Microbial Fitness Research Articles

A metagenomic approach to demystify the anaerobic digestion black box and achieve higher biogas yield: a review.

The increasing reliance on fossil fuels and the growing accumulation of organic waste necessitates the exploration of sustainable energy alternatives. Anaerobic digestion (AD) presents one such solution by utilizing secondary biomass to produce biogas while reducing greenhouse gas emissions. Given the crucial role of microbial activity in anaerobic digestion, a deeper understanding of the microbial community is essential for optimizing biogas production. While metagenomics has emerged as a valuable tool for unravelling microbial composition and providing insights into the functional potential in biodigestion, it falls short of interpreting the functional and metabolic interactions, limiting a comprehensive understanding of individual roles in the community. This emphasizes the significance of expanding the scope of metagenomics through innovative tools that highlight the often-overlooked, yet crucial, role of microbiota in biomass digestion. These tools can more accurately elucidate microbial ecological fitness, shared metabolic pathways, and interspecies interactions. By addressing current limitations and integrating metagenomics with other omics approaches, more accurate predictive techniques can be developed, facilitating informed decision-making to optimize AD processes and enhance biogas yields, thereby contributing to a more sustainable future.

Growth rate as a link between microbial diversity and soil biogeochemistry.

Measuring the growth rate of a microorganism is a simple yet profound way to quantify its effect on the world. The absolute growth rate of a microbial population reflects rates of resource assimilation, biomass production and element transformation-some of the many ways in which organisms affect Earth's ecosystems and climate. Microbial fitness in the environment depends on the ability to reproduce quickly when conditions are favourable and adopt a survival physiology when conditions worsen, which cells coordinate by adjusting their relative growth rate. At the population level, relative growth rate is a sensitive metric of fitness, linking survival and reproduction to the ecology and evolution of populations. Techniques combining omics and stable isotope probing enable sensitive measurements of the growth rates of microbial assemblages and individual taxa in soil. Microbial ecologists can explore how the growth rates of taxa with known traits and evolutionary histories respond to changes in resource availability, environmental conditions and interactions with other organisms. We anticipate that quantitative and scalable data on the growth rates of soil microorganisms, coupled with measurements of biogeochemical fluxes, will allow scientists to test and refine ecological theory and advance process-based models of carbon flux, nutrient uptake and ecosystem productivity. Measurements of in situ microbial growth rates provide insights into the ecology of populations and can be used to quantitatively link microbial diversity to soil biogeochemistry.

Bacteria face trade-offs in the decomposition of complex biopolymers.

Although depolymerization of complex carbohydrates is a growth-limiting bottleneck for microbial decomposers, we still lack understanding about how the production of different types of extracellular enzymes affect individual microbes and in turn the performance of whole decomposer communities. In this work we use a theoretical model to evaluate the potential trade-offs faced by microorganisms in biopolymer decomposition which arise due to the varied biochemistry of different depolymerizing enzyme classes. We specifically consider two broad classes of depolymerizing extracellular enzymes, which are widespread across microbial taxa: exo-enzymes that cleave small units from the ends of polymer chains and endo-enzymes that act at random positions generating degradation products of varied sizes. Our results demonstrate a fundamental trade-off in the production of these enzymes, which is independent of system's complexity and which appears solely from the intrinsically different temporal depolymerization dynamics. As a consequence, specialists that produce either exo- or only endo-enzymes limit their growth to high or low substrate conditions, respectively. Conversely, generalists that produce both enzymes in an optimal ratio expand their niche and benefit from the synergy between the two enzymes. Finally, our results show that, in spatially-explicit environments, consortia composed of endo- and exo-specialists can only exist under oligotrophic conditions. In summary, our analysis demonstrates that the (evolutionary or ecological) selection of a depolymerization pathway will affect microbial fitness under low or high substrate conditions, with impacts on the ecological dynamics of microbial communities. It provides a possible explanation why many polysaccharide degraders in nature show the genetic potential to produce both of these enzyme classes.

Symbiotic bacteria and fungi proliferate in diapause and may enhance overwintering survival in a solitary bee.

Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a highly specialized gut microbiome. Here, we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that A. bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy.

Bayesian inference of relative fitness on high-throughput pooled competition assays.

The tracking of lineage frequencies via DNA barcode sequencing enables the quantification of microbial fitness. However, experimental noise coming from biotic and abiotic sources complicates the computation of a reliable inference. We present a Bayesian pipeline to infer relative microbial fitness from high-throughput lineage tracking assays. Our model accounts for multiple sources of noise and propagates uncertainties throughout all parameters in a systematic way. Furthermore, using modern variational inference methods based on automatic differentiation, we are able to scale the inference to a large number of unique barcodes. We extend this core model to analyze multi-environment assays, replicate experiments, and barcodes linked to genotypes. On simulations, our method recovers known parameters within posterior credible intervals. This work provides a generalizable Bayesian framework to analyze lineage tracking experiments. The accompanying open-source software library enables the adoption of principled statistical methods in experimental evolution.

The actinomycete Kitasatospora sp. SeTe27, subjected to adaptive laboratory evolution (ALE) in the presence of selenite, varies its cellular morphology, redox stability, and tolerance to the toxic oxyanion

The effects of oxyanions selenite (SeO32−) in soils are of high concern in ecotoxicology and microbiology as they can react with mineral particles and microorganisms. This study investigated the evolution of the actinomycete Kitasatospora sp. SeTe27 in response to selenite. To this aim, we used the Adaptive Laboratory Evolution (ALE) technique, an experimental approach that mimics natural evolution and enhances microbial fitness for specific growth conditions. The original strain (wild type; WT) isolated from uncontaminated soil gave us a unique model system as it has never encountered the oxidative damage generated by the prooxidant nature of selenite. The WT strain exhibited a good basal level of selenite tolerance, although its growth and oxyanion removal capacity were limited compared to other environmental isolates. Based on these premises, the WT and the ALE strains, the latter isolated at the end of the laboratory evolution procedure, were compared. While both bacterial strains had similar fatty acid profiles, only WT cells exhibited hyphae aggregation and extensively produced membrane-like vesicles when grown in the presence of selenite (challenged conditions). Conversely, ALE selenite-grown cells showed morphological adaptation responses similar to the WT strain under unchallenged conditions, demonstrating the ALE strain improved resilience against selenite toxicity. Whole-genome sequencing revealed specific missense mutations in genes associated with anion transport and primary and secondary metabolisms in the ALE variant. These results were interpreted to show that some energy-demanding processes are attenuated in the ALE strain, prioritizing selenite bioprocessing to guarantee cell survival in the presence of selenite. The present study indicates some crucial points for adapting Kitasatospora sp. SeTe27 to selenite oxidative stress to best deal with selenium pollution. Moreover, the importance of exploring non-conventional bacterial genera, like Kitasatospora, for biotechnological applications is emphasized.

Horizontal gene transfer is predicted to overcome the diversity limit of competing microbial species

Natural microbial ecosystems harbor substantial diversity of competing species. Explaining such diversity is challenging, because in classic theories it is extremely infeasible for a large community of competing species to stably coexist in homogeneous environments. One important aspect mostly overlooked in these theories, however, is that microbes commonly share genetic materials with their neighbors through horizontal gene transfer (HGT), which enables the dynamic change of species growth rates due to the fitness effects of the mobile genetic elements (MGEs). Here, we establish a framework of species competition by accounting for the dynamic gene flow among competing microbes. Combining theoretical derivation and numerical simulations, we show that in many conditions HGT can surprisingly overcome the biodiversity limit predicted by the classic model and allow the coexistence of many competitors, by enabling dynamic neutrality of competing species. In contrast with the static neutrality proposed by previous theories, the diversity maintained by HGT is highly stable against random perturbations of microbial fitness. Our work highlights the importance of considering gene flow when addressing fundamental ecological questions in the world of microbes and has broad implications for the design and engineering of complex microbial consortia.

Molecular basis of the phenotypic variants arising from a Pseudoalteromonas lipolytica mutator.

Bacterial deficiencies in the DNA repair system can produce mutator strains that promote adaptive microevolution. However, the role of mutator strains in marine Pseudoalteromonas, capable of generating various gain-of-function genetic variants within biofilms, remains largely unknown. In this study, inactivation of mutS in Pseudoalteromonas lipolytica conferred an approximately 100-fold increased resistance to various antibiotics, including ciprofloxacin, rifampicin and aminoglycoside. Furthermore, the mutator of P. lipolytica generated variants that displayed enhanced biofilm formation but reduced swimming motility, indicating a high phenotypic diversity within the ΔmutS population. Additionally, we observed a significant production rate of approximately 50 % for the translucent variants, which play important roles in biofilm formation, when the ΔmutS strain was cultured on agar plates or under shaking conditions. Using whole-genome deep-sequencing combined with genetic manipulation, we demonstrated that point mutations in AT00_17115 within the capsular biosynthesis cluster were responsible for the generation of translucent variants in the ΔmutS subpopulation, while mutations in flagellar genes fliI and flgP led to a decrease in swimming motility. Collectively, this study reveals a specific mutator-driven evolution in P. lipolytica, characterized by substantial genetic and phenotypic diversification, thereby offering a reservoir of genetic attributes associated with microbial fitness.

Genome-wide screening reveals metabolic regulation of stop-codon readthrough by cyclic AMP.

Translational fidelity is critical for microbial fitness, survivaland stress responses. Much remains unknown about the genetic and environmental control of translational fidelity and its single-cell heterogeneity. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified over 20 genes critical for stop-codon readthrough. Most of these identified genes were not previously known to affect translational fidelity. Intriguingly, we show that several genes controlling metabolism, including cyaA and crp, enhance stop-codon readthrough. CyaA catalyzes the synthesis of cyclic adenosine monophosphate (cAMP). Combining RNA sequencing, metabolomicsand biochemical analyses, we show that deleting cyaA impairs amino acid catabolism and production of ATP, thus repressing the transcription of rRNAs and tRNAs to decrease readthrough. Single-cell analyses further show that cAMP is a major driver of heterogeneity in stop-codon readthrough and rRNA expression. Our results highlight that carbon metabolism is tightly coupled with stop-codon readthrough.

Gut microbes predominantly act as living beneficial partners rather than raw nutrients

Animals and their gut microbes mutually benefit their health. Nutrition plays a central role in this, directly influencing both host and microbial fitness and the nature of their interactions. This makes nutritional symbioses a complex and dynamic tri-system of diet-microbiota-host. Despite recent discoveries on this field, full control over the interplay among these partners is challenging and hinders the resolution of fundamental questions, such as how to parse the gut microbes’ effect as raw nutrition or as symbiotic partners? To tackle this, we made use of the well-characterized Drosophila melanogaster/Lactiplantibacillus plantarum experimental model of nutritional symbiosis to generate a quantitative framework of gut microbes’ effect on the host. By coupling experimental assays and Random Forest analysis, we show that the beneficial effect of L. plantarum strains primarily results from the active relationship as symbionts rather than raw nutrients, regardless of the bacterial strain. Metabolomic analysis of both active and inactive bacterial cells further demonstrated the crucial role of the production of beneficial bacterial metabolites, such as N-acetylated-amino-acids, as result of active bacterial growth and function. Altogether, our results provide a ranking and quantification of the main bacterial features contributing to sustain animal growth. We demonstrate that bacterial activity is the predominant and necessary variable involved in bacteria-mediated benefit, followed by strain-specific properties and the nutritional potential of the bacterial cells. This contributes to elucidate the role of beneficial bacteria and probiotics, creating a broad quantitative framework for host-gut microbiome that can be expanded to other model systems.

A Selective Bottleneck During Host Entry Drives the Evolution of New Legume Symbionts.

During the emergence of new host-microbe symbioses, microbial fitness results from the ability to complete the different steps of symbiotic life cycles, where each step imposes specific selective pressures. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts is still poorly known. Here, we characterized the dynamics of phenotypic adaptation to a simplified symbiotic life cycle during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly explained by improved competitiveness for host entry, which outweighed adaptation to within-host proliferation. Whole-population sequencing of bacteria at regular time intervals along this evolution experiment revealed the continuous accumulation of new mutations (fuelled by a transient hypermutagenesis phase occurring at each cycle before host entry, a phenomenon described in previous work) and sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that several adaptive mutations can co-occur in the same cohort. Moreover, all adaptive mutations improved competitiveness for host entry, while only a subset of those also improved within-host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry occurring before within-host proliferation and just after the hypermutagenesis phase in the rhizosphere. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

Gastric microbiota dysbiosis and Helicobacter pylori infection.

Helicobacter pylori (H. pylori) infection is one of the most common causes of gastric disease. The persistent increase in antibiotic resistance worldwide has made H. pylori eradication challenging for clinicians. The stomach is unsterile and characterized by a unique niche. Communication among microorganisms in the stomach results in diverse microbial fitness, population dynamics, and functional capacities, which may be positive, negative, or neutral. Here, we review gastric microecology, its imbalance, and gastric diseases. Moreover, we summarize the relationship between H. pylori and gastric microecology, including non-H. pylori bacteria, fungi, and viruses and the possibility of facilitating H. pylori eradication by gastric microecology modulation, including probiotics, prebiotics, postbiotics, synbiotics, and microbiota transplantation.

Generation and comprehensive analysis of Synechococcus elongatus–Aspergillus nidulans co-culture system for polyketide production

BackgroundArtificial microbial consortia composed of heterotrophic and photoautotrophic organisms represent a unique strategy for converting light energy and carbon dioxide into high-value bioproducts. Currently, the types of desired bioproducts are still limited, and microbial fitness benefit rendered by paired partner generally needs to be intensified. Exploring novel artificial microbial consortia at a laboratory scale is an essential step towards addressing this unmet need. This study aimed to conduct and analyze an artificial consortium composed of cyanobacterium Synechococcus elongatus FL130 with the filamentous fungus Aspergillus nidulans TWY1.1 for producing fungi-derived secondary metabolite of polyketide neosartoricin B.ResultsPolyketide-producing A. nidulans TWY1.1 substantially ameliorated the growth and the survival of sucrose-secreting cyanobacterium S. elongatus FL130 in salt-stressed environments. Besides sucrose, comparable amounts of other carbohydrates were released from axenically cultured FL130 cells, which could be efficiently consumed by TWY1.1. Relative to axenically cultured FL130, less glycogen was accumulated in FL130 cells co-cultured with TWY1.1, and the glycogen phosphorylase gene catalyzing the first step for glycogen degradation had two-fold expression. Different from axenically cultured filamentous fungi, abundant vacuoles were observed in fungal hyphae of TWY1.1 co-cultured with cyanobacterium FL130. Meanwhile, FL130 cells displayed a characteristic pattern of interacting with its heterotrophic partner, densely dispersing along certain hyphae of TWY1.1. Finally, polyketide neosartoricin B was produced from TWY1.1 in FL130-TWY1.1 co-cultures, which was tightly adjusted by nitrogen level.ConclusionOverall, the results thoroughly proved the concept of pairing cyanobacteria with filamentous fungi to build artificial consortia for producing fungi-derived biomolecules.

Case Studies in the Assessment of Microbial Fitness: Seemingly Subtle Changes Can Have Major Effects on Phenotypic Outcomes

Following the completion of an adaptive evolution experiment, fitness evaluations are routinely conducted to assess the magnitude of adaptation. In doing so, proper consideration should be given when determining the appropriate methods as trade-offs may exist between accuracy and throughput. Here, we present three instances in which small changes in the framework or execution of fitness evaluations significantly impacted the outcomes. The first case illustrates that discrepancies in fitness conclusions can arise depending on the approach to evaluating fitness, the culture vessel used, and the sampling method. The second case reveals that variations in environmental conditions can occur associated with culture vessel material. Specifically, these subtle changes can greatly affect microbial physiology leading to changes in the culture pH and distorting fitness measurements. Finally, the last case reports that heterogeneity in CFU formation time can result in inaccurate fitness conclusions. Based on each case, considerations and recommendations are presented for future adaptive evolution experiments.

Nutrient encryption and the diversity of cobamides, siderophores, and glycans.

Encryption makes information available only to those with the decoding key. We propose that microbes, living in a chemical environment, encrypt nutrients, thereby making them available only to those with the decoding enzymes, such as their kin. Examples of encrypted nutrients include cobamides, which are expensive to make and valuable for microbial fitness. Furthermore, we propose that hosts encrypt nutrients to encourage desirable colonizers. For instance, plant root exudates and breast milk oligosaccharides encourage beneficial microbes.

Undecaprenyl phosphate translocases confer conditional microbial fitness

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2–6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

P335 Genome plasticity in Candida albicans : acutting-edge strategy for evolution, adaptation, and survival

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives Candida albicans is the most implicated fungal species that grows as a commensal or opportunistic pathogen in the human host. The genome plasticity is central to its adaptation and has long been of considerable interest. Understanding the factors that affect the genome is crucial for a proper understanding of species and rapid development and adjustment of therapeutic strategies to mitigate their spread.MethodsHere, we present an up-to-date overview of recent genomic studies involving C. albicans and discuss the accumulating evidence that shows how mitotic recombination events, ploidy dynamics, aneuploidy, and loss of heterozygosity (LOH) influence evolution, adaptation, and survival in C. albicans. This review was exclusively based on a thorough literature review on research papers investigating C. albicans and its genome. The Boolean search for relevant literatures was performed in Google scholar, Science direct, PubMed, and Web of science using a combination of the following precise keywords: Candida albicans, Genome plasticity, Parasexual events, Ploidy dynamics, Aneuploidy, LOH, and Mutations. All the papers that relate to the genome and genetics of C. albicans were included in the study.Results Candida albicans has a diploid heterozygous genome that is highly dynamic and can display variation from small to large-scale chromosomal rearrangement and aneuploidy, which have implications for drug resistance, virulence, and pathogenicity. The instability in the genome arising from stress or exposure to the host environment can be detrimental to survival. However, C. albicans can tactically exploit this instability in their genome as a survival strategy. Our investigation showed that there is a need for more studies to understand the regulation and the exact mechanisms that lead to chromosomal instability in C. albicans. It is important to understand how increasing ploidy affects genome stability. It is also important to unravel how tetraploids lose chromosomes to revert to diploids. Whether other ploidy states can be efficiently sustained also remains to be unraveled. Moreover, as the understanding of the pathogenicity of fungi continues to evolve, there is a continuing challenge in grasping the processes and factors that mediate the switching from commensal to the pathogen. It remains unclear whether parasexual processes also operate within natural populations. More studies are needed to appreciate better parasexual recombination events and mutational processes in C. albicans evolution and its link to persistence and infection establishment. The interrelationship between the host and the microbes can promote microbial fitness in the host and help the host acquire some beneficial features such as resistance against several pathogens. However, the long-term consequences on host fitness are largely unknown. This evidence provides an avenue to study evolutionary forces controlling C. albicans fitness level after host exposure.ConclusionThe variation in the genome of C. albicans greatly influences its biology, stress response approaches, and its virulence and resistant potentials. Understanding how C. albicans generate diversity is very crucial as this will help to design strategies mitigating their spread.

Mini Review] Microbial Volatile-mediated Plant Growth, Fitness, and Health Promotion

植物の生育は，地上部葉圏，地下部根圏，維管束輸送系内に生息する微生物の存在によって左右される．これらの微生物は，植物の成長，光合成，澱粉蓄積，開花，ストレス耐性等を促進する多数の揮発性化合物 (VCs) を合成することができる．近年，微生物の培養条件によっては，基本的に植物に有害な植物病原体が植物の生育や機能に有益な場合もあることがわかってきた．VCsはいくつかの代謝経路を脱調節することで生理機能の大きな変化をもたらすことから，作物の持続的生産の強化や環境変化に備えるための新たな戦略としてVCsを利用することが考えられている．しかしながら，VCsが関わる植物-微生物間の化学的相互作用，分子・細胞標的，植物の成長や体力に劇的な変化をもたらすメカニズムやネットワークについてはほとんど知られていないのが現状である．本稿では，VCsによる植物の成長，発達，代謝の変化の基盤となる主要な制御因子とそのメカニズムに焦点を当てる．また，作物収量を向上させるための革新的な農業技術・手法としてVCsシステムの利用に関するアイデアを提供することを目的としている．

Phenotypic and genomic signatures of interspecies cooperation and conflict in naturally occurring isolates of a model plant symbiont.

Given the need to predict the outcomes of (co)evolution in host-associated microbiomes, whether microbial and host fitnesses tend to trade-off, generating conflict, remains a pressing question. Examining the relationships between host and microbe fitness proxies at both the phenotypic and genomic levels can illuminate the mechanisms underlying interspecies cooperation and conflict. We examined naturally occurring genetic variation in 191 strains of the model microbial symbiont Sinorhizobium meliloti, paired with each of two host Medicago truncatula genotypes in single- or multi-strain experiments to determine how multiple proxies of microbial and host fitness were related to one another and test key predictions about mutualism evolution at the genomic scale, while also addressing the challenge of measuring microbial fitness. We found little evidence for interspecies fitness conflict; loci tended to have concordant effects on both microbe and host fitnesses, even in environments with multiple co-occurring strains. Our results emphasize the importance of quantifying microbial relative fitness for understanding microbiome evolution and thus harnessing microbiomes to improve host fitness. Additionally, we find that mutualistic coevolution between hosts and microbes acts to maintain, rather than erode, genetic diversity, potentially explaining why variation in mutualism traits persists in nature.

Anaplasma phagocytophilum Ankyrin A Protein (AnkA) Enters the Nucleus Using an Importin-β-, RanGTP-Dependent Mechanism.

Anaplasma phagocytophilum, a tick-borne obligately intracellular bacterium of neutrophils, causes human granulocytic anaplasmosis. Ankyrin A (AnkA), an effector protein with multiple ankyrin repeats (AR) is injected via type IV-secretion into the host neutrophil to gain access to the nucleus where it modifies the epigenome to promote microbial fitness and propagation. AR proteins transported into the host cell nucleus must use at least one of two known eukaryotic pathways, the classical importin β-dependent pathway, and/or the RanGDP- and AR (ankyrin-repeat)-dependent importin β-independent (RaDAR) pathway. Truncation of the first four AnkA N-terminal ARs (AR1-4), but not other regions, prevents AnkA nuclear accumulation. To investigate the mechanism of nuclear import, we created point mutations of AnkA N-terminal ARs, predicted to interfere with RaDAR protein import, and used importazole, a specific inhibitor of the importin α/β, RanGTP-dependent pathway. Nuclear colocalization analysis shows that nuclear localization of AnkA is unaffected by single AR1-4 mutations but is significantly reduced by single mutations in consecutive ARs suggesting RaDAR protein nuclear import. However, AnkA nuclear localization was also decreased with importazole, and with GTPγS. Furthermore, A. phagocytophilum growth in HL-60 cells was completely suppressed with importazole, indicating that A. phagocytophilum propagation requires a β-importin-dependent pathway. A typical classical NLS overlapping AR4 was subsequently identified suggesting the primacy of the importin-α/β system in AnkA nuclear localization. Whether the mutational studies of putative key residues support RaDAR NLS function or simply reflect structural changes that diminish engagement of an AR-NLS-importin pathway needs to be resolved through careful structure-function studies.