DNA modifications impact natural transformation of Acinetobacter baumannii.

Acinetobacter baumannii is a dangerous nosocomial pathogen, especially due to its ability to rapidly acquire new genetic traits, including antibiotic resistance genes (ARG). In A. baumannii, natural competence for transformation, one of the primary modes of horizontal gene transfer (HGT), is thought to contribute to ARG acquisition and has therefore been intensively studied. However, knowledge regarding the potential role of epigenetic DNA modification(s) on this process remains lacking. Here, we demonstrate that the methylome pattern of diverse A. baumannii strains differs substantially and that these epigenetic marks influence the fate of transforming DNA. Specifically, we describe a methylome-dependent phenomenon that impacts intra- and inter-species DNA exchange by the competent A. baumannii strain A118. We go on to identify and characterize an A118-specific restriction-modification (RM) system that impairs transformation when the incoming DNA lacks a specific methylation signature. Collectively, our work contributes towards a more holistic understanding of HGT in this organism and may also aid future endeavors towards tackling the spread of novel ARGs. In particular, our results suggest that DNA exchanges between bacteria that share similar epigenomes are favored and could therefore guide future research into identifying the reservoir(s) of dangerous genetic traits for this multi-drug resistant pathogen.

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

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 that bacteria 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 affect 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 affect not only 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 data for our work on A. baumannii to data reported for Acinetobacter 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 it differs from the well-studied species A. baylyi.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

Fluorescently Labeled DNA Interacts with Competence and Recombination Proteins and Is Integrated and Expressed Following Natural Transformation of Bacillus subtilis.

During competence, Bacillus subtilis is able to take up DNA from its environment through the process of transformation. We investigated the ability of B. subtilis to take up fluorescently labeled DNA and found that it is able to take up fluorescein-dUTP-, DyLight 550-dUTP-, and DyLight 650-dUTP-labeled DNA. Transformation with labeled DNA containing an antibiotic cassette resulted in uptake of the labeled DNA and also generated antibiotic-resistant colonies. DNA is primarily taken up at the pole, as it can be seen to colocalize with ComFC, which is a component of the competence machinery. The DNA is taken up rapidly and can be seen to localize with (the actively searching form of) RecA. Colocalization with a homologous locus on the chromosome increases over time. Using microfluidics, we observed replacement of the homologous locus and subsequent expression of the integrated labeled and unlabeled DNA, although whether the integrated DNA contains labeled nucleotides needs to be determined conclusively. Integrated DNA in cells with a doubling time of 60 min is expressed on average 6 h 45 min after the addition of DNA and 4 h 45 min after the addition of fresh medium. We also found that the expression of the incoming DNA under these conditions can occur before cell division and, thus, before complete exit from the competence state. Because the competence machinery is conserved among naturally competent bacteria, this method of labeling is also suitable for studying transformation of other naturally competent bacteria.IMPORTANCE We used DNA that was covalently labeled with fluorescent nucleotides to investigate the transformation process of Bacillus subtilis at the molecular level. We show that the labeled DNA colocalizes with components of the competence machinery, the chromosome, and the recombination protein RecA. Using time-lapse microscopy and microfluidics, we visualized, in real-time, the uptake of fluorescently labeled DNA. We found that under these conditions, cell division is not required for the expression of integrated DNA. Because the competence machinery is conserved in naturally competent bacteria, this method can also be used to investigate the transformation process in many other bacterial species.