Biology subjects

de Siqueira, G. M. V.

Publications and source records attributed to de Siqueira, G. M. V..

7 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↗

bioRxiv2025

Discovery and Functional Characterization of Salinity-Responsive Promoters in Pseudomonas putida KT2440

Microbial bioprocesses are associated with environmental advantages, but their freshwater demand remains a challenge. Seawater is abundant and a sustainable alternative; however, high salinity often limits microbial growth and survival. This study aimed to identify biological parts involved in microbial salt stress responses for applications in microbial engineering. Regulatory regions adjacent to genes related to salinity and other industrially relevant conditions in Pseudomonas putida KT2440 were cloned into a reporter plasmid for functional evaluation. We identified and characterized the promoters of PP_1833 and PP_4707 as salt-inducible in KT2440, showing fluorescence levels 1.9- and 2.8-fold greater than a standard strong constitutive promoter (Pj100) under 3.5% NaCl (w/v), respectively, while no activity was detected in Escherichia coli DH10B. These results provide a basis for developing salt-responsive regulatory circuits in P. putida KT2440. For table of contents only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/687801v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1b5214org.highwire.dtl.DTLVardef@1a9bde0org.highwire.dtl.DTLVardef@f0d071org.highwire.dtl.DTLVardef@4c0bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Differences in GenBank and RefSeq annotations may affect genomics data interpretation for Pseudomonas putida KT2440

Annotations of genomic features are cornerstone data that support routine workflows in conventional omics analyses in Pseudomonas putida KT2440 and other organisms. The GenBank and the RefSeq versions of the annotated KT2440 genome are two popular resources widely cited in the literature; yet, they originate from distinct prediction pipelines and possess potentially different biological information that is often overlooked. In this study, we systematically compared the features present in these resources and found that approximately 16% of the total of KT2440 ORFs show differences in their predicted genomic positions across GenBank and RefSeq, despite sharing equivalent locus tag codes. Furthermore, we show that these discrepancies can affect the results of high-throughput analyses by processing a collection of RNAseq expression datasets utilizing both annotations. Our findings provide a comprehensive overview of the current state of available resources for genomics research in P. putida KT2440 and highlight a rarely addressed yet widespread potential pitfall in the literature on this organism, with possible implications for other prokaryotes. ImportanceGenome annotation databases often rely on different statistical models for their function predictions and inherently carry biases propagated into studies using them. This work provides a quantitative assessment of two popular annotation resources for the model bacterium P. putida KT2440 and their influence on data interpretation. As large-scale omics datasets are commonly used to inform experimental decisions, our results aim to promote awareness of the caveats associated with these computational resources and foster reproducibility and transparency in P. putida research.

bioinformatics↗

bioRxiv2024

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. While this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here we examined growth and isoprenol production in a Pseudomonas putida strain pre-tolerized ("PT") background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon source. We found that PT displays impaired growth in minimal medium containing acetate, and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, while a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains, and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in P. putida allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled development of a P. putida bioconversion chassis able to use a more complex carbon stream to produce isoprenol. ImportanceAcetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential of being used together with sugars, especially in microbes with versatile catabolism such as P. putida. However, the use of mixed carbon streams necessitates additional optimization. Further, the use of P. putida for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study we generate acetate tolerant P. putida strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, though independent phenotypes can both be optimized in a given P. putida strain. Using proteomics and whole genome sequencing we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.

microbiology↗

bioRxiv2022

A termination-aware vector design improves heterologous gene expression in Pseudomonas putida

Transcriptional terminators are key players in the flow of genetic information, but are often overlooked in circuit design. In this work, we used the Standard European Vector Architecture (SEVA) as a scaffold to investigate the effects of different terminators in the output of a reporter construct expressed in two bacterial species and found that replacing the conventional T1 and T0 transcriptional terminators of the SEVA vector format with a set of broad-host metagenomic terminators resulted in a significant improvement in the signal of a fluorescent device in Pseudomonas putida KT2440. Interestingly, this new vector design displayed the same performance as pSEVA231 in Escherichia coli DH10B. Our results support the notion that transcriptional terminators may affect the interconnected mesh of biological processes taking place in gene expression, leading to a host-dependent output that should be heeded for optimal circuit expression in different microbial hosts.

synthetic biology↗

bioRxiv2021

Nanopore sequencing provides rapid and reliable insight into microbial profiles of Intensive Care Units

Fast and accurate identification of pathogens is an essential task in healthcare settings. Next generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopores minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units from a hospital in Brazil. While our results point that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

microbiology↗

bioRxiv2020

Turning the screw: engineering extreme pH resistance in Escherichia coli through combinatorial synthetic operons

Adoption of microorganisms as platforms for sustainable biobased production requires host cells to be able to withstand harsh industrial conditions, which are usually far from the ones where these organisms are naturally adapted to thrive. However, novel survival mechanisms unearthed by the study of microbiomes from extreme habitats may be exploited to enhance microbial robustness under the strict conditions needed for different applications. In this work, synthetic biology approaches were used to engineer enhanced acidic tolerance in Escherichia coli under extreme conditions through the characterization of a library of twenty-seven unique operons composed of combinatorial assemblies of three novel genes from an extreme environment and three synthetic ribosome binding sites. The results here presented illustrate the efficacy of combining different metagenomic genes for tolerance in truly synthetic genetic operons, as expression of these gene clusters increased hundred-fold the survival percentage of cells exposed to an acidic shock in minimal media at pH 1.9 under aerobic conditions.

synthetic biology↗