Acinetobacter baylyi are naturally competent soil bacteria. Natural transformation is the acquisition of new genetic material via the uptake of foreign, exogenous DNA. Competence is the physiological state some bacterial species may realize in order for natural transformation to occur. Natural transformation, and therefore competence, is clinically relevant, as natural transformation serves as a chief method by which antibiotic-resistant genes are dispersed amongst the bacterial population. A. baylyi serves as an ideal model organism to model natural transformation. A. baylyi are easy to cultivate in vitro, may be genetically modified with ease, and there is a complete library of single gene deletion mutants available for research use. Our first goal was to test the role of Type IV pilus (T4P) proteins in competence using a novel surface-associated quantitative protocol. From the French collection, we obtained knockout mutants lacking proteins predicted to be important for comprising a T4P or for uptake of DNA across the inner membrane. Transformation of cells on a nutritive agar surface allowed for quantitative determination of transformation efficiency over nine orders of magnitude. Using this method we determined which genes were necessary for competence. Under the conditions we tested, genes absolutely required for transformation in A. baylyi include genes encoding the basal apparatus of a T4P (comM, pilF, pilC, pilU, and pilT), the gene encoding the inner membrane DNA translocation protein (comA), the gene encoding the major pilin (comP), genes encoding minor pilins (pilV, fimT), and the gene encoding the pilus tip protein (comC). Mutations in genes encoding for a periplasmic protein that helps target DNA to the comA channel (comEA), conserved hypothetical protein (CHP), genes encoding for a signal transduction response and regulatory receiver (pilG, pilR, and pilS), and a gene encoding for a minor pilins, comF and comE, resulted in a 2-4 log loss in competence. Using transformation on a surface instead of in liquid, we have discovered that a T4P, including its major pilin, is required for transformation A. baylyi. Further, in order to determine conditions under which A. baylyi are most competent in LB media, a simple broth comprised of three ingredients in an easy ratio, we tested additives to LB broth. Though A. baylyi may be grown in a variety of different medias, our laboratory chooses to grow ADP1 cells in LB because of its ubiquity in bacterial labs, low cost, and high rates of natural transformation. Our experiments address whether we may increase the efficacy of LB by altering growing temperature or infusing it with a variety of experimental additives. Overall, we found that A. baylyi cells are competent in nearly all LB conditions tested, but notably, rates of transformation slightly increase in LB+succinate but drastically decrease in LBK+Fe-deficient.
Competence for natural transformation is the physiological ability of bacteria to take up extracellular DNA. This ability is wielded by over 40 species of bacteria. We hypothesize that competence for natural transformation might be required for the GASP phenotype: a phenomenon by which cells grown to long term stationary phase can out-compete young cells co-incubated with them in stationary phase. In this experiment, we show for the first time that the soil bacteria Acinetobacter baylyi exhibits the GASP phenotype, and that knock out mutants of the gene comP which codes for a prepilin-like protein showed significantly reduced fitness levels during a GASP test when competed against young wild type cells. Knock out mutants of the gene comQ, which codes for a transmembrane protein, showed reduced fitness levels when competed against a young wild type strain in stationary phase but this difference was not significant. Defective GASP responses suggest that natural competence is important for cells to “age” (evolve) normally during long-term stationary phase.
Imaging live bacteria with an atomic force microscope (AFM) is a challenging process, taking into account the lateral forces exerted by the probe on cells that must be immobilized but still immersed in liquid. The Lang-Lostroh labs use AFM to examine Acinetobacter baylyi cells that are competent (able to take up DNA from the environment). In order to image cells while they are competent, it is necessary to find an effective combination of liquid media and sample preparation method while maintaining the cells alive, competent, and attached to the AFM pucks with porcine gelatin. Many combinations were tested using different washing and gelatin immobilization media (distilled deionized water, and phosphate-buffer saline), as well as using different media (distilled deionized water, phosphate-buffer saline, and diverse dilutions of Luria-Bertani broth with and without sodium chloride) during imaging itself. The combinations that presented the most satisfying images of immobilized cells with AFM were submitted to membrane integrity and competence assays. Washing and immobilizing the cells with distilled deionized water and imaging them with 50% Luria-Bertani broth without sodium chloride was the most successful combination. Even though the cells did not show detectable competence ability, they were alive yet inactive since after the imaging procedure they were still able to form colonies on plain Luria-Bertani agar plates. It provides insight on a viability spectrum that must be taken into account when distinguishing cell viability. Further research should focus on testing smoother media transitions with access to food source to avoid osmotic pressure stress and starvation.
Acinetobacter baylyi are highly competent Gram-negative bacteria that are easy to culture in vitro, making them model organisms for studying natural transformation. Natural transformation is a means of genetic exchange for bacteria that occurs by the uptake and incorporation of extracellular DNA into a bacterium’s own genome to enhance fitness and survival. It is hypothesized that hair-like appendages known as type IV pili (T4P) or homologous structures make up the competence machine that facilitates natural transformation, and these appendages are also associated with the bacterial form of movement known as twitching motility. While studies have investigated the role of pilin proteins and general competence proteins in the competence machine, little is known about how the cytoskeleton influences its assembly and function. The cytoskeleton includes shape-determining proteins that specify cell shape and maintain intracellular organization. These proteins might influence natural transformation and twitching motility in A. baylyi because the assembly, extension, and retraction of T4P requires localization of the necessary components, which is facilitated in part by the cytoskeleton. Specifically, I tested the importance of two shape genes, mreC and rodA, on transformation and twitching motility by comparing knockout mutants to wild type A. baylyi cells. The results indicate that RodA plays an important role in natural transformation and twitching motility, as deletion of rodA decreased both processes. The mreC knockout did not have a significant impact on transformation or twitching motility. The data suggests that cellular organization facilitated by shape-determining proteins like RodA impacts the proper assembly and function of the competence and locomotive machine.
Acinetobacter baylyi ADP1 is a gram-negative soil bacterium that exhibits competence and twitching motility. DNA uptake is achieved via the Type IV Pilus competence machine and twitching is performed by Type IV pili. Homologues of Type IV pili proteins are involved in transformation in a variety of bacteria. The similarities between proteins involved in DNA uptake, Type IV pilus systems and type II protein secretion systems suggests that they belong to evolutionary related systems containing cell envelope spanning structures with conserved architecture and core components. As many competence proteins of ADP1 are related to structural subunits and biogensis proteins of Type IV pili, a key question is whether Type IV pili of ADP1 are directly involved in DNA uptake and binding. Or, do the pilin-like components of the transformation system make up a completely different structure? Many bacteria can perform natural transformation; however, our knowledge regarding the structures and mechanisms needed for DNA uptake is scarce. Thus, our research involved determining which genes are needed for competence, which are used for twitching motility and which are possibly involved in both functions in ADP1. In order to test each protein’s role, tdk-kan knock out mutants were created and the mutants were compared to the wild type. An existing library of proteins predicted to encode various parts of the Type IV pilus with knock out genes was used. Our results showed that the majority of tested genes are needed for both competence and twitching, suggesting a physiological relationship. Specifically, mutants with a greater twitching ability were also more competent.
Acinetobacter baylyi is a gram-negative soil dwelling bacterium. The strain ADP1 is highly competent which allows for easy manipulation of its genes. The genes of interest here are a set of the genes encoding a type VI secretion system (T6SS), namely tssG, tssF, tssE, tssB, and tssC. This T6SS is homologous to the bacteriophage tail. The tail of the bacteriophage is used to inject DNA into a bacterial cell. Therefore, we hypothesized that the T6SS could be used to release DNA into the environment. Over the course of this research, we investigated ADP1 bacterial cells that contained knockouts of the various parts of the T6SS. The knockouts were used to determine which, if any, of the parts of the T6SS play a role in DNA release. We also tested the mutants for differences in growth rate and survival in long-term stationary phase (LTSP). LTSP is a phase in which 99% of the cells die off and the remaining 1% begin to eat waste and dead cells to survive. We examined survival in LTSP because another T6SS gene, vgrG (ACIAD0167), is known to be expressed during LTSP (Lostroh and Voyles, 2010). The gene is also necessary for survival during LTSP (Stanley and Lostroh, 2010) and twitching motility (Nguyen and Lostroh, 2013). We discovered that the core T6SS genes are not needed for a normal growth rate during exponential phase or for DNA release. However, we were unable to definitively determine if tssG, tssF, tssE, tssB, and tssC are necessary for survival during LTSP due to poor survival rates of the wild-type and mutants, likely caused by evaporation of water over the course of the experiments. Further research will be performed to determine which secretion system if any is responsible for DNA release and if tssG, tssF, tssE, tssB, and tssC are necessary for survival during LTSP.
Acinetobacter baylyi strain ADP1 is a gram-negative bacterium normally studied because of its high competence for genetic transformation and its ability to catabolize plant-derived aromatic compounds. A previous study has identified that the gene cluster ACIAD1969-ACIAD1952 contains genes that may be responsible for potassium tellurite resistance, as well as other proteins that are “hypothetical.” Our goal was to use bioinformatics to investigate this gene cluster and to determine whether it played a role in potassium tellurite resistance as well as twitching motility. Our results indicate that the gene cluster is actually composed of four different operons that play a role in tellurite resistance. We also found that the gene cluster was most likely inherited from horizontal gene transfer, as it is not found in any other Acinetobacter strains. Furthermore, all genes except ACIAD1956, ACIAD1962 and ACIAD1964 are responsible for potassium tellurite resistance in ADP1 and all mutants exhibit twitching motility defects. Our results indicate that the genes in the gene cluster ACIAD1969-ACIAD1952 encode proteins and should no longer be considered “hypothetical.”
Acinetobacter baylyi ADP1is a naturally competent, non-pathogenic soil bacterium used for the study of natural transformation. Natural transformation is the ability to acquire extracellular DNA and use that DNA as new genetic material. Here, we tested the impact of monovalent cations on transformation efficiency by comparing transformation using LB agar, which contains Na+ ions, to transformation using LBK agar, which contains instead K+ ions. We found no difference in transformation efficiencies using these two types of solid media during transformation. Next, we intended to test the effects of divalent cations on transformation efficiency. But, rates of transformation were so high on both LB and LBK that we first needed to find conditions under which transformation efficiency was <0.1% in order to be able to detect whether the addition of cations would have a positive effect on transformation efficiency. Thus, we reduced the amount of DNA used for transformation, but doing so did not reduce the transformation efficiency. Then, we altered the conditions under which we measured transformation efficiency by adding DNase, which degrades extracellular DNA, in order to restrict the time of DNA availability. However, these manipulations did not reduce the transformation efficiency either. Control experiments verified that the wild type, non-transformed cells were sensitive to the antibiotic chloramphenicol. This control is important because the donor DNA confers resistance to chloramphenicol. Unfortunately, the desired conditions of experimental room were never obtained, and thus a effective comparison of transformation rates due to divalent cation presence was conducted. We conclude that ADP1 cells are extremely competent under all conditions tested.
Acinetobacter baylyi ADP1 has been studied in laboratories because of their competence for natural transformation and ability to adapt to different environmental conditions. A previous study has found 30 different genes in A. baylyi ADP1 that are induced by starvation during the long term stationary phase. ACIAD0167 is one of them, encoding a Vgr-like protein. The goal of this study was to test whether ACIAD0167 and other genes in its operon (ACIAD0166, ACIAD0168 and ACIAD0169) are required for twitching motility, or for surviving stressful conditions including heat shock, desiccation and DNA damage. Our study found that ACIAD0167 and the other three genes in the operon play a role in twitching motility in A. baylyi but apparently not in other phenotypes. The likely first gene in the operon, ACIAD0166, was cloned into wild-type ADP1 and over-expression of the gene caused a smaller twitching zone than one produced by wild type cells, further implicating the role of ACIAD0167-containing operon in twitching motility. These results indicate that ACIAD0166, ACIAD0167, ACIAD0168, and ACIAD0169 genes encode proteins, and should no longer be considered “hypothetical” genes. We also found a novel link among ACIAD0167, twitching motility and the type VI secretion system (T6SS). ACIAD0167 is found in the STRING network to be associated with genes involved the T6SS, whose structure resembles an inverted bacteriophage tail.