Evidence of Correlation between Membrane Phase Transition and Clonogenicity in Dehydrating Acinetobacter baumannii: A Combined Micro-Raman and AFM Study.

The Gram-negative bacterium Acinetobacter baumannii is one of the most resilient multidrug-resistant pathogens in hospitals. Among Gram-negative bacteria, it is particularly resistant to dehydration (anhydrobiosis), and this feature allows A. baumannii to persist in hospital environments for long periods, subjected to unfavorable conditions. We leverage the combination of µ-Raman spectroscopy and atomic force microscopy (AFM) to investigate the anhydrobiotic mechanisms in A. baumannii cells by monitoring the membrane (both inner and outer membranes) properties of four A. baumannii strains during a 16-week dehydration period and in response to temperature excursions. We noted that the membranes of A. baumannii remained intact during the dehydration period despite undergoing a liquid-crystal-to-gel-phase transition, accompanied by changes in the mechanical properties of the membrane. This was evident from the AFM images, which showed the morphology of the bacterial cells alongside modifications of their superficial mechanical properties, and from the alteration in the intensity ratio of µ-Raman features linked to the CH3 and CH2 symmetric stretching modes. Furthermore, employing a universal power law revealed a significant correlation between this ratio and bacterial fitness across all tested strains. Additionally, we subjected dry A. baumannii to a temperature-dependent experiment, the results of which supported the correlation between the Raman ratio and culturability, demonstrating that the phase transition becomes irreversible when A. baumannii cells undergo different temperature cycles. Besides the relevance to the present study, we argue that µ-Raman can be used as a powerful nondestructive tool to assess the health status of bacterial cells based on membrane properties with a relatively high throughput.

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa.

The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral ß-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in Escherichia coli proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than E. coli, the opportunistic human pathogen Pseudomonas aeruginosa. Bioinformatic analyses revealed that P. aeruginosa possesses seven AsmA-like proteins. Deletion of asmA-like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in P. aeruginosa, including a novel AsmA-like protein (PA4735) that is not present in E. coli. Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM. IMPORTANCE: Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

Phage-mediated colistin resistance in Acinetobacter baumannii.

AIMS: Antimicrobial resistance is a global threat to human health, and Acinetobacter baumannii is a paradigmatic example of how rapidly bacteria become resistant to clinically relevant antimicrobials. The emergence of multidrug-resistant A. baumannii strains has forced the revival of colistin as a last-resort drug, suddenly leading to the emergence of colistin resistance. We investigated the genetic and molecular basis of colistin resistance in A. baumannii, and the mechanisms implicated in its regulation and dissemination. METHODS: Comparative genomic analysis was combined with genetic, biochemical, and phenotypic assays to characterize Φ19606, an A. baumannii temperate bacteriophage that carries a colistin resistance gene. RESULTS: Ф19606 was detected in 41% of 523 A. baumannii complete genomes and demonstrated to act as a mobile vehicle of the colistin resistance gene eptA1, encoding a functional lipid A phosphoethanolamine transferase. The eptA1 gene is coregulated with its chromosomal homolog pmrC via the PmrAB two-component system and confers colistin resistance when induced by low calcium and magnesium levels. Resistance selection assays showed that the eptA1-harbouring phage Ф19606 promotes the emergence of spontaneous colistin-resistant mutants. CONCLUSIONS: Φ19606 is an unprecedented example of a self-transmissible phage vector implicated in the dissemination of colistin resistance.

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging.

IMPORTANCE: By harnessing the versatility of fluorescence microscopy and super-resolution imaging, bacteriologists explore critical aspects of bacterial physiology and resolve bacterial structures sized beyond the light diffraction limit. These techniques are based on fluorophores with profitable photochemical and tagging properties. The paucity of available far-red (FR)-emitting dyes for bacterial imaging strongly limits the multicolor choice of bacteriologists, hindering the possibility of labeling multiple structures in a single experiment. The set of FR fluorophores characterized in this study expands the palette of dyes useful for microbiologists, as they can be used for bacterial LIVE/DEAD staining and for tagging the membranes of viable Escherichia coli and Bacillus subtilis cells. The absence of toxicity makes these dyes suitable for live-cell imaging and allows monitoring of bacterial membrane biogenesis. Moreover, a newly synthesized FR-fluorophore can be employed for imaging bacterial membranes with stimulated emission depletion microscopy, a super-resolution technique capable of increasing the resolving power of conventional microscopes.

Pathogenicity and virulence of Acinetobacter baumannii: Factors contributing to the fitness in healthcare settings and the infected host.

Acinetobacter baumannii is a common cause of healthcare-associated infections and hospital outbreaks, particularly in intensive care units. Much of the success of A. baumannii relies on its genomic plasticity, which allows rapid adaptation to adversity and stress. The capacity to acquire novel antibiotic resistance determinants and the tolerance to stresses encountered in the hospital environment promote A. baumannii spread among patients and long-term contamination of the healthcare setting. This review explores virulence factors and physiological traits contributing to A. baumannii infection and adaptation to the hospital environment. Several cell-associated and secreted virulence factors involved in A. baumannii biofilm formation, cell adhesion, invasion, and persistence in the host, as well as resistance to xeric stress imposed by the healthcare settings, are illustrated to give reasons for the success of A. baumannii as a hospital pathogen.

Fighting bacterial pathogens with carbon nanotubes: focused review of recent progress.

The fast and global spread of bacterial resistance to currently available antibiotics results in a great and urgent need for alternative antibacterial agents and therapeutic strategies. Recent studies on the application of nanomaterials as antimicrobial agents have demonstrated their potential for the management of infectious diseases. Among the diverse palette of nanomaterials currently used in biomedical applications, carbon nanotubes (CNTs) have gained massive interest given their many valuable properties, such as high thermal and electrical conductivity, tensile strength, flexibility convenient aspect ratio, and low fabrication costs. All these features are augmented by facile conjugation with functional groups. CNTs are currently available in many configurations, with two main categories being single-walled and multi-walled CNTs, depending on the number of rolled-up single-layer carbon atoms sheets making up the nanostructure. Both classes have been identified over the past years as promising antibacterial agents but the current level of understanding of their efficiency still harbors many pending questions. This mini-review surveys recent progress on the topic of antibacterial effects of CNTs and examines the proposed mechanisms of action(s) of different CNT typologies, placing the main focus on past studies addressing the antibacterial activity on Staphylococcus aureus and Escherichia coli, two prototypical Gram-positive and Gram-negative pathogens, respectively.

An image processing-based quantification of gram variability in Acinetobacter baumannii.

Gram staining differentiates bacteria as gram-positive and gram-negative, depending on their cell wall constituents, and coloring cells in violet and pink, respectively. Sometimes, a subpopulation of the same bacterial species assumes different colors, ranging from pink to violet, for reasons that are not completely understood yet. We analyze conventional brightfield images and use an automated pipeline to count pink and violet cells. The ImageJ-based processing algorithm quantifies the gram variability in Acinetobacter baumannii ACICU in the stationary phase of growth with a percentage of 66% pink cells. Different bacterial strains and cell growth stages have been considered. RESEARCH HIGHLIGHTS: Gram staining differentiates bacteria into gram-positive (violet) and gram-negative (pink). Gram variability represents an inhomogeneous distribution of pink and violet cells within the same species. We developed an ImageJ-based pipeline for the quantification of Gram variability in Acinetobacter baumannii.

Bio-physical mechanisms of dehydrating membranes of Acinetobacter baumannii linked to drought-resistance.

Acinetobacter baumanni, is an opportunistic nosocomial multi-drug resistant bacterium, which represents a threat for human health. This pathogen is able to persist in intensive care units thanks to its extraordinary resistance towards dehydration, whose mechanisms are unknown and enable it to easily spread through surfaces, contaminating also medical devices. In this article we reveal, with a multimodal approach, based on µ-R Spectroscopy, Gas Chromatography coupled to Mass Spectroscopy, Atomic Force Microscopy and Fluorescence Recovery After Photobleaching, the bio-physical mechanisms that the membrane of two A. baumannii strains undergoes during dehydration. Showing a substantial decoupling of the phase transition from liquid crystalline to gel phase from evidence of cell lysis. Such decoupling may be the core of the resistance of A. baumannii against dehydration and highlights the different ability to resist to drought between strains.

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa.

DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the ß clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

Antibacterial alkylguanidino ureas: Molecular simplification approach, searching for membrane-based MoA.

The ever-faster rise of antimicrobial resistance (AMR) represents a major global Public Health challenge. New chemical entities with innovative Modes of Action (MoAs) are thus desirable. We recently reported the development of a novel class of broad-spectrum bactericidal agents, the AlkylGuanidino Ureas (AGU). Due to their polycationic structure, they likely target bacterial membranes. In order to better understand their MoA, we synthesized a library of AGU derivatives by structural simplification of selected hit compounds and developed specific assays based on membrane models by means of both analytical and computational techniques. Cell-based assays provided experimental evidence that AGUs disrupt bacterial membranes without showing hemolytic behavior. Hence, we herein report a thorough chemical and biological characterization of a new series of AGUs obtained through molecular simplification, allowing the rational design of potent antibacterial compounds active on antibiotic-resistant strains.

Genome diversity of domesticated Acinetobacter baumannii ATCC 19606T strains.

Acinetobacter baumannii has emerged as an important opportunistic pathogen worldwide, being responsible for large outbreaks for nosocomial infections, primarily in intensive care units. A. baumannii ATCC 19606T is the species type strain, and a reference organism in many laboratories due to its low virulence, amenability to genetic manipulation and extensive antibiotic susceptibility. We wondered if frequent propagation of A. baumannii ATCC 19606T in different laboratories may have driven micro- and macro-evolutionary events that could determine inter-laboratory differences of genome-based data. By combining Illumina MiSeq, MinION and Sanger technologies, we generated a high-quality whole-genome sequence of A. baumannii ATCC 19606T, then performed a comparative genome analysis between A. baumannii ATCC 19606T strains from several research laboratories and a reference collection. Differences between publicly available ATCC 19606T genome sequences were observed, including SNPs, macro- and micro-deletions, and the uneven presence of a 52 kb prophage belonging to genus Vieuvirus. Two plasmids, pMAC and p1ATCC19606, were invariably detected in all tested strains. The presence of a putative replicase, a replication origin containing four 22-mer direct repeats, and a toxin-antitoxin system implicated in plasmid stability were predicted by in silico analysis of p1ATCC19606, and experimentally confirmed. This work refines the sequence, structure and functional annotation of the A. baumannii ATCC 19606T genome, and highlights some remarkable differences between domesticated strains, likely resulting from genetic drift.

A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Pseudomonas aeruginosa Siderophore Pyochelin.

The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production in vivo, PCH quantification in biological samples is problematic due to analytical complexity, requiring extraction from large volumes and time-consuming purification steps. Here, the construction of a bioluminescent whole cell-based biosensor, which allows rapid, sensitive, and single-step PCH quantification in biological samples, is reported. The biosensor was engineered by fusing the promoter of the PCH biosynthetic gene pchE to the luxCDABE operon, and the resulting construct was inserted into the chromosome of the ΔpvdAΔpchDΔfpvA siderophore-null P. aeruginosa mutant. A bioassay was setup in a 96-well microplate format, enabling the contemporary screening of several samples in a few hours. A linear response was observed for up to 40 nM PCH, with a lower detection limit of 1.64 ± 0.26 nM PCH. Different parameters were considered to calibrate the biosensor, and a detailed step-by-step operation protocol, including troubleshooting specific problems that can arise during sample preparation, was established to achieve rapid, sensitive, and specific PCH quantification in both P. aeruginosa culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing P. aeruginosa strains on a solid medium.

Phylogenomic analysis and characterization of carbon monoxide utilization genes in the family Phyllobacteriaceae with reclassification of Aminobacter carboxidus (Meyer et al. 1993, Hördt et al. 2020) as Aminobacter lissarensis comb. nov. (McDonald et al. 2005).

The monotypic carboxydophilic genus Carbophilus has recently been transferred to the genus Aminobacter within the family Phyllobacteriaceae, and Carbophilus carboxidus was renamed Aminobacter carboxidus (comb. nov.) [Hördt et al. 2020]. Due to the poor resolution of the 16S rRNA gene-based phylogeny, an extensive phylogenomic analysis of the family Phyllobacteriaceae was conducted, with particular focus on the genus Aminobacter. Whole genome-based analyses of Phyllobacteriaceae type strains provided evidenced that the genus Aminobacter forms a monophyletic cluster, clearly demarcated from all other members of the family. Close relatedness between A. carboxidus DSM 1086T and A. lissarensis DSM 17454T was inferred from core proteome phylogeny, shared gene content, and multilocus sequence analyses. ANI and GGDC provided genetic similarity values above the species demarcating threshold for these two type strains. Metabolic profiling and cell morphology analysis corroborated the phenotypic identity between A. carboxidus DSM 1086T and A. lissarensis DSM 17454T. Search for the presence of carbon monoxide dehydrogenase (CODH) genes in Phyllobacteriaceae genomes revealed that the form II CODH is widespread in the family, whereas form I CODH was detected in few Mesorhizobium type strains, and in both A. carboxidus DSM 1086T and A. lissarensis DSM 17454T. Results of phylogenomic, chemotaxonomic, and morphological investigations, combined with the presence of similarly arranged CODH genes, indicate that A. carboxidus DSM 1086T and A. lissarensis DSM 17454T are distinct strains of the same species. Hence A. carboxidus is a later subjective heterotypic synonym of A. lissarensis.

Generation of Genetic Tools for Gauging Multiple-Gene Expression at the Single-Cell Level.

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple-gene expression at the single-cell level has limited the understanding of phenotypic heterogeneity. To investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple-gene expression at the single-cell level has been generated. This tool, named pRGC, consists of a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single-cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population without the need for image processing for spectral cross talk correction. In addition, two pRGC variants have been generated for either (i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome that is suitable for long-term experiments in the absence of antibiotic selection or (ii) replication in bacterial genera other than Pseudomonas The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple-gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity.IMPORTANCE Within a bacterial population, single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial subpopulations with distinct phenotypes. The analysis of gene expression at the single-cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation, and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple-gene expression at the single-cell level by fluorescence microscopy without the need for advanced image-processing procedures. A proof of concept has been provided by investigating iron uptake and iron storage gene expression in response to iron availability in P. aeruginosa.

Growth Phase- and Desiccation-Dependent Acinetobacter baumannii Morphology: An Atomic Force Microscopy Investigation.

Acinetobacter baumannii has emerged as a major bacterial pathogen during the past three decades. The majority of the A. baumannii infections occur in hospitals and are caused by strains endowed with high desiccation tolerance, which represents an essential feature for the adaptation to the nosocomial environment. This work aims at investigating the desiccation response of the multidrug-resistant A. baumannii strain ACICU as a function of the bacterial growth phase and oxygen availability, by correlating bacterial survival with shape alterations. The three-dimensional morphological analysis of bacteria was carried out by atomic force microscopy (AFM), following the evolution of bacterial shape descriptors, such as the area, volume, roughness of individual cell membranes, and the cell cluster roughness, which exhibited peculiar and distinctive behavior as a function of the growth conditions. AFM images of A. baumannii ACICU cells revealed the prevalence of the coccoid morphology at all growth stages, with a tendency to reduce their size in the stationary phase, accompanied by a higher survival rate to air-drying. Moreover, cells harvested from the logarithmic phase featured a larger volume and resulted to be more sensitive to desiccation compared to the cells harvested at later growth stages. In addition, oxygen deprivation caused a significant decrease in cellular size and was associated with the formation of pores in the cell membrane, accompanied by a relative reduction in culturability after desiccation. Morphological plasticity and multidrug resistance may contribute to desiccation tolerance and therefore to persistence in the hospital setting.

SSNOMBACTER: A collection of scattering-type scanning near-field optical microscopy and atomic force microscopy images of bacterial cells.

BACKGROUND: In recent years, a variety of imaging techniques operating at nanoscale resolution have been reported. These techniques have the potential to enrich our understanding of bacterial species relevant to human health, such as antibiotic-resistant pathogens. However, owing to the novelty of these techniques, their use is still confined to addressing very particular applications, and their availability is limited owing to associated costs and required expertise. Among these, scattering-type scanning near field optical microscopy (s-SNOM) has been demonstrated as a powerful tool for exploring important optical properties at nanoscale resolution, depending only on the size of a sharp tip. Despite its huge potential to resolve aspects that cannot be tackled otherwise, the penetration of s-SNOM into the life sciences is still proceeding at a slow pace for the aforementioned reasons. RESULTS: In this work we introduce SSNOMBACTER, a set of s-SNOM images collected on 15 bacterial species. These come accompanied by registered Atomic Force Microscopy images, which are useful for placing nanoscale optical information in a relevant topographic context. CONCLUSIONS: The proposed dataset aims to augment the popularity of s-SNOM and for accelerating its penetration in life sciences. Furthermore, we consider this dataset to be useful for the development and benchmarking of image analysis tools dedicated to s-SNOM imaging, which are scarce, despite the high need. In this latter context we discuss a series of image processing and analysis applications where SSNOMBACTER could be of help.

STED nanoscopy of KK114-stained pathogenic bacteria.

Super-resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine-like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram-positive and Gram-negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114-staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells.

Geometrical-optics approach to measure the optical density of bacterial cultures using a LED-based photometer.

We develop a suitable geometrical-optics approach and demonstrate that it is possible to measure the optical density (OD) of bacterial cultures using a light emitting diode (LED)-based photometer. We measure both attenuation and spot-size variation, and we compensate for diffraction and stray-light impairment related to the incoherent source and large detection area. The approach is validated for different concentrations of two bacterial species, Escherichia coli and Staphylococcus aureus, that present different shapes and clustering organization.

Geometrical-optics approach to increase the accuracy in LED-based photometers for point-of-care testing.

A geometrical-optics approach is proposed to increase the accuracy in photometric measurements, using a point-of-care testing (POCT) LED-based sensor. Due to stray-light effects, the measurement accuracy depends on the dimension of the CMOS area, where the radiation is detected. We propose two image processing approaches and evaluate the influence of the sensor area. In addition, we demonstrate that with the same measurement, both absorption coefficient and refractive index can be determined, measuring the beam attenuation and the spot-size enlargement due to ray refraction.

New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors.

The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model.IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.