R..

10 recordsLinked to original sources

bioRxiv2026

Strain handling exposes IS-mediated recurrent genome reduction at a dynamic accessory locus in Komagataeibacter rhaeticus

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.

microbiology↗

bioRxiv2026

Engineering the bacterial cellulose-forming surface as a programmable protein recruitment interface

Synthetic biology has advanced microorganisms to be programmed as production hosts, but its application to bacteria that inherently assemble extracellular materials remains limited. Komagataeibacter spp., natively synthesizes cellulose at the bacterial cell surface, creating a material-forming interface that has not been used as a programmable recruitment platform. Here we establish cell-surface display in Komagataeibacter intermedius and show that this interface can recruit defined proteins, making functionalization part of cellulose formation. By engineering LppOmpA, we displayed a fluorescent protein and genetically encoded capture modules (SpyTag and SilkTag) to selectively capture catcher-fused protein cargos onto K. intermedius cell surface. Recruitment of silk-derived structural protein before cellulose production generated silk-associated fibrous structures within the pellicles, with retained cargo signal after washing. The resulting biocomposite showed reorganized fibre-network morphology, increased surface hydrophobicity, mesoscale ordering, and improved wet-state compressive strength. Wild-type cells exposed to same conditions did not reproduce these changes, demonstrating that material properties arise from surface-directed recruitment rather than protein exposure alone. This work demonstrates the material-forming bacterial surface as a programmable engineering interface for organizing extracellular proteins, providing a general strategy for engineering living materials.

bioengineering↗

bioRxiv2025

Engineering signal peptide-mediated recombinant protein secretion in Komagataeibacter rhaeticus for biological Engineered Living Material applications

BackgroundBacterial nanocellulose (BC), produced by Komagataeibacter species, is an ideal scaffold for biological Engineered Living Materials (bioELMs) research. Current BC functionalization strategies often rely on secondary microbial hosts or post-production enzyme immobilization, limiting the scalability and modularity required for programmable bioELMs. Establishing a single-chassis system capable of simultaneous biopolymer synthesis and in situ functionalization remains a primary objective in bioELM research. This study addresses the need by benchmarking signal peptide-mediated protein translocation in K. rhaeticus iGEM, a model bacterium for BC-based bioELMs, enabling a synthetic biology framework for single-chassis based biomaterial functionalization. ResultsGenome-wide analysis confirmed the presence of a complete Sec translocation machinery in K. rhaeticus. Through liquid chromatography-tandem mass spectrometry and SignalP 5.0 prediction, native signal peptides were identified and evaluated alongside previously characterized heterologous signal peptides using {beta}-lactamase and mScarlet as cargo proteins. Protein translocation was found to depend on signal peptide identity, cargo type, and expression mode. Fluorescence imaging revealed cytoplasmic, polar, and peripheral localization patterns, confirming functional engagement with the native translocation machinery. A key limitation identified was the retention of recombinant proteins within the periplasm, restricting extracellular availability. Despite this, signal peptide-mediated translocation enabled the incorporation of enzymatic activity into BC during biosynthesis. A post-growth osmotic shock-release strategy increased measurable enzymatic activity by 30%, demonstrating a practical route to overcome this physiological bottleneck while maintaining the biomaterial production capacity. ConclusionsThis study benchmarks signal peptide-dependent protein translocation in K. rhaeticus and identifies periplasmic retention as a key constraint for extracellular protein release. By linking protein translocation to in situ BC functionalization, this work establishes a synthetic biology framework that supports the development of K. rhaeticus as a single-chassis platform for the scalable production of functionalized bioELMs.

bioengineering↗

bioRxiv2025

Exploring Genome-Wide Mutation Dynamics and Bacterial Cellulose Impairment in Komagataeibacter intermedius Cultivated Under Agitation Stress

BackgroundBacterial cellulose (BC), natively synthesized by Komagataeibacter spp., is a biodegradable biomaterial with superior mechanical properties. However, under agitated cultivation, cellulose producing strain (Cel +) often transition to non-producing mutants (Cel -), restricting scalability and hindering widespread use. Agitation-associated shear stress, elevated oxygen levels, and genetic mutations have been linked to the emergence of the Cel- phenotype. A genome-wide investigation that considers population heterogeneity and dynamics is essential to reveal the mutational landscape and evolutionary processes driving this phenotypic shift. ResultsOver successive rounds of agitated cultivation, the strain transitioned to a planktonic state, losing BC production. Whole-genome sequencing revealed both structural variations (SVs) and non-structural variations (NSVs). Contrary to previous reports, SVs, including insertion sequences (IS) mediated junction events, did not affect genes related to BC synthesis. Instead, the accumulation and positive selection of NSVs, such as frameshift and replication slippage events, in key BC-related genes, strongly correlated with the loss of cellulose synthesis ConclusionsThis study provides the first genome-wide population-level analysis revealing mutational dynamics underlying the BC phenotypic switch in Komagataeibacter spp. under agitated conditions. We show that BC-related gene mutations are not solely driven by SVs, with NSVs emerging as equally critical contributors. Furthermore, the genomic evidence implicates broader involvement of quorum sensing and cyclic dimeric guanosine monophosphate signaling, suggesting both genetic and regulatory factors underlying the disruption of BC synthesis. These findings highlight the importance of examining genetic heterogeneity with population to understand phenotypic adaptation for strain improvement strategies for scalable BC production.

microbiology↗

bioRxiv2025

Integrated Multi-analytical Framework for Comprehensive Characterization of Lignocellulosic Hydrolysates

Lignocellulosic hydrolysates are promising second-generation feedstocks for sustainable biomanufacturing. However, biomass compositional complexities and a lack of analytical framework, particularly for lignin fraction (LF), have limited holistic characterization and impeded downstream valorization. In this study, we present an integrated multi-analytical framework for in-depth compositional profiling of spruce sawdust hydrolysate (SSH). Combining high-performance anion-exchange chromatography and liquid chromatography, sugars and inhibitors were quantified, with monomeric arabinose (78.8{+/-}0.6 g/L) and acetic acid (14.9{+/-}0.1 g/L) as predominant components, respectively. Additionally, we developed a workflow to quantify and evaluate lignin structures and interlinkages, determining factors in biomanufacturing using a unique spectroscopic-chromatographic approach enabling a comprehensive understanding of the LF. UV-Vis spectroscopy quantified a total aromatic content of 58.2{+/-}0.1g/L, while quantitative 31P NMR clarified the hydroxyl group distribution in the LF. 1H-13C HSQC NMR provided structural identification of lignin units and interconnecting linkages, and gel permeation chromatography showed the molecular weight distribution. Gas chromatography-mass spectrometry identified and quantified 38 lignin-derived monomers and extractives, with benzoic acid (2.7{+/-}0.2 mg/L) as the most abundant. This integrated approach provides holistic biomass compositional insights and cross-validation across methods, overcomes the limitations of a single technique, and delivers a robust workflow to evaluate the biomass suitability for bio-valorization. ABSTRACT GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/675780v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@db6eceorg.highwire.dtl.DTLVardef@47b9b3org.highwire.dtl.DTLVardef@f0a85aorg.highwire.dtl.DTLVardef@1bcb66c_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISThis study develops an integrated multi-analytical workflow to comprehensively profile biomass hydrolysate for downstream valorization.

bioengineering↗

bioRxiv2024

Modulating Bacterial Nanocellulose Crystallinity through Post-Transcriptional Repression in Komagataeibacter xylinus

Bacterial nanocellulose (BC), a versatile and biodegradable polymer, has been extensively studied as an alternative biomaterial for various applications. For biomedical and packaging uses, where precise control over nanocellulose structure is essential, existing literature describes BC structural modification processes that involve additives or additional steps. With the aim to develop a programmable method to control the bulk microstructure, we developed sRNA-based post-transcriptional repression cassettes that allows precise regulation of the crystalline phase of BC. Before investigating the effects of post-transcriptional repression of bcsD, bcsZ, and ccpA genes, known to influence BC crystallinity, we validated the specificity of the sRNA repression cassette by targeting a genome-integrated red fluorescent protein, mRFP1. The observed growth inhibition (>80%) caused by overexpressed Hfq RNA chaperone in Komagataeibacter xylinus was alleviated ([≤] 23%) by its removal, resulting in a 43% reduction in relative mRFP1 expression. By varying the design of the repression cassette and the inducer concentrations, we successfully modulated the repression of the target genes [with relative expression reductions of 6%-34% for bcsD, 8%-24% for bcsZ, and 2%-20% for ccpA, as confirmed by qRT-PCR]. These gene repression levels led to statistically significant changes in the amorphous content of the BC microstructure, as demonstrated by X-ray diffraction and wide-angle X-ray scattering analysis.

bioengineering↗

bioRxiv2022

Characterization and genome analysis of a psychrophilic methanotroph representing a ubiquitous Methylobacter spp. cluster in boreal lake ecosystems

Lakes and ponds are considered as a major natural source of CH4 emissions, particularly during the ice-free period in boreal ecosystems. Aerobic methane oxidizing bacteria (MOB), which utilize CH4 using oxygen as an electron acceptor, are one of dominant microorganisms in the CH4-rich water columns. The metagenome-assembled genomes (MAGs) have revealed the genetic potential of MOB from boreal aquatic ecosystems for various microaerobic/anaerobic metabolic functions; however, the experimental validation of the process has not been succeeded. Additionally, psychrophilic (i.e., cold loving) MOB isolates and their CH4 oxidizing process have rarely been investigated. In this study, we isolated, provided taxonomic description, and analyzed the genome of Methylobacter sp. S3L5C, a psychrophilic MOB, from a boreal lake in Finland. Based on phylogenomic comparisons to MAGs, Methylobacter sp. S3L5C represented a ubiquitous cluster of Methylobacter spp. in boreal aquatic ecosystems. At optimal temperatures (3-12 {degrees}C) and pH (6.8-8.3), the specific growth rates () and CH4 utilization rate were in the range of 0.018-0.022 h-1 and 0.66-1.52 mmol l-1 d-1, respectively. In batch cultivation, the isolate could produce organic acids and the concentrations were elevated after replenishing CH4 and air into headspace. The highest concentrations of 4.1 mM acetate, 0.02 mM malate and 0.07 mM propionate were observed at the end of the cultivation period under the optimal operational conditions. The results herein highlight the key role of Methylobacter spp. in regulating CH4 emissions and their potential to provide CH4-derived organic carbon compounds to surrounding heterotrophic microorganisms in cold ecosystems.

microbiology↗

bioRxiv2021

A two-staged bacterial process coupling methanotrophic and heterotrophic bacteria for 1-alkene production from methane

Methane (CH4) is a sustainable carbon feedstock source for aerobic CH4-oxidizing bacteria (methanotrophs) to produce value-added chemicals. Under substrate-limited (e.g., CH4, oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as a feedstock for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Types I and II methanotrophs, i.e., Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8-7.0% of consumed CH4-carbon while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7-1.8% of consumed CH4-carbon For the first time, the organic acids-rich spent media of methanotrophs were successfully used for 1-alkene production using engineered Acinetobacter baylyi ADP1 tesA-undA cells. The highest yield of 1-undecene was obtained from spent medium of M. tundripaludum SV96 at 68.9 {+/-} 11.6 mol mol Csubstrate-1.

microbiology↗

bioRxiv2021

Nitrate enhances anaerobic oxidation of methane in boreal lake sediments

The identity of electron acceptors in promoting anaerobic oxidation of methane (AOM) in the sediments of boreal lakes is currently unknown. Here, we studied the AOM rate of sediment slurries collected from three profundal stations of a nitrate-rich, oligo-mesotrophic, boreal lake (Lake Paajarvi, Finland), under varying nitrate concentrations using 13C-labelling. Furthermore, vertical profiles of the sediment and porewater geochemistry, and the microbial communities (16S rRNA gene and shotgun metagenomic sequencing) were analyzed. Despite geochemical data indicating that simultaneous consumption of nitrate and methane took place at the sediment layers chosen for incubations, AOM rate was not enhanced by nitrate amendments at either of the stations. AOM rate was much higher at the shallow Station 1 (0.9-6.8 nmol C cm-3 d-1) with high contents of labile phytoplankton-derived organic matter, than at the deeper stations, Station 2 (0-0.3 nmol C cm-3 d-1) and 3 (0-0.2 nmol C cm-3 d-1). Accordingly, a higher relative abundance of methanotrophic archaea (Candidatus Methanoperedens) and bacteria (Methylococcales) were observed in the layers chosen for incubations at Station 1 than at the other stations. Besides nitrate, the geochemical profiles indicated that AOM was potentially coupled with iron or sulfate reduction at all stations. Furthermore, putative nitrite-reducing methanotrophs (Ca. Methylomirabilis) were the most abundant methanotrophs above the incubation layer at Station 2 and 3, which suggests that nitrite reduction also plays a role in driving AOM in the study lake. We conclude that AOM is not uniquely coupled to nitrate reduction in sediments of nitrate-rich, oligo-mesotrophic, boreal lakes.

ecology↗

bioRxiv2018

Metabolic pairing of aerobic and anaerobic production in a one-pot batch cultivation

BackgroundThe versatility of microbial metabolic pathways enables their utilization in vast number of applications. However, the electron and carbon recovery rates, essentially constrained by limitations of cell energetics, are often too low in terms of process feasibility. Cocultivation of divergent microbial species in a single process broadens the metabolic landscape and thus, the possibilities for more complete carbon and energy utilization.\n\nResultsIn this study, we integrated the metabolisms of two bacteria, an obligate anaerobe Clostridium butyricum and an obligate aerobe Acinetobacter baylyi ADP1. In the process, a glucose-negative mutant of A. baylyi ADP1 first deoxidized the culture allowing C. butyricum to grow and produce hydrogen from glucose. In the next phase, ADP1 produced long chain alkyl esters utilizing the by-products of C. butyricum, namely acetate and butyrate.\n\nConclusionsThe cocultivation of strictly anaerobic and aerobic bacteria allowed the production of both hydrogen gas and long-chain alkyl esters in a simple one-pot batch process. The study demonstrates the potential of metabolic pairing using designed microbial consortia for optimal electron and carbon recovery.

bioengineering↗