Selective release of a deoxyribonucleic acid-binding factor from the surface of competent pneumococci.

Methods are described that resulted in the selective release of deoxyribonucleic acid (DNA)-binding factor from the surface of competent pneumococci. The same methods caused a parallel inactivation of the DNA-binding capacity of the extracted bacteria. Genetically or physiologically incompetent pneumococci did not yield binding factor upon exposure to the same methods. The solubilized binding factor appeared to be a protein; it could be assayed by a membrane filter binding procedure. The binding factor had properties reminiscent of those of the DNA receptors of transformable pneumococci (Seto et al., 1975).     

Intraspecies and Interspecies Transformation Reactions in Pneumococcus and Streptococcus

The efficiency of transformation of pneumococcus and a strain of viridans streptococcus (strain D) to streptomycin resistance is influenced by the species in which the mutation to resistance occurred, as well as by the species in which the mutated gene has been replicated. Pneumococcus and streptococcus strain D transform in higher frequency with DNA that has been replicated in bacteria of the same species than with DNA from the heterologous species. However, the difference between the frequencies of interspecific and intraspecific transformation is much greater with pneumococcus as receptor than with streptococcus. In addition pneumococcus transforms in higher frequency with wholly homologous (pneumococcal) DNA than with DNA from pneumococci that have replicated the streptococcal Sm^r gene. Pneumococcus is transformed in lower frequency by wholly heterologous (streptococcal) DNA than by DNA from streptococci that have replicated the pneumococcal Sm^r gene. Streptococcus behaves similarly in that wholly homologous (streptococcal) DNA transforms it more efficiently than when the transforming fragment contains a pneumococcal moiety. Streptococcus is transformed in the same or lower frequency by wholly heterologous (pneumococcal) DNA than by DNA from pneumococci that have replicated the streptococcal Sm^r gene. When erythromycin resistance was used as genetic marker instead of streptomycin resistance, similar results were found.     

Competence-induced cells of Streptococcus pneumoniae lyse competence-deficient cells of the same strain during cocultivation

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.     

Effect of short interspersed element sequences on the integration and expression of a reporter gene in the preimplantation-stage mouse embryos

Based on the assumption that foreign DNA sequences may have increased chance of integration into the host genome if they are flanked by high copy-numbered genomic sequences such as SINEs (short interspersed elements), we investigated the integration frequency of Lac Z reporter gene flanked by a fused B1/B2 in an in vivo system using pronuclear microinjection technique in the mouse. The SINE-flanked DNA showed a 4-fold increased integration frequency of the reporter gene than the control DNA (63% vs. 16%). Moreover, the level of beta-galactosidase expression, estimated from the X-Gal staining intensity in transgenic embryos, was greatly higher in SINE-carrying DNA. These results suggest that the SINE sequences can serve a very useful tool in improving the efficiency of current transgenic animal technology.     

Deletion between direct repeats in T7 DNA stimulated by double-strand breaks.

An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 was used to study genetic deletions between directly repeated sequences. The frequency of deletion was highest under conditions in which the DNA was actively replicating. Deletion frequency increased markedly with the length of the direct repeat both in vitro and in vivo. When a T7 gene was interrupted by 93 bp of nonsense sequence flanked by 20-bp direct repeats, the region between the repeats was deleted in about 1 out of every 1,600 genomes during each round of replication. Very similar values were found for deletion frequency in vivo and in vitro. The deletion frequency was essentially unaffected by a recA mutation in the host. When a double-strand break was placed between the repeats, repair of this strand break was often accompanied by the deletion of the DNA between the direct repeats, suggesting that break rejoining could contribute to deletion during in vitro DNA replication.     

APPLICATION OF THE MEMBRANE FILTER FOR THE QUANTITATIVE STUDY OF TRANSFORMATIONS WITH PARTICULAR REFERENCE TO PHENOTYPIC EXPRESSION OF AN ERYTHROMYCIN-RESISTANCE MUTATION.

Iyer, V. N. (University of Rochester, Rochester, N.Y.). Application of the membrane filter for the quantitative study of transformations with particular reference to phenotypic expression of an erythromycin-resistance mutation. J. Bacteriol. 84:326-330. 1962.-A technique using membrane filters for quantitative studies of transformation of pneumococci is described and some possible applications discussed. This method has been found amenable to the study of the integration and phenotypic expression of an erythromycin-resistance mutation of pneumococci which is characterized by a low frequency of transformation and delayed expression. The expression of survival occurs prior to the expression of colony-forming ability in the presence of erythromycin.     

Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis

Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT-PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence-specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum-sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction - from the pneumococcal point of view - between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm-like state.     

Alveolar macrophage apoptosis contributes to pneumococcal clearance in a resolving model of pulmonary infection

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.     

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.     

Induction of normal levels of genetic transformation in a class of endonuclease-defective mutants of Pneumococci.

Pneumococcal mutants were isolated that showed no zones of DNA hydrolysis around the bacterial colonies, even after prolonged incubation on the surface of agar plates containing DNA and methylgreen. Lysates of such mutants contained only very low levels of endonuclease activity; the mutants were also defective in genetic transformation even though they could still bind DNA to their surface. However, the same mutants could be made to undergo normal, high frequency genetic transformation by treatment with the activator protein, under appropriate conditions. The same treatment caused no detectable increase in the endonuclease level. Poor transformability of these mutants seems to be related to an inhibitory factor(s) released into the growth medium. Activation in the presence of this factor(s) leads to an abnormal (non-productive) DNA adsorption-both in mutant and in wild type pneumococci.     

Processive action of T4 endonuclease V on ultraviolet-irradiated DNA.

The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular DNa. Form I ColE1 DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule were treated with T4 endonuclease V and analysed by agarose gel electrophoresis. At all dimer frequencies examined, the production of form III DNA was linear with time and the double-strand scissions were made randomly on the ColE1 DNA genome. Since the observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of the DNA population while some DNA molecules contained no scissions. When DNA containing an average of 25 dimers per circle was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimmer sites in some molecules before additional strand scissions were produced in other DNA molecules. The results support a processive model for the interaction of T4 endonuclease V with UV-irradiated DNA.     

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.     

Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells.     

Mutation in Pneumococcus Type III Affecting Multiple Cistrons Concerned with the Synthesis of Capsular Polysaccharide

The genetic behavior in transformation reactions of 20 noncapsulated mutants of pneumococcus type III suggests that each has a single-site mutation in the locus controlling the synthesis of uridine diphosphate glucose (UDPG) dehydrogenase. Each strain is capable of yielding transformants of the binary capsular type SI-III when exposed to deoxyribonucleic acid (DNA) from type I cells. One additional mutant reacted differently and behaved as if it were a multisite mutant with the mutation affecting both the locus for UDPG dehydrogenase and that controlling the synthesis of high molecular weight type III capsular polysaccharide. When exposed to DNA from type I pneumococci, this strain yielded transformants which were genotypically binary but which expressed only the type I capsular phenotype.     

Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho-epithelial cells in vivo

The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.     

Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases beta and eta in vitro

DNA polymerases beta and eta are among the few eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Our laboratory has previously established that both polymerases misincorporated dTTP with high frequency across from cisplatin- and oxaliplatin-GG adducts. This decrease in polymerase fidelity on platinum-damaged DNA could lead to in vivo mutations, if this base substitution were efficiently elongated. In this study, we performed a steady-state kinetic analysis of the steps required for fixation of dTTP misinsertion during translesion synthesis past cisplatin- and oxaliplatin-GG adducts by pol beta and pol eta. The efficiency of translesion synthesis by pol eta past Pt-GG adducts was very similar to that observed for this polymerase when the template contains thymine-thymine dimers. This finding suggested that pol eta could play a role in translesion synthesis past platinum-GG adducts in vivo. On the other hand, translesion synthesis past platinum-GG adducts by pol beta was much less efficient. Translesion synthesis by pol eta is likely to be predominantly error-free, since the probability of correct insertion and extension by pol eta was 1000-2000-fold greater than the probability of incorrect insertion and extension. Our results also indicated that for pol eta the frequency of misincorporation is the same across from the 3'G and the 5'G of the platinum-GG adducts for both cisplatin and oxaliplatin adducts. On the other hand, pol beta is more likely to misinsert at the 3'G of the adducts and misinsertion occurs at higher frequency for oxaliplatin-GG than for cisplatin-GG adducts.     

Studies on Transformations of Hemophilus influenzae : IV. Linked and unlinked transformations

Unlinked transformations were demonstrated to occur by varying the multiplicity of DNA molecules taken up by competent cells. The number of doubles was directly proportional to the product of the frequency of singles for varying concentrations of cells. The kinetics of transformation to doubles and the effect of DNA concentration on double transformations were consistent with the concept that the cell must take up two molecules of DNA in order to be doubly transformed. Linked markers, on the other hand, were a constant fraction of the single transformation for variations in DNA or cell concentration, or time. The kinetics of transformation of linked markers was the same as for the kinetics of single transforming factors. It was, therefore, concluded that linked transformations involve interaction between the cell and a molecule of DNA carrying both markers. The frequency of transformation was found to be the same from resistance to sensitivity as from sensitivity to resistance for the markers streptomycin (S) and cathomycin (C). Purified DNAs, in general, show lower levels of linkage than crude DNA preparations, and for some crude preparations all the S markers were linked to C, suggesting that some dispersion, at least, was a result of DNA preparation. The inactivation of linked markers by heat, ultraviolet, and DNAase was studied.