Stimulation of bacterial DNA transformation by cattle saliva: implications for using genetically modified plants in animal feed

To investigate the likelihood of DNA transfer from genetically modified plants (GMP) to bacteria, a rescue plasmid system for Streptococcus gordonii was modified. It was applied to monitor the DNA transformation into oral and intestinal bacteria in cattle. Transformation and recombination frequency of S. gordonii was dependent on the length of the transformed DNA. Beside horse serum, cow saliva also rendered the cells competent for DNA uptake. Competence induction was completely abolished by the addition of liquid from maize silage. Competence was partially suppressed by the addition of rumen liquid. In order to study native bacteria, 724 colonies sensitive to the antibiotics were isolated from either silage or the saliva and rumen of cows. Using horse serum, silage liquid, cow saliva or rumen liquid for competence induction, the isolates failed to integrate linearized pMK110 DNA and restore antibiotic resistance. Only 6 of the colonies obtained from the teeth of a silage-fed cow were sensitive to the antibiotics. Two isolates were related to Staphylococcus warneri. They could be transformed with the model plasmid pMK110 after induction by horse serum. DNA transformation, however, was not stimulated by incubation with cattle saliva, silage or rumen liquid. The response to competence-stimulating factors seems to vary between different bacterial species. These results suggest that the probability of DNA uptake from silage of GMPs is very low.     

Expression of Green Fluorescent Protein in Streptococcus gordonii DL1 and Its Use as a Species-Specific Marker in Coadhesion with Streptococcus oralis 34 in Saliva-Conditioned Biofilms In Vitro

Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter, PhppA, which is situated upstream of the chromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp (′gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-′gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the PhppA-′gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement of S. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.     

Marker Rescue Studies of the Transfer of Recombinant DNA to Streptococcus gordonii In Vitro, in Foods and Gnotobiotic Rats

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10⁻⁶ to 5.8 × 10⁻⁷ transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 × 10⁻⁹). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10⁻¹⁰), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10⁻¹¹. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.     

Transformation of group F streptococci by plasmid DNA.

When the Challis strain of Streptococcus sanguis was transformed by the 17 megadalton beta plasmid from Streptococcus faecalis strain DS5, the plasmid underwent a 1.5 megadalton deletion (LeBlanc & Hassell, 1976). Furthermore, the covalently closed circular (CCC) plasmid DNA isolated from Challis transformants was rapidly converted to a linear form which did not possess any detectable transforming activity. To obtain stable CCC plasmid DNA a competent culture of a Lancefield group F streptococcus, strain DL8 (ATCC 12393), was used as a recipient of beta plasmid DNA. The plasmid DNA isolated from group F transformants exhibited the same configuration and size characteristics as the DS5 beta plasmid, and the CCC configuration was stable upon storage. CCC plasmid DNA from a group F transformant was biologically active and, when added to competent cultures of strain DL8, transformed them at frequencies about 100-fold greater than did beta plasmid DNA from DS5. This suggests the existence of a restriction--modification system in strain DL8.     

Formation of Single-Stranded DNA during DNA Transformation of Neisseria gonorrhoeae

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.     

Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.     

Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [³H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca²⁺-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ.     

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl₂ solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl₂ solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.     

Isolation and characterization of coaggregation-defective (Cog−) mutants ofStreptococcus gordonii DL1 (Challis)

Streptococcus gordonii DL1 (Challis) bears coaggregation-mediating surface adhesins which recognize galactoside-containing surface polysaccharides onStreptococcus oralis 34,Streptococcus oralis C104, andStreptococcus SM PK509. Fifty-nine spontaneously-occurring coaggregation-defective (Cog^–) mutants ofS. gordonii DL1 unable to coaggregate with partner streptococci were isolated. Six representative Cog^– mutants were characterized by their coaggregation properties with fourActinomyces naeslundii strains (T14V, PK947, PK606, PK984),Veillonella atypica PK1910, andPropionibacterium acnes PK93. The six representative Cog^– mutants showed altered coaggregation with their streptococcal partners,A. naeslundii PK947, andP. acnes PK93. Based on the coaggregation phenotypes of these mutants, a model for the lactose-inhibitable coaggregation betweenS. gordonii DL1 and its partner bacteria is proposed. The potential use of these mutants in studies of oral biofilms is discussed.     

The high prevalence of herpes simplex virus type 1 DNA in human trigeminal ganglia is not a function of age or gender

The purpose of this study was to determine the presence and copy numbers of herpes simplex virus type 1 (HSV-1) DNA in human trigeminal ganglia (TG) with respect to age, gender, and postmortem interval (PMI). Human TG (n = 174, obtained from the Oregon Brain Bank, with data on age, gender, and PMI) were analyzed for HSV-1 DNA copies (HSV-1 DNA polymerase gene) by using real-time PCR. We found that 89.1% (131/147) of subjects and 90.1% (155/174) of TG contained HSV-1 DNA. The copy numbers of HSV-1 DNA in the positives ranged from very high (>10⁶) to very low (5). These data confirm and strengthen our previous findings that subjects were positive for HSV-1 DNA in tears (46/50; 92%) and saliva (47/50; 94%). These TG data and tear and saliva data demonstrated considerable variability in copy numbers of HSV-1 DNA per subject. Statistical analysis showed no significant relationship between gender and copy number, age and copy number, or PMI and copy number for each pair of variables. A factorial analysis of gender, age, and PMI with respect to copy number also showed no statistical significance. This is the first study that provides statistical analysis that documents that the prevalence of HSV-1 DNA in the human TG is not a function of either gender or age.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.     

The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model.     

Natural Competence and the Evolution of DNA Uptake Specificity

Many bacteria are naturally competent, able to actively transport environmental DNA fragments across their cell envelope and into their cytoplasm. Because incoming DNA fragments can recombine with and replace homologous segments of the chromosome, competence provides cells with a potent mechanism of horizontal gene transfer as well as access to the nutrients in extracellular DNA. This review starts with an introductory overview of competence and continues with a detailed consideration of the DNA uptake specificity of competent proteobacteria in the Pasteurellaceae and Neisseriaceae. Species in these distantly related families exhibit strong preferences for genomic DNA from close relatives, a self-specificity arising from the combined effects of biases in the uptake machinery and genomic overrepresentation of the sequences this machinery prefers. Other competent species tested lack obvious uptake bias or uptake sequences, suggesting that strong convergent evolutionary forces have acted on these two families. Recent results show that uptake sequences have multiple “dialects,” with clades within each family preferring distinct sequence variants and having corresponding variants enriched in their genomes. Although the genomic consensus uptake sequences are 12 and 29 to 34 bp, uptake assays have found that only central cores of 3 to 4 bp, conserved across dialects, are crucial for uptake. The other bases, which differ between dialects, make weaker individual contributions but have important cooperative interactions. Together, these results make predictions about the mechanism of DNA uptake across the outer membrane, supporting a model for the evolutionary accumulation and stability of uptake sequences and suggesting that uptake biases may be more widespread than currently thought.     

An Effective Strategy, Applicable to Streptococcus salivarius and Related Bacteria, To Enhance or Confer Electroporation Competence

Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable Streptococcus salivarius strain, ATCC 25975, could be transformed reproducibly at a level of 10⁵ transformants per μg of DNA. The technique was used to introduce a plasmid into other strains of S. salivarius, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria.     

Evaluation of biological and physical protection against nuclease degradation of clay-bound plasmid DNA

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.     

Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a clustered DNA damage site

The formation of clustered DNA damage sites is a unique feature of ionizing radiation. Recent studies have shown that the repair of lesions within clusters may be compromised, but little is understood about the mutagenic consequences of such damage sites. Using a plasmid-based method, damaged DNA containing uracil positioned at 1–5 bp separations from 8-oxo-7,8-dihydroguanine on the complementary strand was transfected into wild-type Escherichia coli or into strains lacking the DNA glycosylases Fpg and MutY. Mutation frequencies were found to be significantly higher for clustered damage sites than for single lesions. The loss of MutY gave a large relative increase in mutation frequency and a strain lacking both Fpg and MutY showed even higher mutation frequencies, up to nearly 40% of rescued plasmid. In these strains, the mutation frequency decreases with increasing spacing of the uracil from the 8-oxo-7,8-dihydroguanine site. Sequencing of plasmid DNA carrying clustered damage, following rescue from bacteria, showed that almost all of the mutations are GC→TA transversions. The data suggest that at clustered damage sites, depending on lesion spacing, the action of Fpg is compromised and post-replication processing of lesions by MutY is the most important mechanism for protection against mutagenesis.     

DNA Immunization with Japanese Encephalitis Virus Nonstructural Protein NS1 Elicits Protective Immunity in Mice

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a zoonotic pathogen that is prevalent in some Southeast Asian countries and causes acute encephalitis in humans. To evaluate the potential application of gene immunization to JEV infection, we characterized the immune responses from mice intramuscularly injected with plasmid DNA encoding JEV glycoproteins, including the precursor membrane (prM) plus envelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice survived after a lethal JEV challenge, whereas when immunized with the plasmid expressing NS1, 90% of the mice survived after a lethal challenge. As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effect of the plasmid DNA. Despite having no detectable neutralizing activity, the NS1 immunization elicited a strong antibody response exhibiting cytolytic activity against JEV-infected cells in a complement-dependent manner. By contrast, immunization with a construct expressing a longer NS1 protein (NS1′), containing an extra 60-amino-acid portion from the N terminus of NS2A, failed to protect mice against a lethal challenge. Biochemical analyses revealed that when individually expressed, NS1 but not NS1′ could be readily secreted as a homodimer in large quantity and could also be efficiently expressed on the cell surface. Interestingly, when NS1 and NS1′ coexisted in cells, the level of NS1 cell surface expression was much lower than that in cells expressing NS1 alone. These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.     

Degradation of Escherichia coli chromosomal and plasmid DNA in serum

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281 (pBR322) harbouring small plasmids (mol. wt 5.5 X 10(6) and 2.6 X 10(6] in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.