Biology subjects

Kroeger, C.

Publications and source records attributed to Kroeger, C..

7 recordsLinked to original sources

bioRxiv2026

Contributions of plasmid p1AB5075-encoded antibiotic resistance genes to multidrug resistance of Acinetobacter baumannii AB5075

Infections with multidrug resistant Acinetobacter baumannii are considered a threat to human and animal health. The widely studied A. baumannii strain AB5075 displays a high degree of antibiotic resistance. In this study, we experimentally validated that antibiotic resistance is largely mediated by resistance genes located on plasmid p1AB5075. We used a p1AB5075-deficient AB5075 strain to assess individual contributions of p1AB5075-encoded antibiotic resistance genes by ectopically (over-)expressing each gene in the {Delta}p1AB5075 background. By determining individual contributions of seven p1AB5075-encoded antibiotic resistance genes, we show individual and overlapping roles of genes for aminoglycoside resistance and uncover the importance of extended-spectrum {beta}-lactamase blaGES-11 for cephalosporin resistance in A. baumannii AB5075. We discovered that aminoglycoside N-acetyltransferase aaC(6)-Ib3 (aacA4), which was considered a potential pseudogene in A. baumannii AB5075, to be functional providing broad resistance to gentamicin, kanamycin, amikacin, streptomycin and tobramycin when overexpressed in A. baumannii AB5075. Because p1AB5075 is transferrable to a wide range of environmental and clinical A. baumannii strains and non-baumannii Acinetobacter species, the relevance of our findings extends beyond A. baumannii AB5075.

microbiology↗

bioRxiv2026

Methylome and transcriptome mapping reveal miniscule DNA methyltransferase regulons in Salmonella enterica serovar Typhimurium

DNA methylation is a regulator of bacterial gene expression and adaptation, influencing traits such as virulence and antimicrobial resistance. The dynamic nature of DNA methylation enables rapid responses to changing environments and is a source of heterogeneity in bacterial populations. However, condition-dependent DNA methylation and consequences for transcriptional output remain poorly understood. We applied Oxford Nanopore sequencing to profile DNA methylation during exponential growth and late stationary phase of Salmonella enterica serovar Typhimurium and integrated these data with transcriptomic analyses. We found that each DNA methyltransferase (MTases) exhibits a distinct activity pattern across growth stages, which could not be explained by transcriptional levels of the corresponding enzymes. As predicted, DNA methylation patterns determined by regulatory MTases were dynamic across growth conditions whereas methylation patterns of MTases belonging to R-M systems were comparatively stable. We identified growth stage-specific methylation patterns for all studied MTases and correlations between methylation states and gene expression patterns. Together, these findings chart DNA methylation networks in the epigenetic regulation of bacterial physiology. Author summaryDNA methylation in bacteria is best known for its role protecting DNA from endonucleases, such as restriction-modification, and coordinating chromosome replication and mutation repair, yet DNA methylation also regulates gene expression and cell physiology. Previous studies primarily examined bacterial DNA methylation at single time points or in limited genomic regions, providing only a partial view of its biological significance. In this study, we used Oxford Nanopore sequencing to compare DNA methylation patterns in Salmonella enterica during exponential growth and late stationary phase then integrated these data with corresponding gene expression profiles. We identified numerous methylation target motifs, all of which demonstrated constitutively methylated or unmethylated regions. This systems-level analysis clarifies the role of DNA methylation in bacterial adaptation across growth stages and demonstrates the utility of Oxford Nanopore sequencing for genome-wide methylation profiling.

microbiology↗

bioRxiv2025

DNA uptake and twitching motility are controlled by the small RNA Arp through repression of pilin translation in Acinetobacter baumannii

Acinetobacter baumannii is a major opportunistic pathogen capable of natural transformation, a process driven by type IV pili (T4P) that facilitates horizontal gene transfer and accelerates the spread of antimicrobial resistance. While the transcriptional regulation of T4P is increasingly understood, post-transcriptional mechanisms controlling pilus assembly remain unexplored. Here, we identify and characterise a small RNA, Arp (Acinetobacter repressor of pilin), as a post-transcriptional repressor of T4P-mediated functions in A. baumannii. In a previous Hi-GRIL-seq experiment, we detected specific ligation events between Arp and the ribosome binding site of the pilA mRNA, encoding the major pilin subunit PilA. In-line probing and translational reporter assays revealed that Arp represses pilA translation by sequestering the Shine-Dalgarno sequence and the first 17 codons of the mRNA. Overexpression of Arp significantly impairs DNA uptake and twitching motility, two hallmark T4P-dependent phenotypes. Together, our findings identify a native A. baumannii sRNA that modulates natural competence by targeting pilin synthesis, revealing a new regulatory layer that could be exploited to disrupt horizontal gene transfer in multidrug-resistant strains. Significance StatementAcinetobacter baumannii is a multidrug-resistant WHO #1 priority pathogen that acquires antibiotic resistance genes through natural transformation, a process dependent on type IV pili (T4P). This work reveals Arp, the first native post-transcriptional repressor of natural competence in A. baumannii, uncovering a novel regulatory layer that modulates horizontal gene transfer. The widespread presence of arp in pathogenic Acinetobacter strains suggests that sRNA is an important regulator in those organisms. Furthermore, these findings broaden our understanding of RNA-based regulation in this priority pathogen and open potential avenues for interfering with antibiotic resistance dissemination.

microbiology↗

bioRxiv2025

Development of two compatible plasmids to assess sRNA-mediated post-transcriptional regulation in Acinetobacter baumannii

Post-transcriptional regulation can be mediated by small, regulatory RNAs in bacteria, which can act by base-pairing to a target messenger RNA. The discovery and mechanistic validation of base-pairing sRNAs in multidrug resistant Acinetobacter baumannii has been hampered by the lack of genetic tools to assess RNA-RNA interactions. Here, we created two compatible plasmids for A. baumannii, which addresses this need. The newly designed plasmids validated the known Aar sRNA-carO mRNA, and a new interaction of sRNA44 and the mRNA of the biofilm-associated protein Bap. The new plasmid system should accelerate the mechanistic characterisation of small, regulatory RNAs in A. baumannii. IMPACT STATEMENTMulti-drug resistance of pathogenic microorganisms is one of the greatest challenges for modern medicine. Carbapenem-resistant Acinetobacter baumannii are considered a highly critical organism, yet we are only beginning to understand its physiology and mechanisms of gene regulation. Post-transcriptional regulation by base-pairing, small RNAs is an understudied area, partly because of the lack of genetic tools to investigate them. In this study, we developed a 2-plasmid system to assess sRNA-mRNA interactions, which will greatly accelerate the discovery and validation of small, regulatory RNAs and their target molecules. DATA SUMMARYPlasmid sequences of pAMCK14-sRNA44 and pAMCK18-Bap have been made available in GenBank of National Center for Biotechnology Information (accession numbers PV916437 and PV916438).

microbiology↗

bioRxiv2024

Unveiling the Power of High-Dimensional Cytometry Data with cyCONDOR

High-dimensional cytometry (HDC) is a powerful technology for studying single-cell phenotypes in complex biological systems. Although technological developments and affordability have made HDC broadly available in recent years, technological advances were not coupled with an adequate development of analytical methods that can take full advantage of the complex data generated. While several analytical platforms and bioinformatics tools have become available for the analysis of HDC data, these are either web-hosted with limited scalability or designed for expert computational biologists, making their use unapproachable for wet lab scientists. Additionally, end-to-end HDC data analysis is further hampered due to missing unified analytical ecosystems, requiring researchers to navigate multiple platforms and software packages to complete the analysis. To bridge this data analysis gap in HDC we developed cyCONDOR, an easy-to-use computational framework covering not only all essential steps of cytometry data analysis but also including an array of downstream functions and tools to expand the biological interpretation of the data. The comprehensive suite of features of cyCONDOR, including guided pre-processing, clustering, dimensionality reduction, and machine learning algorithms, facilitates the seamless integration of cyCONDOR into clinically relevant settings, where scalability and disease classification are paramount for the widespread adoption of HDC in clinical practice. Additionally, the advanced analytical features of cyCONDOR, such as pseudotime analysis and batch integration, provide researchers with the tools to extract deeper insights from their data. We used cyCONDOR on a variety of data from different tissues and technologies demonstrating its versatility to assist the analysis of high dimensionality data from preprocessing to biological interpretation.

bioinformatics↗

bioRxiv2023

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi, and we performed an intensive comparative genomic analysis of 608 isolates obtained from fever surveillance at the Queen Elizabeth Hospital, Blantyre between 1996 and 2018. We discovered that following the upsurge of the well-characterised S. Typhimurium ST313 lineage 2 from 1999 onwards, two new multidrug-resistant sublineages designated 2.2 and 2.3, emerged in Malawi in 2006 and 2008, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineage 2.2 or 2.3. To identify factors that characterised the emergence of the prevalent ST313 sublineage 2.2, we performed genomic and functional analysis of two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). Comparative genomic analysis showed that the chromosome of ST313 lineage 2 and sublineage 2.2 were broadly similar, only differing by 29 SNPs and small indels and a 3kb deletion in the Gifsy-2 prophage region that spanned the sseI pseudogene. Lineage 2 and sublineage 2.2 have unique plasmid profiles that were verified by long read sequencing. The transcriptome was initially explored in 15 infection-relevant conditions and within macrophages. Differential gene expression was subsequently investigated in depth in the four most important in vitro growth conditions. We identified up-regulation of SPI2 genes in non-inducing conditions, and down-regulation of flagellar genes in D37712, compared to D23580. Following phenotypic confirmation of transcriptional differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed-growth in minimal media. We speculate that this competitive advantage is contributing to the continuing presence of sublineage 2.2 in Malawi.

bioinformatics↗

bioRxiv2021

Food or physics: plankton communities structured across Gulf of Alaska eddies

Oceanic features, such as mesoscale eddies that entrap and transport water masses, create heterogeneous seascapes to which biological communities may respond. To date, however, our understanding of how internal eddy dynamics influence plankton community structuring is limited by sparse sampling of eddies and their associated biotic communities. In this paper, we used 10 years of archived Continuous Plankton Recorder (CPR) data (2002-2013) associated with 9 mesoscale eddies in the Northeast Pacific/Gulf of Alaska to test the hypothesis that eddy origin and rotational direction determines the structure and dynamics of entrained plankton communities. Using generalized additive models and accounting for confounding factors (e.g., timing of sampling), we found peak diatom abundance within both cyclonic and anticyclonic eddies near the eddy edge. Zooplankton abundances, however, varied with distance to the eddy center/edge by rotational type and eddy life stage, and differed by taxonomic group. For example, the greatest abundance of small copepods was found near the center of anticyclonic eddies during eddy maturation and decay, but near the edge of cyclonic eddies during eddy formation and intensification. Distributions of copepod abundances across eddy surfaces were not mediated by phytoplankton distribution. Our results therefore suggest that physical mechanisms such as internal eddy dynamics exert a direct impact on the structure of zooplankton communities rather than indirect mechanisms involving potential food resources.

ecology↗