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DNA is the genetic code found inside all living cells and its molecular stability can also be utilized outside the cell. While extracellular DNA (eDNA) has been identified as a structural polymer in bacterial biofilms, whether it persists stably throughout development remains unclear. Here, we report that eDNA is temporarily invested in the biofilm matrix before being reclaimed later in development. Specifically, by imaging eDNA dynamics within undomesticated Bacillus subtilis biofilms, we found eDNA is produced during biofilm establishment before being globally degraded in a spatiotemporally coordinated pulse. We identified YhcR, a secreted Ca 2+ -dependent nuclease, as responsible for eDNA degradation in pellicle biofilms. YhcR cooperates with two other nucleases, NucA and NucB, to reclaim eDNA for its phosphate content in colony biofilms. Our results identify extracellular nucleases that are crucial for eDNA reclamation during biofilm development and we therefore propose a new role for eDNA as a dynamic metabolic reservoir., (© 2024. The Author(s).)

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion., Competing Interests: Declaration of interests S.D.G. and L.O.B. are founders, shareholders, and members of the scientific advisory board of Clarametyx Biosciences, Inc., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications., Competing Interests: The authors have declared that no competing interests exist.

Moraxella catarrhalis is a host-adapted bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease. This study characterises the conserved M. catarrhalis extracellular nuclease, a member of the ββα metal finger family of nucleases, that we have named NucM. NucM shares conserved sequence motifs from the ββα nuclease family, including the DRGH catalytic core and Mg 2+ co-ordination site, but otherwise shares little primary sequence identity with other family members, such as the Serratia Nuc and pneumococcal EndA nucleases. NucM is secreted from the cell and digests linear and circular nucleic acid. However, it appears that a proportion of NucM is also associated with the cell membrane and acts as an entry nuclease, facilitating transformation of M. catarrhalis cells. This is the first example of a ββα nuclease in a Gram negative bacteria that acts as an entry nuclease. In addition to its role in competence, NucM affects cell aggregation and biofilm formation by M. catarrhalis, with ΔnucM mutants having increased biofilm biomass. NucM is likely to increase the ability of cells to survive and persist in vivo, increasing the virulence of M. catarrhalis and potentially affecting the behaviour of other pathogens that co-colonise the otorhinolaryngological niche.

SbcC-SbcD are the bacterial orthologs of Mre11-Rad50, a nuclease complex essential for genome stability, normal development, and viability in mammals. In vitro, these enzymes degrade long DNA palindromic structures. When inactivated along with ExoI in Escherichia coli , or Sae2 in eukaryotes, palindromic amplifications arise and propagate in cells. However, long DNA palindromes are not normally found in bacterial or human genomes, leaving the cellular substrates and function of these enzymes unknown. Here, we show that during the completion of DNA replication, convergent replication forks form a palindrome-like structural intermediate that requires nucleolytic processing by SbcC-SbcD and ExoI before chromosome replication can be completed. Inactivation of these nucleases prevents completion from occurring, and under these conditions, cells maintain viability by shunting the reaction through an aberrant recombinational pathway that leads to amplifications and instability in this region. The results identify replication completion as an event critical to maintain genome integrity and cell viability, demonstrate SbcC-SbcD-ExoI acts before RecBCD and is required to initiate the completion reaction, and reveal how defects in completion result in genomic instability., Competing Interests: The authors declare no conflict of interest.

Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading- and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement., (© 2016 The Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Bacterial biofilms endure high concentrations of biocides, and new strategies for biofilm control must therefore replace or complement the use of antibiotics, for example, by targeting the extracellular matrix to cause dispersal or increased antimicrobial susceptibility. Extracellular DNA (eDNA) is a matrix component of most biofilms, and is therefore an attractive target. Enzymatic degradation of eDNA can prevent, disperse, or sensitize biofilm to antimicrobials, but cheaper production is required to realize large-scale application. Replacing mammalian DNase with bacterial nucleases could offer a path to lower production costs. Alternatively, eDNA could be targeted by disrupting its interactions with other matrix components. As new knowledge about eDNA-binding matrix components comes to light, exciting opportunities for targeting the biofilm matrix via eDNA are emerging., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The release of extracellular DNA molecules (eDNA) contributes to various biological processes, such as biofilm formation, virulence, and stress tolerance. The quantity of eDNA released by bacteria is usually regulated by extracellular nucleases that are secreted by different systems. In this study, we show that high concentrations of eDNA inhibit the growth of two strains of Deinococcaceae, Deinococcus radiodurans, and Deinococcus radiopugnans, but have no effect on other selected organisms, such as Escherichia coli. In D. radiodurans, an extracellular nuclease was shown to be secreted through the Sec pathway. Disruption of one member of this pathway, SecD/F, inhibited cell growth, suggesting that the Sec pathway plays an important role in growth rate. However, the Sec pathway mutant exhibited a greater deficiency in growth rate compared with the extracellular nuclease mutant, indicating that the pathway not only secretes the extracellular nuclease, but has other unknown functions as well.

DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

Biofilm formation is a critical component to the lifestyle of many naturally occurring cellulose-degrading microbes. In this work, cellular aggregation and biofilm formation of Clostridium phytofermentans, a cellulolytic anaerobic bacterium, was investigated using a combination of microscopy and analytical techniques. Aggregates included thread-like linkages and a DNA/protein-rich extracellular matrix when grown on soluble cellobiose. Similar dense biofilms formed on the surface of the model cellulosic substrate Whatman no. 1 filter paper. Following initially dispersed attachment, microcolonies of ~500 µm diameter formed on the filter paper after 6 days. Enzymic treatment of both the biofilm and cellular aggregates with DNase and proteinase resulted in significant loss of rigidity, pointing to the key role of extracellular DNA and proteins in the biofilm structure. A high-throughput biofilm assay was adapted for studying potential regulators of biofilm formation. Various media manipulations were shown to greatly impact biofilm formation, including repression in the presence of glucose but not the β(1→4)-linked disaccharide cellobiose, implicating a balance of hydrolytic activity and assimilation to maintain biofilm integrity. Using the microtitre plate biofilm assay, DNase and proteinase dispersed ~60 and 30 % of mature biofilms, respectively, whilst RNase had no impact. This work suggests that Clostridium phytofermentans has evolved a DNA/protein-rich biofilm matrix complementing its cellulolytic nature. These insights add to our current understanding of natural ecosystems as well as strategies for efficient bioprocess design., (© 2014 The Authors.)

Quinolones trap the covalent gyrase-DNA complex in Escherichia coli, leading to cell death. Processing activities for trapped covalent complex have not been characterized. A mutant strain lacking SbcCD nuclease activity was examined for both accumulation of gyrase-DNA complex and viability after quinolone treatment. Higher complex levels were found in ΔsbcCD cells than in wild-type cells after incubation with nalidixic acid and ciprofloxacin. However, SbcCD activity protected cells against the bactericidal action of nalidixic acid but not ciprofloxacin.

Free extracellular DNA provides nutrition to bacteria and promotes bacterial evolution by inducing excessive mutagenesis of the genome. To understand the influence of extracellular DNA fragments on D. radiodurans, we investigated cell growth and survival after extracellular DNA or dNMPs treatment. The results showed that the extracellular DNA fragments inhibited the growth of D. radiodurans. Interestingly, dGMP, a DNA component, enhanced D. radiodurans tolerance to H(2)O(2) and gamma-radiation significantly. Further experiments indicated that extracellular dGMP stimulated the activity of one catalase (KatA, DR1998), and induced gene transcription including the extracellular nuclease (drb0067). When this only extracellular nuclease gene (drb0067) in D. radiodurans was deleted, the mutant strain showed more sensitive to H(2)O(2) and gamma-radiation than the wild type strain. These results suggest that DRB0067 plays an important role in oxidative stress resistance. Taken together, we proposed a new anti-oxidation mechanism in D. radiodurans. This mechanism acts to increase expression levels of DRB0067 which then secretes active nuclease to degrade extracellular DNA fragments. The extracellular nuclease has a two-fold benefit, creating more free dNTPs for further cell protection and the removal of extracellular DNA fragments.

Extragenic sequences in genomes, such as microRNA and CRISPR, are vital players in the cell. Repetitive extragenic palindromic sequences (REPs) are a class of extragenic sequences, which form nucleotide stem-loop structures. REPs are found in many bacterial species at a high copy number and are important in regulation of certain bacterial functions, such as Integration Host Factor recruitment and mRNA turnover. Although a new clade of putative transposases (RAYTs or TnpA(REP)) is often associated with an increase in these repeats, it is not clear how these proteins might have directed amplification of REPs. We report here the structure to 2.6 Å of TnpA(REP) from Escherichia coli MG1655 bound to a REP. Sequence analysis showed that TnpA(REP) is highly related to the IS200/IS605 family, but in contrast to IS200/IS605 transposases, TnpA(REP) is a monomer, is auto-inhibited and is active only in manganese. These features suggest that, relative to IS200/IS605 transposases, it has evolved a different mechanism for the movement of discrete segments of DNA and has been severely down-regulated, perhaps to prevent REPs from sweeping through genomes.

Eukaryotic cells sterilize the cytosol by using autophagy to route invading bacterial pathogens to the lysosome. During macrophage infection with Mycobacterium tuberculosis, a vacuolar pathogen, exogenous induction of autophagy can limit replication, but the mechanism of autophagy targeting and its role in natural infection remain unclear. Here we show that phagosomal permeabilization mediated by the bacterial ESX-1 secretion system allows cytosolic components of the ubiquitin-mediated autophagy pathway access to phagosomal M. tuberculosis. Recognition of extracelluar bacterial DNA by the STING-dependent cytosolic pathway is required for marking bacteria with ubiquitin, and delivery of bacilli to autophagosomes requires the ubiquitin-autophagy receptors p62 and NDP52 and the DNA-responsive kinase TBK1. Remarkably, mice with monocytes incapable of delivering bacilli to the autophagy pathway are extremely susceptible to infection. Our results reveal an unexpected link between DNA sensing, innate immunity, and autophagy and indicate a major role for this autophagy pathway in resistance to M. tuberculosis infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae., (© 2011 Blackwell Publishing Ltd.)

Staphylococcus aureus spreads on soft agar surfaces and forms giant colonies. Here, we examined the inhibitory role of extracellular DNA on the colony spreading activity. The double-deletion mutation of nuc1 and nuc2, which encode secretory nucleases, increased extracellular DNA and showed a decreased ability to form giant colonies. The addition of DNase I or micrococcal nuclease to the soft agar restored the ability of the nuc1-nuc2 double mutant to form giant colonies. In addition, the promoter activities of nuc1 and nuc2 in the wild-type strain were elevated in the peripheral region of the giant colony. These findings suggest that the digestion of extracellular DNA by secretory nucleases is required for the colony spreading activity of S. aureus., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Transformation requires specialized proteins to facilitate the binding and uptake of DNA. The genes of the Bacillus subtilis comG operon (comGA-G) are required for transformation and to assemble a structure, the pseudopilus, in the cell envelope. No role for the pseudopilus has been established and the functions of the individual comG genes are unknown. We show that among the comG genes, only comGA is absolutely required for DNA binding to the cell surface. ComEA, an integral membrane DNA-binding protein plays a minor role in the initial binding step, while an unidentified protein which communicates with ComGA must be directly responsible for binding to the cell. We show that the use of resistance to DNase to measure 'DNA uptake' reflects the movement of transforming DNA to a protected state in which it is not irreversibly associated with the protoplast, and presumably resides outside the cell membrane, in the periplasm or associated with the cell wall. We suggest that ComGA is needed for the acquisition of DNase resistance as well as for the binding of DNA to the cell surface. Finally, we show that the pseudopilus is required for DNA uptake and we offer a revised model for the transformation process., (© 2011 Blackwell Publishing Ltd.)

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Helicobacter pylori is a genetically diverse bacterial species, owing in part to its natural competence for DNA uptake that facilitates recombination between strains. Inter-strain DNA recombination occurs during human infection and the H. pylori genome is in linkage equilibrium worldwide. Despite this high propensity for DNA exchange, little is known about the factors that limit the extent of recombination during natural transformation. Here, we identify restriction-modification (R-M) systems as a barrier to transformation with homeologous DNA and find that R-M systems and several components of the recombination machinery control integration length. Type II R-M systems, the nuclease nucT and resolvase ruvC reduced integration length whereas the helicase recG increased it. In addition, we characterized a new factor that promotes natural transformation in H. pylori, dprB. Although free recombination has been widely observed in H. pylori, our study suggests that this bacterium uses multiple systems to limit inter-strain recombination., (© 2010 Blackwell Publishing Ltd.)

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3' to 5' DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7-11.2-kbp linear duplex DNAs at a rate of ∼250 bp s(-1), while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp(-1). Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the "leading" AdnB motor propagates a Y-fork by translocation along the 3' DNA strand, ahead of the "lagging" AdnA motor domain. By tracking the resection of the 5' and 3' strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5' strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3' strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.

Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing the nonpalindromic 10- or 12-nucleotide sequence 5'-ATGCCGTCTGAA-3' (additional semiconserved nucleotides are underlined), termed the DNA uptake sequence (DUS10 or DUS12). Here we investigated the effects of the DUS on transformation and DNA uptake for several laboratory strains of N. gonorrhoeae. We found that all strains showed efficient transformation of DUS containing DNA (DUS10 and DUS12) but that the level of transformation with DNA lacking a DUS (DUS0) was variable in different strains. The DUS-enhanced transformation was 20-fold in two strains, FA1090 and FA19, but was approximately 150-fold in strains MS11 and 1291. All strains tested provide some level of DUS0 transformation, and DUS0 transformation was type IV pilus dependent. Competition with plasmid DNA revealed that transformation of MS11 was enhanced by the addition of excess plasmid DNA containing a DUS while FA1090 transformation was competitively inhibited. Although FA1090 was able to mediate much more efficient transformation of DNA lacking a DUS than was MS11, DNA uptake experiments showed similar levels of uptake of DNA containing and lacking a DUS in FA1090 and MS11. Finally, DNA uptake was competitively inhibited in both FA1090 and MS11. Taken together, our data indicate that the role of the DUS during DNA transformation is variable between strains of N. gonorrhoeae and may influence multiple steps during transformation.

Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of gamma-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.

Extracellular DNA can play a structural role in the microbial environment. Here evidence is presented that an environmental isolate of Acidovorax temperans utilises extracellular DNA for intercellular and cell-surface attachment and that Type IV pili and electrostatic interactions play a role in this interaction. Preliminary attempts to isolate and purify extracellular polysaccharides from A. temperans strain CB2 yielded significant amounts of DNA raising the question of whether this molecule was present as a structural component in the extracellular matrix. The role of DNA in attachment was indicated by experiments in which the addition of DNase to liquid medium inhibited the attachment of Acidovorax to glass wool. A Tn5 insertional mutant, lacking Type IV pili, was unable to initiate attachment. Addition of DNase caused rapid detachment of bound cells, but no detachment occurred when proteinase, RNase or inactivated DNase were used. Addition of MgCl(2) also caused significant detachment, supporting the possible mechanistic role of electrostatic interactions in the attachment process. Although attachment was apparent in early to mid-log phase growth, surprisingly DNA was not detected in the culture supernatant until late stationary phase and coincided with an appreciable loss of cell viability. This suggests that during log-phase growth attachment is mediated by eDNA that is released in low quantities and/or is highly localised within the extracellular matrix and also that stationary phase DNA release through widespread cell lysis may be a separate and unrelated event.

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this "reckless" DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.

Fur (ferric uptake regulator) is a prokaryotic transcriptional regulator that controls a large number of genes mainly related to iron metabolism. Several Fur homologues with different physiological roles are frequently found in the same organism. The genome of the filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 codes for three different fur genes. FurA is an essential protein involved in iron homoeostasis that also modulates dinitrogen fixation. FurA interacts with haem, impairing its DNA-binding ability. To explore functional differences between Fur homologues in Anabaena, factors affecting their regulation, as well as some biochemical characteristics, have been investigated. Although incubation of FurB with haem severely hinders its ability to interact with DNA, binding of haem to FurC could not be detected. Oxidative stress enhances the transcription of the three fur genes, especially that of furB and furC. In addition, overexpression of FurA and FurB in Escherichia coli increases survival when the cells are challenged with H(2)O(2) or Methyl Viologen (paraquat), a superoxide-anion-generating reagent. When present in saturating concentrations, FurB exhibits unspecific DNA-binding activity and protects DNA from cleavage produced by hydroxyl radicals or DNaseI. On the basis of these results, we suggest that, whereas at low concentrations FurB would act as a member of the Fur family, at saturating concentrations FurB protects DNA, showing a DNA-protection-during-starvation-like behaviour.

The minimal length of integrated homologous donor DNA tracks in Acinetobacter baylyi transformation and factors influencing the location and length of tracks were determined. Donor DNA contained the nptII gene region (kanamycin resistance, KmR). This region carried nine approximately evenly spaced silent nucleotide sequence tags and was embedded in heterologous DNA. Recipient cells carried the normal nptII gene with a central 10 bp deletion (kanamycin-sensitive). The Km(R) transformants obtained had donor DNA tracks integrated covering on average only 4.6 (2-7) of the nine tags, corresponding to about 60 % of the 959 nt homologous donor DNA segment. The track positions were biased towards the 3' end of nptII. While the replication direction of recipient DNA did not affect track positions, inhibited transcription (by rifampicin) shifted the beginning of tracks towards the nptII promoter. Absence of the RecJ DNase decreased the length of tracks. Absence of SbcCD DNase increased the integration frequency of the 5' part of nptII, which can form hairpin structures of 43-75 nt, suggesting that SbcCD DNase interferes with hairpins in transforming DNA. In homology-facilitated illegitimate recombination events during transformation (in which a homologous DNA segment serves as a recombinational anchor to facilitate illegitimate recombination in neighbouring heterologous DNA), on average only about half of the approximately 800 nt long tagged nptII anchor sequences were integrated. From donor DNA with an approximately 5000 nt long homologous segment having the nptII gene in the middle, most transformants (74 %) had only a part of the donor nptII integrated, showing that short track integration occurs frequently also from large homologous DNA. It is discussed how short track integration steps can also accomplish incorporation of large DNA molecules.

The extracellular DNA pool in marine sediments is the largest reservoir of DNA of the world oceans and it potentially represents an archive of genetic information and gene sequences involved in natural transformation processes. However, no information is at present available for the gene sequences contained in the extracellular DNA and for the factors that influence their preservation. In the present study, we investigated the depurination and degradation rates of extracellular DNA in a variety of marine sediment samples characterized by different ages (up to 10,000 years) and environmental conditions according to the presence, abundance and diversity of prokaryotic gene sequences. We provide evidence that depurination of extracellular DNA in these sediments depends upon the different environmental factors that act synergistically and proceeds at much slower rates than those theoretically predicted or estimated for terrestrial ecosystems. These findings suggest that depurination in marine sediments is not the main process that limits extracellular DNA survival. Conversely, DNase activities were high suggesting a more relevant role of biologically driven processes. Amplifiable prokaryotic 16S rDNA sequences were present in most benthic systems analysed, independent of depurination and degradation rates and of the ages of the sediment samples. Additional molecular analyses revealed that the extracellular DNA pool is characterized by relatively low-copy numbers of prokaryotic 16S rDNA sequences that are highly diversified. Overall, our results suggest that the extracellular DNA pool in marine sediments represents a repository of genetic information, which can be used for improving our understanding of the biodiversity, functioning and evolution of ecosystems over different timescales.

Bacterial superantigens are potent stimulators of the immune system. In this study, we expressed recombinant superantigens, which were then affinity purified and used for growth curves and DNase activity assays. Overexpression of Mycoplasma arthritidis-derived superantigen in Escherichia coli reduced bacterial growth. This is unique, as staphylococcal enterotoxin A and toxic shock syndrome toxin-1, expressed in the same vector system, showed no growth impairment. The observed growth inhibition was caused by the DNase activity of recombinant M. arthritidis-derived superantigen, thus describing the first superantigen showing enzymatic activity, which may be a result of the separate evolution of this toxin.

The release of chromosomal DNA into culture media has been reported for several naturally transformable bacterial species, but a direct link between competence development and the liberation of DNA is generally lacking. Based on the analysis of strains with mutations in competence-regulatory genes and the use of conditions favouring or preventing competence, we provide evidence that DNA release is triggered by the induction of competence in Streptococcus pneumoniae. Kinetic analyses revealed that whereas competence was maximal 20 min after addition of competence-stimulating peptide, and then decreased, the amount of liberated DNA continued to increase and reached a maximum in stationary phase, when cells are no longer competent for DNA uptake. These data are not consistent with the proposal that release of DNA by a fraction of the population is coordinated with uptake by the remainder. Moreover, we observed that an unidentified DNase was specifically induced or released in competent cultures, and that together with the major pneumococcal endonuclease, EndA, it could degrade released DNA. Nearby complete abolition of release in a mutant lacking both the major autolysin, LytA, and the autolytic lysozyme, LytC, indicated that DNA liberation occurs by LytA-LytC-dependent cell lysis. These observations suggest that competence-dependent DNA release is one facet of a more general phenomenon of sensitization to autolysis that reaches its maximum in stationary phase.

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA. The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 A resolution. Vvn has a novel mixed alpha/beta topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases; however, a known 'betabetaalpha-metal' motif is identified in the central cleft region. The crystal structure of the mutant Vvn-DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by approximately 20 degrees. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme-substrate and enzyme-product structures observed in the mutant Vvn-DNA crystals, a catalytic mechanism is proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.

Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3- micro m-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 x 10(3) to 3.1 x 10(5) genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 x 10(1) and 1.3 x 10(4) genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.

Rearrangements between tandem sequence homologies of various lengths are a major source of genomic change and can be deleterious to the organism. These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats. Molecular genetic analysis of repetitive sequence instability in Escherichia coli has provided several clues to the underlying mechanisms of these rearrangements. We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing. We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination. Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms.

DNases were shown to be present in the extracellular fraction of Frankia strains R43 and CcI3. In spite of this, DNA was found in both the extracellular and cell wall fractions of these strains, and it was shown that extracellular DNA was resistant to the DNases secreted into the culture medium of both Frankia strains. Furthermore, Southern blot analysis under high stringency conditions revealed the chromosomal origin of the cell wall-adsorbed DNA (CW-DNA). Mobility gel band shift assays suggested that the extracellular DNA and the CW-DNA are engaged in complexes with other molecules, most likely proteins, which are probably responsible for the enzymatic resistance observed against extracellular DNase activities. In addition, it was shown that lysis of a small proportion of the cells in the exponential growth phase may account for the DNA being released into the supernatant and adsorbed to the cell wall.

Mycobacterium phlei (M. phlei) DNA inhibits cancer cell division but is susceptible to degradation by DNase. Chitosan forms nanoparticulate polyelectrolyte complexes with DNA, and may thus reduce nuclease degradation. We have characterized chitosan-DNA nanoparticle formation, determined DNase susceptibility, and evaluated their antiproliferative activity. Nanoparticle diameter initially decreased with increasing phosphate charge density. However nanoparticle diameter increased above 6 micromol of phosphate. Particle aggregation occurred at 16.2 micromol phosphate and was related to reduced surface charge. Incorporation of DNA within chitosan nanoparticles significantly decreased degradation by DNase. The ability of M. phlei DNA-chitosan nanoparticles to inhibit melanoma cell division was determined relative to M. phlei DNA and a cationic liposomal M. phlei DNA formulation. M. phlei DNA had antiproliferative activity (MTT reduction, IC50 = 0.9 mg/ml) without intrinsic cytotoxicity (LDH release, ED50 > 50 microg/ml). Cationic polyphosphate chitosan nanoparticles were inert (antiproliferative IC50 > 1 mg/ml, ED50 > 1 mg/ml). M. phlei DNA-chitosan nanoparticles were 20-fold more potent than M. phlei DNA. Cationic DOTAP/DOPE liposomes were cytostatic (IC50 = 49 microg/ml) and cytotoxic (ED50 = 87 microg/ml), and complexation of M. phlei DNA resulted in a significant reduction of antiproliferative activity. Chitosan nanoparticles may therefore be appropriate delivery vehicles for M. phlei DNA.

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5'-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.

A 24 triplet TGG.CCA repeat array shows length- and orientation-dependent propagation when present in the plasmid pUC18. When TGG(24) is present as template for leading-strand synthesis, plasmid recovery is normal in all strains tested. However, when it acts as template for lagging-strand synthesis, plasmid propagation is seriously compromised. Plasmids carrying deletions in the 5' side of this sequence can be isolated and products carrying 15 TGG triplets do not significantly interfere with plasmid propagation. Mutations in sbcCD, mutS and recA significantly improve the recovery of plasmids with TGG(24) on the lagging-strand template. These findings suggest that TGG(24) can fold into a structure that can interfere with DNA replication in vivo but that TGG(15) cannot. Furthermore, since the presence of the MutS and SbcCD proteins are required for propagation interference, it is likely that stabilisation of mismatched base pairs and secondary structure cleavage are implicated. In contrast, there is no correlation of triplet repeat expansion and deletion instability with predicted DNA folding. These results argue for a dissociation of the factors affecting DNA fragility from those affecting trinucleotide repeat expansion-contraction instability.

DNA-degrading activity from anaerobic samples of bovine ruminal fluid, primary anaerobic digestor wastewater, freshwater sediments, and marine sediments was observed in the presence of 5 mM EDTA. Nuclease activity experiments involved exposing salmon chromosomal DNA to the environmental samples in 50 mM pH 7.2 buffer, incubating at 37 degrees C, and subjecting the products to electrophoresis. The same stock and concentration of EDTA used in these assays (5 mM) completely inhibited commercial grade DNase. Nuclease activity in two of the samples, ruminal fluid and wastewater, was further characterized. DNA degradation in the ruminal sample was significantly reduced when EDTA or citrate concentrations were increased to 50 mM or above. DNA degradation activity in ruminal fluid was associated with material that passed through a 0.22-micron filter, but wastewater activity was associated with material retained by a 3-micron filter. Degradation activity in the wastewater was resistant to heat pretreatment, whereas the rumen activity was heat-labile (70 degrees C, 60 min). These results demonstrated the biochemical complexity of these two environments and that high molecular weight DNA has a short half-life in these anaerobic environments.

HBsu, the Bacillus subtilis homolog of the Escherichia coli HU proteins and the major chromosomal protein in vegetative cells of B. subtilis, is present at similar levels in vegetative cells and spores ( approximately 5 x 10(4) monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the alpha/beta-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with alpha/beta-type SASP on the nucleoid, suggesting that HBsu could modulate alpha/beta-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered alpha/beta-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing alpha/beta-type SASP-mediated positive supercoiling, and greatly ameliorated the alpha/beta-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the alpha/beta-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of alpha/beta-type SASP on the properties of DNA in the developing and dormant spore.

We suggest here that Porphyromonas gingivalis DNA may function as a virulence factor in periodontal disease through expression of inflammatory cytokine. The bacterial DNA markedly stimulated in a dose-dependent manner interleukin-6 (IL-6) production by human gingival fibroblasts. The stimulatory action was eliminated by treatment with DNase but not RNase. The stimulatory effect was not observed in the fibroblasts treated with eucaryotic DNAs. The bacterial DNA also stimulated in dose- and treatment time-dependent manners the expression of the IL-6 gene in the cells. In addition, the stimulatory effect was eliminated when the DNA was methylated with CpG motif methylase. Interestingly, a 30-base synthetic oligonucleotide containing the palindromic motif GACGTC could stimulate expression of the IL-6 gene and production of its protein in the cells. Furthermore, the synthetic oligonucleotide-induced expression of this cytokine gene was blocked by pyrrolidine dithiocarbamate and N-acetyl-L-cystine, potent inhibitors of transcriptional factor NF-kappaB. Gel mobility shift assay showed increased binding of NF-kappaB to its consensus sequence in the synthetic oligonucleotide-treated cells. Also, using specific antibody against p50 and p65, which compose NF-kappaB, we showed the consensus sequence-binding proteins to be NF-kappaB. These results are the first to demonstrate that the internal CpG motifs in P. gingivalis DNA stimulate IL-6 expression in human gingival fibroblasts via stimulation of NF-kappaB.

Hairpin structures can inhibit DNA replication and are intermediates in certain recombination reactions. We have shown that the purified SbcCD protein of Escherichia coli cleaves a DNA hairpin. This cleavage does not require the presence of a free (3' or 5') DNA end and generates products with 3'-hydroxyl and 5'-phosphate termini. Electron microscopy of SbcCD has revealed the "head-rod-tail" structure predicted for the SMC (structural maintenance of chromosomes) family of proteins, of which SbcC is a member. This work provides evidence consistent with the proposal that SbcCD cleaves hairpin structures that halt the progress of the replication fork, allowing homologous recombination to restore DNA replication.

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (non-proteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean +/- standard deviation = 3,890 +/- 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.

Natural transformation of the soil bacterium Pseudomonas stutzeri JM300 in a non-sterile brown earth microcosm was studied. For this purpose, the microcosm was loaded with purified DNA (plasmid or chromosomal DNA, both containing a high-frequency-transformation marker, his+, of the P. stutzeri genome), the non-adsorbed DNA was washed out with soil extract and then the soil was charged with competent cells (his-1). Both chromosomal and plasmid transformants were found among the P. stutzeri cells recovered from the soil. The number of plasmid transformants increased in a linear fashion with the amount of DNA added [10-600 ng (0.7 g soil)-1]. The observed efficiency of transformation, the time course of transformant formation and the complete inhibition of transformation by DNase I, when added to the soil, were similar to that seen in optimized transformations in nutrient broth. Addition of cells as late as 3 d after loading the soil with plasmid DNA still yielded 3% of the initial transforming activity. This suggests that nucleases indigenous to the soil destroyed the transforming DNA, but at a rate allowing considerable DNA persistence. Transformants were also obtained when intact P. stutzeri cells were introduced into the soil to serve as plasmid DNA donors. Apparently, DNA was released from the cells, adsorbed to the soil material and subsequently taken up by recipient cells. The results indicate that competent cells of P. stutzeri were able to find access to and take up DNA bound on soil particles in the presence of micro-organisms and DNases indigenous to the soil.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.

Rheograms of Alcaligenes eutrophus (NCIMB 40529) and Escherichia coli (C90 NCIMB 10616) cells lysed by sodium dodecyl sulphate were compared before and after a variety of heat shock regimes. It was found that unheated cells produced a very characteristic shear thickening rheogram which could be destroyed by DNase treatment. Cells which had been subjected to heat shock produced rheograms very similar to DNase digested material. We thus suggest that the rheogram is largely due to the presence of intact DNA molecules. The extent and nature of the heat shock affected the shape of the rheogram of the SDS lysed material. Heat shock of cells after SDS lysis did not appear to significantly damage the DNA. Storage of the cells at 10 degrees C before heat shock considerably reduced the shear thinning effect of subsequent heat shock at 90 degrees C. We attribute the shear thinning effect of the heat shock to the action of nucleases which are activated and then depolymerise the DNA molecules.