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.
Cell morphology in bacteria is a crucial factor for survival in that it affects evading predators and acquiring nutrition leading to growth and development. In order to grow, bacteria divide through binary fission to create two daughter cells identical in size, shape, and genome. One of the main contributors to accurate and efficient cell division is the Min system. Composed of proteins, MinC, MinD, and MinE, the following work together to place the FtsZ ring in the middle of the cell for septum formation. While the Min system and its effects have been heavily studied in other model organisms, little is known about its function and mechanism in the Gram-negative soil bacteria, Acinetobacter baylyi (ADP1). Bioinformatics tools such as sequence alignments, protein and operon predictions demonstrated evolutionary similarities between ADP1 and other rod-shaped organisms such as Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. In this study, we utilized ADP1’s high transforming capabilities to create individual knockout mutants of minC, minD, and minE. Based on bioinformatics, it is predicted that the min mutants would exert similar compromised growth and division morphologies of filamentation and minicell production as seen in other organisms. To test this hypothesis, cells were imaged under atomic force microscopy (AFM), and we acquired detailed nanoscopic data that showcased many filamented cells with few minicells. Features such as indents, side, and through bites also appeared on the surface of the mutant cells. Indents are shallow dips that appear on the cell surface, while bites are deeper features that either depress through the width of the cell (through bite) or asymmetrically along the side of the cell (side bite). Due to the mutation in division, bites and indent distribution were random as expected in the min mutants. This preliminary finding into the morphology effects of the Min system provides further insight into the complex mechanism of bacterial cell division.
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 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 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.”