MicroDiv2010 Author: Wang, Harris H. A mini-project report for the 2010 Microbial Diversity Course at the Marine Biology Laboratory in Woods Hole, MA, USA DNA subsistence, natural competency, and horizontal gene transfer in marine and soil organisms in the environment Harris H. Wang, Ph.D. Department of Genetics Harvard Medical School, Boston, MA, USA Address: 77 Avenue Louis Pasteur, NRB Room 238, Boston, MA 02115, USA Email: [email protected] Phone: 617-432-6976 Course Instructors: Prof. Dan Buckley and Prof. Steve Zinder Date: July 27, 2010 Page 1 MicroDiv2010 Author: Wang, Harris H. ABSTRACT DNA is the universal genetic material of cells, but can potentially serve also as a source of nutrients as it is rich in carbon, nitrogen, and phosphate. Utilization of DNA as nutrition requires importing the large DNA molecules into the cell through mechanisms akin to those of natural competency. This study aimed to enrich and isolate DNA-subsisting bacteria that are also naturally competent for their ability to uptake long pieces of DNA and utilize their genetic information through integration into the chromosome. DNA-subsisting bacteria were isolated both from soil and marine environments over the course of 3 weeks. Various physiological characteristics were measured including the potential for natural transformation. These enrichments and isolations are useful for developing new model organisms for which new genetic systems can be easily developed by reducing the barrier of introducing exogenous DNA into cells through natural mechanisms. Page 2 MicroDiv2010 Author: Wang, Harris H. INTRODUCTION DNA is conventionally viewed as the essential information-carrying genetic material common to all organisms that exist in the intracellular space of cells. However, dissolved DNA is also prevalent in the environment, existing up to 0.2-50 micrograms per gram of soil and 0.2-44 micrograms per liter of marine water [1]. DNA is particularly rich in phosphate which is known to be a nutritional limitation in marine environments. It has been shown that up to 25% of all phosphates in some freshwater ecosystems are found in dissolved DNA [2]. Furthermore, DNA has a high turnover rate, which makes it an important factor in the carbon, nitrogen, and phosphate recycling system in nature [3, 4]. However, few evidences are found in the literature for the existence of oligotrophic organisms that can utilization DNA for nutrition in the environment. A survey of marine water revealed Vibrionales and Altermonodalecea that can subsist on DNA [5], but no such studies have been done for terrestrial bacteria. On the other hand, there is a wealth of literature describing the general phenomenon of natural competence in which extracellular DNA is taken up into the cell by membrane transporters [6]. These naturally competent cells are putatively thought to uptake extracellular DNA as a means for acquisition of novel trait that are encoded by the heterologous DNA. More likely than not, similar organisms tend to share more similarly useful genes, which is certainly the explanation for why some organisms such as H. influenza specifically uptake exogenous DNA containing sequence motifs similar to its own genome [7]. Genetic experiments have shown that competence genes are important in the utilization of DNA as nutrition in long-term stationary phase E. coli [8], providing further evidence that DNA utilization and natural competency may be linked in environmental organisms. Understanding DNA recycling in the environment is an important step towards understanding the role of DNA not only as a source of genetic material but also as a source of nutrition and structural material as is sometimes found in biofilms. The ability to scavenge DNA for nutrition may also be associated with predation and cannibalism phenotypes of competing organisms under nutrient limiting environments. This study aims to investigate the prevalence of microbes that can subsist on DNA as it sole carbon source in both soil and marine environments by enrichment, isolation, molecular phylogenic analysis, and physiological measurements for DNA catabolism. Furthermore, we test the hypothesis that bacteria subsisting on extracellular DNA are more likely to be naturally competent and may be enriched for their ability to incorporate extracellular DNA into its genome. Since DNA transformation is one of the biggest barriers to developing genetic systems for new organisms, isolation techniques for naturally competent cells from the environment may be a useful strategy to accelerate development of recombinant technologies for these more tractable environmental organisms. MATERIALS AND METHODS Field sampling. Marine samples were obtained from the mouth of Trunk River which flows to the Atlantic Ocean near Falmouth, MA, USA. About 50ml water samples were taken at a depth of 0.5m. Soil samples were obtained from organic rich soils along a nearby hiking trail. Media. For liquid media, NH4Cl (10 mM), Na2SO4 (1 mM), KH2PO4/K2HPO4 (1 mM), MOPS (10 mM), trace elements, and dissolved DNA were added to FW base (for cultivation of soil samples) or SW base (for cultivation of marine samples) to make FW broth (FWB) or SW broth (SWB), pH to 7.2 with NaOH, autoclaved for 15 minutes at 122 °C, and supplemented with pre- sterilized vitamin mix. The FW Base contains NaCl (17.1 mM), MgCl2 6H2O (1.97 mM), CaCl2 2H2O (0.15 mM), and KCl (6.71 mM) dissolved in MilliQ water. The SW base contains NaCl Page 3 MicroDiv2010 Author: Wang, Harris H. (342.2 mM), MgCl2 6H2O (14.8mM), CaCl2 2H2O (1.0 mM), and KCl (6.71 mM). Noble agar (15g/L) was added to the pre-autoclaved solution for making agar plates. Filter sterilized antibiotics were added as necessary: chloramphenicol (25 ug/ml), kanamycin (50 ug/ml), gentamycin (15 ug/ml), spectromycin (50 ug/ml), tetracycline (20 ug/ml), and ampicillin (100 ug/ml). Two forms of dissolved DNA were used: low-molecular weight (LMW) herring sperm DNA (Sigma D7290) and high molecular weight (HMW) double stranded DNA (salmon testes, sodium salt; Sigma D-1626) were used at final concentrations of 0.15 g/L to 1 g/L. The dissolved DNA is the sole carbon sources in the growth media. Salt Water Complete (SWC) rich medium was used for marine organism and nutrient Broth rich medium was used for soil organisms. Enrichment. Liquid culture enrichment for bacteria that subsistent on DNA was performed using either FW media for soil sample or SWB media for marine sample supplemented HMW DNA. Simultaneously, control inoculums of FW or SWB media without dissolved DNA were also made. FW enrichment of soil sample was made by addition of 0.5g soil into FW media, vigorous vortexing for 5 minutes, settling for 1 hr, and inoculation of 100ul of the supernatant into 3mL of FW media with or without DNA. SW enrichment of marine sample was made by direct inoculation of 100ul of marine sample into SW media with or without DNA. Final DNA concentration in the enrichment was at 0.5-0.6 g/L. Samples were grown in the dark at 30 °C in a shaking incubator. Isolation. Clones were isolated on FW or SW agar plates with and without dissolved DNA (0.15-0.5g/L). Isolates were obtained first on LMW DNA plates, but was subsequently switched to HMW DNA plates because subsistence on longer DNA fragments will increase the likelihood of enriching for naturally competent bacteria. Plates were incubated at 30 °C in the dark. Visible colonies generally formed in 3-5 days. Growth curves, DNA quantification, microscopy. Growth curve experiments were conducted by measuring the optical density (OD 600nm) of enrichments at every ~12 hrs, blanked to a standard. Simultaneously, the extracellular DNA concentration in the growth media was determined on an UV-Vis Spectrophotometer (NanoDrop 2000c, Thermo Scientific, USA) by measuring the absorbance at 260 nm. Quality of the DNA reading was determined by the A260/280 ratio, which general varied between 1.8 and 2.0. Images of cell suspensions were taken on a Zeiss epifluorescent microscope (Axio) using the AxioCam MRc imaging system. Clone library construction and phylogenetic identity of isolates. To determine the identity of individual organisms in the enrichment cultures, clone libraries of 16S rRNA genes were constructed from a genomic preparation (Ultraclean Soil DNA Isolation Kit, Mo-Bio Inc., USA) of the enrichment (1 ml sample), using bacterial PCR primers 8F and 1492R. The resulting PCR product was cloned into a vector for sequencing using the Invitrogen TOPO TA cloning Kit. A total of 96 clones were picked for each sample set. Four total sample sets were sent for Sanger DNA sequencing (Marine Biology Laboratory, Sequencing Facility): 1) enrichment on SWB media on HWM DNA, 2) enrichment on SWB media without DNA as control, 3) enrichment on FW media with HMW DNA and 4) enrichment on FW without DNA as control. Sequences were analyzed using the RDP web application (http://rdp.cme.msu.edu/). For identification of individual isolates (marine samples), individual colonies were picked into a 20ul lysis buffer 0.05% Triton X-100 and boiling at 105 °C for 5 minutes. One ul of the cell lysis product was used as template for PCR of the 16S rRNA gene using universal 8F and 1492R bacterial primers. The PCR consisted of initial denaturation at 95 °C for 5 min, 30 cycles of [46 °C annealing for 30 sec, 72 °C extension for 90 sec] and final extension at 72 °C for 5 min. The Page 4 MicroDiv2010 Author: Wang, Harris H. PCR products were screened by gel electrophoresis to confirm successful PCR amplification, treated by ExoSAP-IT kit, and sequenced by Sanger DNA sequencing. DNA utilization plate assay. Utilization of DNA was determined on agar plates following a previous described method [5].