GrainGenes
A Database for Triticeae and Avena
Chakrabarti et al., pp. 25-28
II. 6 The analysis of barley genomes. I: The problem that the DNA of bacteria may contribute to the DNA in extracts of barley tissues derived from germinated seeds.
T. Chakrabarti, C. H. Doy and N. C. Subrahmanyam, Genetics Department, Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2601, Australia. "R"
A major aim of this laboratory is to investigate the nature of the DNA from a number of barley species and to determine the degree of homology or divergence between the genomes (Chakrabarti, Doy and Subrahmanyam, 1978, elsewhere this issue). Before this could be done it was necessary to evaluate whether associated micro-organisms contribute a portion of the DNA.
There is an ever increasing literature which shows that many kinds of bacteria may co-exist in adventitious or meaningful associations with plants. Meaningful or not, these associations, depending on the extent of the 'contamination', could confuse the fine analysis of plant DNA. The possibility of the presence of contaminant bacteria is also a vital one for the evaluation of claims of others for the integration of bacterial DNA into plant DNA to form an end-to-end hybrid which is then replicated. Such claims have been made following the treatment of barley seeds with Micrococcus lysodeikticus DNA, for example, Ledous & Huart (1969, 1975) and depend largely on the detection by analytical centrifugation in neutral CsCl gradients of DNA's of buoyant density in between that of barley main band DNA (1.702 g cm-3) and the heavier M. lysodeikticus DNA (1.731 gcm-3). Others (Kleinhofs, 1975 and Kleinhofs et al. 1975) have investigated this and suggest that the more likely explanation is that the intermediate peaks are due to DNA from contaminating bacteria. We have therefore taken the matter of possible 'contaminating' bacteria into particular account.
All DNA was prepared essentially according to the method of Marmur (1963).
Working mainly with extracts of roots from germinating seeds of Hordeum vulgare L. cv. Clipper, at least four kinds of bacteria (not yet classified) were isolated by plating on Luria agar (one of the standard rich media that culture most bacteria). The total count was about 109 bacteria g-1 wet wt. tissue. Similar results were obtained with extracts of shoots, except that the counts were lower.
Seed sterilisation by hypochlorite was grossly inadequate whilst a method using HgCl2 (Williams et al. 1974) and a method using 50% H2SO4 (Kleinhofs et al. 1975) were both detrimental to germination. A method depending on treating seed with 1% x/v AgNO3 and allowing to dry (Halsall and Forrester, 1977 and Halsall personal communication) reduced the bacterial count to less than 106 g-1 wet wt. tissue (more than 3 orders of magnitude).
Routinely, seeds were germinated for 7 days at room temperature on Luria agar. This medium supports the rapid growth at room temperature of all the isolated bacteria (large colonies overnight) yet, with an occasional exception (plate discarded), no microbial growth was observed from roots and shoots lying in intimate contact with the medium. Without AgNO3 treatment the plant material was overgrown by microbial growth.
It is concluded that the bacteria surviving AgNO3 treatment, and therefore found in extracts of tissue, are within, rather than on the surface, of the plant material. Counts show that these bacteria multiply with the growth of the seedling. One wonders whether the presence of presumptive internal bacteria indicates something other than a casual contaminant relationship.
Working cleanly (no contaminants added from tools or solutions) and germinating on sterile filter paper but without adequate surface sterilization of seeds, up to 30% of the total DNA, on analytical centrifugation in neutral CsCl, distributed as a multicomponent system on the heavy side of mainband barley DNA (buoyant density 1.700 + 0.001 g cm-3 ). Four kinds of bacteria were isolated and their DNA's found to be of buoyant density (neutral CsCl) 1.700 (identical to main band barley DNA), 1.714, 1.719 and 1.721. After AgNO3 sterilisation no heavy DNA was detected in neutral CsCl.
It was concluded that the heavy DNA was entirely due to contaminant bacteria. The claim for hybrid bacterial-plant DNAs is based on similar heavy buoyant densities. We therefore concur with the suggestion (Kleinhofs et al. 1975) that the claim for hybrid bacterial-plant DNA is based on inadequate evidence. The validity of all such claims would depend in part on the level and nature of any bacterial contamination. It should therefore be noted that the presence of bacteria can be readily monitored and minimised to a level that does not show as heavy 'satellites' in the usual neutral CsCl analytical centrifugation.
For the purpose of our work the AgNO3 treatment reduces bacterial DNA to about 0.1% of the total. This is reduced to less than 0.025% after preparative density gradient centrifugation and is confined to the bacterial DNA coincident with main band. The bacterial DNA is not displaced in Ag+/CsSO4 gradients. It is highly unlikely that bacterial DNA can be responsible for the satellites we report elsewhere in this issue (Chakrabarti et al. 1978).
References:
Chakrabarti, T., C. H. Doy and N. C. Subrahmanyam, 1978. The analysis of barley genomes. 2. A molecular approach. Barley Genetics Newsletter 8:
Halsall, D. M. and R. I. Forrester, 1977. Effects of certain cations on the formation and infectivity of Phytophthora zoospores. 1. Effect of calcium, magnesium, potassium, and iron ions. Canadian J. Microbiology, 23: 994-1001.
Ledoux, L. and R. Huart, 1969. Fate of exogenous bacterial deoxyribonucleic acids in barley seedlings. J. Mol. Biol. 43: 243-262.
Ledoux, L. and R. Huart, 1975. Integration and replication of bacterial DNA in barley root cells. Arch Internationel de Physiologie et de Biochemie, 83: 196-197.
Kleinhofs, A. 1975. DNA-hybridization studies of the fate of bacterial DNA in plants. In: Genetic manipulations with plant materials. Edit. Ledoux, L., Plenum, New York and London. 461-477.
Kleinhofs, A., F. C. Eden, M-D Chilton and A. J. Bendich, 1975. On the question of the integration of exogenous bacterial DNA into plant DNA. Proc. Nat. Acad. Sci. USA, 72: 2748-2752.
Marmur, J. 1963. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. In: Methods in Enzymology VI, Edit. Colowick, S. P. and Kaplan, N. O. 726-738.
Ranjekar, P. K., D. Pallotta and J. G. Lafontaine, 1976. Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum). Biochim. Biophys. Acta. 425: 30-40.
Williams, K. L., S. Nurmi and L. M. Birt, 1974. Developmental and biochemical characteristics of sterile culture of the blowfly Lucilia cuprini. Lab. Animals 8: 177-187.