bInfections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC 90 of tigecycline from 25 g/ml in the parental strains to 3.1 g/ml in the ⌬adeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains. O ne of the greatest global health problems results from the limited treatment options to fight bacterial infections caused by multidrug-resistant (MDR) organisms. The group of ESKAPE organisms that is comprised of Enterobacter spp., Staphylococcus aureus/epidermidis, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis/faecium is considered to cause the vast majority of, often untreatable, nosocomial infections (1). Among these ESKAPE pathogens A. baumannii is most difficult to treat due to its multiple intrinsic and acquired resistance mechanisms that resulted in the development of MDR, extensively drug resistant (XDR), or even pan-drug-resistant (PDR) phenotypes (2-5).Bacteria have evolved multiple ways to evade antibiotic-mediated cell death, such as (i) enzymatic modification/cleavage of the antibiotic (e.g., beta-lactams), (ii) modification/protection of the antibiotic target (e.g., fluoroquinolones), or (iii) reduction of the intracellular concentration by antibiotic efflux or reduced influx (e.g., tetracyclines) (6). The expression of such defense mechanisms may require an extensive metabolic investment, often leading to a reduced fitness of these resistant bacteria in the absence of the external selection pressure (7). To overcome these ecol...

Summary The identification of the virulence factors of plant‐pathogenic bacteria has relied on the testing of individual mutants on plants, a time‐consuming process. Transposon sequencing (Tn‐seq) is a very powerful method for the identification of the genes required for bacterial growth in their host. We used this method in a soft‐rot pathogenic bacterium to identify the genes required for the multiplication of Dickeya dadantii in chicory. About 100 genes were identified showing decreased or increased fitness in the plant. Most had no previously attributed role in plant–bacterium interactions. Following our screening, in planta competition assays confirmed that the uridine monophosphate biosynthesis pathway and the purine biosynthesis pathway were essential to the survival of D. dadantii in the plant, as the mutants ∆ carA , ∆ purF , ∆ purL , ∆ guaB and ∆ pyrE were unable to survive in the plant in contrast with the wild‐type (WT) bacterium. This study also demonstrated that the biosynthetic pathways of leucine, cysteine and lysine were essential for bacterial survival in the plant and that RsmC and GcpA were important in the regulation of the infection process, as the mutants ∆ rsmC and ∆ gcpA were hypervirulent. Finally, our study showed that D. dadantii flagellin was glycosylated and that this modification conferred fitness to the bacterium during plant infection. Assay by this method of the large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.

SigB is the main stress gene regulator in Listeria monocytogenes affecting the expression of more than 150 genes and thus contributing to multiple-stress resistance. Despite its clear role in most stresses, its role in oxidative stress is uncertain, as results accompanying the loss of sigB range from hyperresistance to hypersensitivity. Previously, these differences have been attributed to strain variation. In this study, we show conclusively that unlike for all other stresses, loss of sigB results in hyperresistance to H 2 O 2 (more than 8 log CFU ml ؊1 compared to the wild type) in aerobically grown stationary-phase cultures of L. monocytogenes strains 10403S and EGD-e. Furthermore, growth at 30°C resulted in higher resistance to oxidative stress than that at 37°C. Oxidative stress resistance seemed to be higher with higher levels of oxygen. Under anaerobic conditions, the loss of SigB in 10403S did not affect survival against H 2 O 2 , while in EGD-e, it resulted in a sensitive phenotype. During exponential phase, minor differences occurred, and this result was expected due to the absence of sigB transcription. Catalase tests were performed under all conditions, and stronger catalase results corresponded well with a higher survival rate, underpinning the important role of catalase in this phenotype. Furthermore, we assessed the catalase activity in protein lysates, which corresponded with the catalase tests and survival. In addition, reverse transcription-PCR (RT-PCR) showed no differences in transcription between the wild type and the ⌬sigB mutant in various oxidative stress genes. Further investigation of the molecular mechanism behind this phenotype and its possible consequences for the overall phenotype of L. monocytogenes are under way. IMPORTANCESigB is the most important stress gene regulator in L. monocytogenes and other Gram-positive bacteria. Its increased expression during stationary phase results in resistance to multiple stresses. However, despite its important role in general stress resistance, its expression is detrimental for the cell in the presence of oxidative stress, as it promotes hypersensitivity against hydrogen peroxide. This peculiar phenotype is an important element of the physiology of L. monocytogenes, and it might help us explain the behavior of this organism in environments where oxidative stress is present. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis, a serious and potentially lethal foodborne illness (1). Despite its low incidence, listeriosis has a high mortality rate (30%), making it the most deadly foodborne disease in the United Kingdom and the United States, as it claims more lives than any other foodborne pathogen (1, 2). One of the key attributes that makes L. monocytogenes such a successful pathogen is its ability to survive and persist in a wide range of harsh environments both outside and within the human host (3). One of the most important stresses L. monocytogenes has to withstand, in order to survive and cause disease, is oxi...

15Soft rot enterobacteria (Dickeya and Pectobacterium) are major pathogens that provoke (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/126896 doi: bioRxiv preprint first posted online Apr. 12, 2017; 3 proteomic can now be used but they only allow the identification of genes induced during the 40 infection process and non-induced genes may be missed. Tn-seq is a very powerful method to 41 identify genes required for bacterial growth in their host. We used for the first time this