Biology subjects

Kakouridis, A.

Publications and source records attributed to Kakouridis, A..

6 recordsLinked to original sources

bioRxiv2026

Mapping Genome-wide Transcription Factor Binding Sites in two Rhodanobacter strains Isolated from Extreme Environments

Identification of target genes for bacterial transcription factors (TFs) provide a deeper understanding of bacterial response to environmental conditions. We focused on two Rhodanobacter sp. isolates from the subsurface of the U.S. Department of Energy Oak Ridge Reservation (ORR), an environment characterized by high-concentration mixed-waste contamination plumes. Bacterial strains in this subsurface ecosystem experience changing and complex environmental conditions, including variable nutrient and oxygen availability, fluctuating pH, nitrate and metals. We examined 91 DNA-binding TFs from 17 TF families in two Rhodanobacter strains, R. denitrificans FW104-10B01 and R. thiooxydans FW510-R12, using both DNA affinity purification sequencing (DAP-seq) and comparative genomics. Target genes regulated by 31 TFs were identified. Despite being isolated from different wells, the two strains shared a highly similar core regulatory network, with orthologous TFs regulating nearly identical sets of genes. These conserved networks governed essential survival functions, including nutrient uptake (e.g., phosphate), carbohydrate metabolism, motility, and stress responses to heavy metals and oxidative environment. TF-binding sequence motifs were also inferred for each of the 31 TFs. Key results include the elucidation of a global regulator Clp which regulates a large number of genes, particularly those involved in biofilm formation and motility. Functional validation through RT-qPCR confirmed that Clp activates the expression of genes critical for type IV pilin structure and flagellar regulation. Our work highlights the advantages of synergistically employing DAP-seq and comparative genomics, which enabled us to corroborate findings and obtain a larger view of the regulatory landscape of these non-model denitrifying bacteria.

microbiology↗

bioRxiv2023

Desiccated cyanobacteria serve as efficient plasmid DNA carrier in space flight

Effective transport of biological systems as cargo during space travel is a critical requirement to use synthetic biology and biomanufacturing in outer space. Bioproduction using microbes will drive the extent to which many human needs can be met in environments with limited resources. Vast repositories of biological parts and strains are available to meet this need, but their on-site availability requires effective transport. Here, we explore an approach that allows DNA plasmids, a ubiquitous synthetic biology part, to be safely transported to the International Space Station and back to the Kennedy Space center, without low-temperature or cryogenic stowage. Our approach relied on the cyanobacterium Nostoc punctiforme PC73102, that is naturally tolerant to prolonged desiccation. Desiccated N. punctiforme was able to carry the non-native pSCR119 plasmid as intracellular cargo safely to space and back. Upon return to the laboratory, the extracted plasmid showed no DNA damage or additional mutations and could be used as intended to transform the model Synbio host Escherichia coli to bestow Kanamycin resistance. This proof-of-concept study provides the foundation for a ruggedized transport host for DNA to environments where there is a need to reduce equipment and infrastructure for biological parts stowage and storage.

microbiology↗

bioRxiv2023

Arbuscular mycorrhiza convey significant plant carbon to a diverse hyphosphere microbial food web and mineral-associated organic matter

O_LIArbuscular mycorrhizal fungi (AMF) transport substantial plant carbon (C) that serves as a substrate for other soil organisms, a precursor of soil organic matter (SOM), and a driver of soil microbial dynamics. Using two-chamber microcosms where an air gap isolated AMF from roots, we 13CO2-labeled Avena barbata for six weeks and measured. the C Rhizophagus intraradices transferred to SOM and hyphosphere microorganisms. C_LIO_LINanoSIMS imaging, IRMS, 13C NMR, and SOM density fractionation showed hyphae and roots had similar 13C enrichment. AMF transferred 0.77 mg C per g of soil (increasing total C by 2%); 33% was found in occluded or mineral-associated pools, primarily as carbohydrates. C_LIO_LIIn the AMF hyphosphere, there was no overall change in community diversity but 36 bacterial ASVs significantly changed in relative abundance. With stable isotope probing (SIP)-enabled shotgun sequencing, we found taxa from the Solibacterales, Sphingobacteriales, Myxococcales and Nitrososphaerales (ammonium oxidizing archaea) were highly enriched in AMF-imported 13C (>20 atom%). Mapping 13C-enriched metagenome-assembled genomes to total ASVs showed at least 92 bacteria and archaea were significantly 13C-enriched. C_LIO_LIOur results illustrate the quantitative impact of hyphal C transport on the formation of potentially protective SOM pools and indicate microbial roles in the AMF hyphosphere soil food web. C_LI

microbiology↗

bioRxiv2022

HT-SIP: A semi-automated Stable Isotope Probing pipeline identifies interactions in the hyphosphere of arbuscular mycorrhizal fungi

BackgroundLinking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive to meet this goal, Stable Isotope Probing--SIP--remains the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, active microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated DNA-SIP pipeline to support well-replicated, temporally-resolved amplicon or metagenomics experiments that enable studies of dynamic microbial communities over space and time. To test this pipeline, we assembled SIP-metagenome assembled genomes (MAGs) from the hyphosphere zone surrounding arbuscular mycorrhizal fungi (AMF), in combination with a 13CO2 plant labelling study. ResultsOur semi-automated pipeline for DNA fractionation, cleanup, and nucleic acid quantification of SIP density gradients requires six times less hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients and reduced variation compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We then tested this pipeline on samples from a highly-constrained soil microhabitat with significant ecological importance, the AMF fungal hyphosphere. Processing via our quantitative SIP pipeline confirmed the AMF Rhizophagus intraradices and its associated microbiome were highly 13C enriched, even though the soils overall enrichment was only 1.8 atom% 13C. We assembled 212 13C-enriched hyphosphere MAGs, and the hyphosphere taxa that assimilated the most AMF-derived 13C (range 10-33 atom%) were from the phlya Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia oxidizing archaeon genus Nitrososphaeara. ConclusionsOur semi-automated SIP approach decreases operator time and errors and improves reproducibility by targeting the most labor-intensive steps of SIP--fraction collection and cleanup. Here, we illustrate this approach in a unique and understudied soil microhabitat--generating MAGs of active microbes living in the AMF hyphosphere (without plant roots). Their phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

microbiology↗

bioRxiv2021

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3){Delta} pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

biochemistry↗

bioRxiv2020

Routes to Roots: Direct Evidence of Water Transport by Arbuscular Mycorrhizal Fungi to Host Plants

Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with 80% of surveyed land plant species and are well-recognized for accessing and transferring nutrients to plants1. Yet AMF also perform other essential functions, notably improving plant-water relations2. Some research attributes the role of AMF in plant-water relations solely to enhancing plant nutrition and osmoregulation for plants partnered with AMF3,4,5, while indirect evidence suggests AMF may transport water to plants1,6,7. Here, we used isotopically-labeled water and a fluorescent dye to directly track and quantify water transport by AMF to plants in a greenhouse experiment. We specifically assessed whether AMF can access water in soil unavailable to plants and transport it across an air gap to host plants. Plants grown with AMF that had access to a physically separated 18O-labeled water source transpired twice as much, and this transpired water contained three times as much label compared to plants with AMF with no access to the separated labeled water source. We estimated that water transported by AMF could explain 46.2% of the water transpired. In addition, a fluorescent dye indicated that water was transported via an extracytoplasmic hyphal pathway.

microbiology↗