Fluorescence-based detection of natural transformation in drug resistant <i>Acinetobacter baumannii</i>

Godeux, Anne-Sophie; Lupo, Agnese; Haenni, Marisa; Guette-Marquet, Simon; Wilharm, Gottfried; Laaberki, Maria-Halima; Charpentier, Xavier

doi:10.1101/262311

Cited by 4 publications

(4 citation statements)

References 46 publications

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“…However, these selection methods have limitations because they depend on the counting of colony forming units, which can lead to the overestimation of transformation efficiencies, due to inefficient separation of transformed from non-transformed daughter cells and nongenetic inheritance of antibiotic resistance ( Dalia and Dalia, 2019 ; Domenech et al, 2018 ; Ephrussi-Taylor, 1962 ; Ephrussi-Taylor, 1958 ; Figure 3—figure supplement 1 ). In order to overcome these concerns and analyze successful recombination events during transformation at the single-cell level, we developed a fluorescence-based reporter system inspired by a system previously used to observe natural transformation in S. pneumoniae ( Bergé et al, 2013 ) and other bacterial species ( Boonstra et al, 2018 ; Corbinais et al, 2016 ; Godeux et al, 2018 ). To do so, we utilized a fluorescent donor strain in which the gene encoding the abundant histone-like protein HlpA (aka HU) was fused in frame with the gene encoding the red fluorescent protein mScarlet-I integrated at the native hlpA locus at 169° on the circular chromosome ( Keller et al, 2019 ) (strain VL1780) ( Figure 3A ).…”

Section: Resultsmentioning

confidence: 99%

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

Kurushima

Campo

Raaphorst

et al. 2020

eLife

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The spread of antimicrobial resistance and vaccine escape in the human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. Here, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. We find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation and because a single stranded donor DNA replaces the original allele, transformation efficiency has an upper threshold of approximately 50% of the population. The fixed mechanism of transformation results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome.

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Section: Resultsmentioning

confidence: 99%

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

Kurushima

Campo

Raaphorst

et al. 2020

eLife

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“…The few studies on transformation in A. baumannii have focused mainly on mild competence inducers such as serum albumin and Ca 2+ , on transforming materials, and the pH (10)(11)(12).…”

Section: Introductionmentioning

confidence: 99%

“…However, transforming protocols vary wildly between studies (13)(14)(15), including the use of different solidifying agents for transformation scoring on surfaces (16). Additionally, only few isolates of this species, such as the strains A118 (13) and M2 (recently reclassified as A. nosocomialis) (15,17), were previously found to be naturally competent, though recent studies are showing that a plethora of clinical and wildlife/livestock A. baumannii isolates are likewise naturally transformable (12,14,18). Therefore, the process of natural competence in A. baumannii needs to be better studied and recorded.…”

Section: Introductionmentioning

confidence: 99%

Pilus Production in Acinetobacter baumannii Is Growth Phase Dependent and Essential for Natural Transformation

Vesel

Blokesch

2021

J Bacteriol

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Acinetobacter baumannii is a severe threat to human health as a frequently multidrug-resistant hospital-acquired pathogen. Part of the danger from this bacterium comes from its genome plasticity and ability to evolve quickly by taking up and recombining external DNA into its own genome in a process called natural competence for transformation. This mode of horizontal gene transfer is one of the major ways pathogens can acquire new antimicrobial resistances and toxic traits. Because these processes in A. baumannii are not well studied, we herein characterized new aspects of natural transformability in this species that include the species’ competence window. We uncovered a strong correlation with a growth–phase-dependent synthesis of a type IV pilus (TFP), which constitutes the central part of competence-induced DNA-uptake machinery. We used bacterial genetics and microscopy to demonstrate that the TFP is essential for the natural transformability and surface motility of A. baumannii, whereas pilus-unrelated proteins of the DNA-uptake complex do not impact the motility phenotype. Furthermore, TFP biogenesis and assembly is subject to input from two regulatory systems that are homologous to Pseudomonas aeruginosa, namely the PilSR two-component system and the Pil-Chp chemosensory system. We demonstrated that these systems not only impact the piliation status of cells but also their ability to take up DNA for transformation. Importantly, we report on discrepancies between TFP biogenesis and natural transformability within the same genus by comparing A. baumannii to data reported for A. baylyi, the latter of which served for decades as a model for natural competence. IMPORTANCE Rapid bacterial evolution has alarming negative impacts on animal and human health, which can occur when pathogens acquire antimicrobial resistance traits. As a major cause of antibiotic-resistant opportunistic infections, A. baumannii is a high priority health threat, which has motivated renewed interest in studying how this pathogen acquires new, dangerous traits. In this study, we deciphered a specific time window in which these bacteria can acquire new DNA, and correlated that with its ability to produce the external appendages that contribute to the DNA acquisition process. These cell appendages function doubly for motility on surfaces and for DNA uptake. Collectively, we showed that A. baumannii is similar in its TFP production to Pseudomonas aeruginosa, though differs from the well-studied species A. baylyi.

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“…Acinetobacter species like A. baumannii and A. nosocomialis have recently become an urgent medical threat due to their prevalence in hospital-acquired infections and their capacity to acquire antibiotic resistance genes, a process that is achieved in part by T4aP-mediated DNA uptake 12,13 . Acinetobacter baylyi is the most naturally transformable species reported to date 14 , with up to 50% of cells undergoing natural transformation in ideal settings.…”

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confidence: 99%

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

Ellison

Dalia

Klancher

et al. 2020

Preprint

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Bacteria employ extracellular appendages called type IV pili (T4P) to interact with their environment. T4P are essential for diverse microbial behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility1. While T4P have been studied extensively, our understanding of these nanomachines largely comes from work on a few model species. Here, we develop Acinetobacter baylyi as a new model organism to study T4P and uncover several unreported mechanisms of T4P regulation. First, using recently-developed T4P-labeling methods2,3, we demonstrate that A. baylyi T4P are synthesized on one side of the cell body along the long axis of the cell, and we uncover that this pattern is dependent on components of a conserved chemosensory pathway. Second, we overturn the current dogma that T4P extension occurs through the action of a single, highly conserved ATP-hydrolyzing motor (ATPase) called PilB by showing that T4P synthesis in A. baylyi is dependent on two partially redundant and phylogenetically distinct motors, PilB and PilB2. Third, we uncover a small protein inhibitor of T4P synthesis that specifically inhibits PilB but not PilB2 activity. Together, these results demonstrate novel mechanisms of T4P regulation, which have broad implications for the unexplored diversity of T4P biology in microbial species.

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Fluorescence-based detection of natural transformation in drug resistant Acinetobacter baumannii

Cited by 4 publications

References 46 publications

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

Pilus Production in Acinetobacter baumannii Is Growth Phase Dependent and Essential for Natural Transformation

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

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