Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Mycobacterium tuberculosis

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Myobacterium tuberculosis. J. Bacteriol. 91:2146-2154. 1966.-The ribosomal fraction of the attenuated strain, H37Ra, of Mycobacterium tuberculosis was treated with trypsin alone, ethylenediaminetetraacetic acid (EDTA) alone, EDTA and pancreatic ribonuclease, or with trypsin and ribonuclease. After each of these treatments, the ribosomal fractions were injected intraperitoneally into male CF-1 mice to test their capacity to produce an immune response to infection with virulent tubercle bacilli, strain H37Rv. Removal of protein with trypsin left the immunogenicity unchanged; EDTA alone reduced immunogenicity in the smaller vaccinating doses; EDTA plus ribonuclease reduced the immunogenicity by approximately 50% in the highest (1.0 mg) vaccinating dose; ribonuclease alone, after treatment with trypsin, reduced immunogenicity also approximately 50%. A crude mycobacterial ribonucleic acid (RNA) was prepared by extraction of the ribosomal fraction with alcohol. This RNA preparation was as effective in producing an immune response as the ribosomal fraction from which it was prepared, unless the RNA was partially or completely degraded during the preparation. The effect of ribonuclease on the immunogenicity of the RNA was similar to that obtained with the ribosomal fractions, except that ribonuclease completely destroyed the immunogenicity of a partially degraded RNA. RNA appears to be an essential part of an immunizing substance in attenuated tubercle bacilli, which produces a high degree of immunity in mice; 50 mug (dry weight) will protect approximately 80% of the mice, and as little as 0.5 mug will protect approximately 30% of the mice. Mycobacterial RNA not incorporated in Freund's incomplete adjuvant was nonimmunogenic. Yeast RNA incorporated in Freund's incomplete adjuvant was not immunogenic.     

Ribonuclease H: a Ubiquitous Activity in Virions of Ribonucleic Acid Tumor Viruses

Ten ribonucleic acid (RNA) tumor viruses grown in five different host cell species and three non-oncogenic viruses from three different virus groups have been examined for ribonuclease H content. Three different substrates were used to assay ribonuclease H: calf thymus [(3)H]RNA-deoxyribonucleic acid (DNA) hybrid prepared with denatured calf thymus DNA and Escherichia coli DNA-directed RNA polymerase, (3)H-polydenylic acid [(3)H-poly(A)] complexed to polydeoxythymidylic acid [poly(dT)], and (3)H-polyuridylic acid [(3)H-poly(U)] complexed to polydeoxyadenylic acid [poly(dA)]. All ten RNA tumor viruses contained ribonuclease H activity which degraded the RNA of both the calf thymus hybrid and poly(A)-poly(dT), whereas only the ribonuclease H in the Moloney strain of murine sarcoma-leukemia virus and in RD-feline leukemia virus hydrolyzed the RNA strand of poly(U)-poly(dA). No appreciable ribonuclease H activity was detected in influenza, Sendai, or vesicular stomatitis virus. The ribonuclease H and RNA-directed DNA polymerase activities in Moloney murine sarcoma-leukemia virus were inseparable by phosphocellulose chromatography or glycerol gradient centrifugation, but appeared to be partially separated by diethylaminoethyl-cellulose chromatography.     

Immunobiological studies with mycobacterial ribonucleic acid-protein complex: Part I--Immune responses and protective role against experimental tuberculosis.

Ribonucleic acid (RNA) isolated from M. tuberculosis H37Ra was found to be native in nature as determined by hyperchromicity studies using ribonuclease. Mycobacterial RNA-protein (Myc. RNA-P) when injected as RNA-P-FIA complexes induced weak humoral immune responses and strong cell-mediated immune (CMI) responses which were directed against Myc. RNA. Protection comparable to BCG was induced in mice immunized with RNA-FIA complexes against LD50 dose of M. tuberculosis as monitored by increased survival rates, decreased lung density, root specific lung weight (RSLW) and by decreased viable counts of M. tuberculosis in lung, liver and spleen of immunized mice. Enzymatic degradation studies revealed Myc. RNA component to specifically mediate protection while the protein component was found ineffective.     

Involvement of Protein and Ribonucleic Acid in the Association of Deoxyribonucleic Acid with Other Cell Components of Escherichia coli

Cells of Escherichia coli were labeled with precursors of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein, lysed with detergent, and examined by starch-block electrophoresis and CsCl density gradient centrifugation. A large amount of the DNA was seen to remain at positions of low electrophoretic mobility and light density along with tryptophan and arginine-containing proteins and some RNA. Addition of labeled, phenol-extracted DNA to unlabeled cells prior to lysis and electrophoresis showed that only a small amount of the DNA became associated during or after lysis. Sonic treatment of a lysate removed most of the DNA to a position of electrophoretic mobility and density similar to that of free DNA, whereas pronase and ribonuclease released only a part of the DNA. We concluded that binding of DNA to cell membranes or other cell components occurs in the cell prior to lysis and involves protein and probably a specific type of RNA.     

Factors Affecting Immunogenic Activity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations

By following careful procedures, mycobacterial ribosomal fractions and ribonucleic acid (RNA) prepared by ethyl alcohol precipitation were obtained which have immunogenic activities similar to the viable attenuated H37Ra cells of Mycobacterium tuberculosis from which they were obtained. This comparison was based on the amount of ribonucleic acid (RNA) present. These preparations consisted of approximately 63% RNA and 37% protein; no deoxyribonucleic acid or polysaccharide was detected by chemical tests. A high correlation was found between the immunogenic activity of a preparation and the per cent increase in hyperchromicity at 260 nm of a ribonuclease-hydrolyzed portion. Final concentrations of sodium dodecyl sulfate higher than 0.25% when used for the preparation of the ribosomal fractions and RNA resulted in significantly lower immune responses and greater variation between experiments. This was not related to the amount of protein present. The stability of the ribosomal and RNA preparations was tested under a variety of conditions. The need for a good protective adjuvant again was shown since mouse serum readily hydrolyzed the RNA. Equal immunity was obtained after immunization by the intraperitoneal and subcutaneous routes; however, no immune response was obtained when the intravenous route was used. Preliminary results with RNA prepared with phenol showed that it was more easily degraded during preparation. This resulted in a lower immune response than was obtained with the RNA prepared with ethyl alcohol.     

Lysosomal Physiology in Tetrahymena. II. Effect of Culture Age and Temperature on the Extracellular Release of 3 Acid Hydrolases*

Tetrahymena cultures were grown from a single inoculum and collected on 3 successive days corresponding to the log, transitional, and early stationary phases of growth. Cells were washed and incubated for 5 hr in a dilute salt solution. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed, and extracellular activities corrected for proteolytic degradation. A marked increase in the cellular content of acid phosphatase and significant decreases in α-glucosidase and ribonuclease occurred with advancing culture age. The intracellular changes in enzyme activities during incubation were roughly similar for cells of all ages. Protein content did not change appreciably during incubation. Extracellular A₂₅₅ release, monitored as an indication of the loss of RNA breakdown products, was at a minimum during incubation of transition cells. Significant quantities of all 3 acid hydrolases were released from cells of all ages except for ribonuclease from transition cells. The release of acid phosphatase and α-glucosidase was approximately proportional to the initial cellular content of these enzymes for cells of different ages and in log cells the effect of temperature on the rates of release was described by the Arrhenius equation. Release of ribonuclease, however, was not proportional to its intracellular content nor did it vary with temperature according to the Arrhenius equation. The results suggest that acid phosphatase and α-glucosidase are released via a first-order process.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

DNA, RNA, and protein biosynthesis in cells of Yersinia pseudotuberculosis at various cultivation temperatures

Y. pseudotuberculosis cells cultivated at temperatures of 37 degrees C and 8 degrees C were found to be capable of incorporating exogenic precursors into DNA, RNA and protein. The linear growth of thymidine incorporation occurred during 8 hours of cultivation at 37 degrees C, then the amount of the incorporated label decreased. At 8 degrees C the level of thymidine incorporation into DNA gradually increased for 80 hours and longer, but not reaching the level of incorporation observed at 37 degrees C. The incorporation of uridine into RNA of Y. pseudotuberculosis cells reached its maximum after 4 hours of cultivation at 37 degrees C, at a lower temperature of cultivation the incorporation of uridine into bacterial cells was almost linear, though slower, and lasted for 20 hours. The content of radioactive alanine in Y. pseudotuberculosis protein increased during 16 hours of cultivation at a high temperature, while at 8 degrees C the growth of the incorporation level lasted for at least 40 hours. For all precursors under study the incorporation rate into the cell biopolymers at the initial stages of cultivation was higher at 37 degrees C, than at a lower temperature.     

Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.     

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.     

Immunizing Capacity of Viable and Killed Attenuated Mycobacterial Cells Against Experimental Tuberculous Infection

The relationship of the dose of vaccine to the immune response was determined in CF-1 mice vaccinated intraperitoneally with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and in mice vaccinated with cells of the same strain killed by autoclaving at 121 C for 15 min. The results showed, in terms of increased resistance to tuberculous infection, that the immune response with both living and killed cells was dependent upon the dose of vaccine, whereas only the living cells were dependent upon the time of challenge after vaccination. The dose response curves show dramatically that viable cells, which do not multiply in vivo, are several hundred times more effective immunizing agents against tuberculous infection than are autoclaved cells. Viable 2-week-old H37Ra cells were far more immunogenic than viable 4-week-old cells. Autoclaved 2-week-old cells, however, were no more immunogenic than autoclaved 4-week-old cells. H37Ra cells killed by boiling (98 C), exposure to 65 C for 30 min, treating with 2% phenol, or by being dried with acetone also lost most of their capacity to immunize mice. The effect of adjuvant on the immune response of mice to tuberculous infection was tested by incorporating both viable and autoclaved cells in Freund's incomplete adjuvant. We found that this vehicle had little or no effect on the immunizing capacity of either viable or heat-killed mycobacterial cells. The relationship of all the findings to the specificity of the immune response to tuberculosis is discussed.     

Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification

Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-kappaB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome-lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.     

Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate. J. Bacteriol. 91:2139-2145. 1966.-Ribosomal fractions of Mycobacterium tuberculosis, strain H37Ra, were prepared by treatment of the intracellular particulate fraction with 0.25 or 0.5% sodium dodecylsulfate (SDS) followed by centrifugation at 144,700 x g for 3 hr. This procedure has greatly simplified the preparation of ribosomal fractions and has given fractions composed of approximately 50% ribonucleic acid (RNA) and 15 to 20% protein. When incorporated into Freund's incomplete adjuvant and injected intraperitoneally into CF-1 mice, the SDS ribosomal fractions were more immunogenic than the particulate fractions from which they were prepared. They were as much as 100 times more immunogenic than ribosomal fractions prepared by differential centrifugation, 1 mug (dry weight) per mouse being sufficient for the induction of some immunity. However, none of these ribosomal preparations, in comparable doses, was as immunogenic as the living cells from which they were prepared. It was also shown that the addition of 10(-4)m MgCl(2) to the final diluent increased immunogenic activity, whereas larger concentrations (10(-3)m) reduced immunogenic activity. Preparation of the ribosomal fraction from ruptured cells in one continuous process during the course of 1 day increased the activity. Two-week-old H37Ra cells contained more RNA and were more immunogenic than the older cultures which have been used in the past.     

Relationships between nucleic acid,nitrogen, and growth rate of tobacco cells in suspension culture

Summary Changes in the amount of nucleic acid and nitrogen, and the relationships between these amounts and the growth rate of tobacco cells (Nicotiana tabacum L. cv. Bright Yellow-2) at different initial nitrogen concentrations in the medium, were examined in batch cultures. During culture in basal medium, the amount of intracellular nucleic acid expressed per unit of dry biomass was 36.3 mg RNA g^–1 cell and 8.1 mg DNA g^–1 cell at the beginning of batch culture. These values increased 2.5 fold for RNA and 1.5 fold for DNA during the exponential growth phase and then gradually decreased with the decline in the growth rate. Similar changes were also observed in the medium containing less nitrogen. The specific growth rate, (day^–1), of the culture corresponded to the magnitude of the intracellular RNA content (mg RNA g^–1 cell), and the linear relationship, RNA=38+23 was obtained. In addition, there were remarkable positive correlations between the total and protein nitrogen, and during the cultures. The mononucleotide composition of total RNA (AMP+UMP)/(GMP+CMP) which was suggested to be a convenient index of metabolic activity was nearly constant (0.78 to 0.80) during tobacco cell culture in the basal medium.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

Competence for Deoxyribonucleic Acid Uptake and Deoxyribonuclease Action External to Cells in the Genetic Transformation of Diplococcus pneumoniae

A mutant of Diplococcus pneumoniae that apparently does not require activator can become competent for uptake of deoxyribonucleic acid (DNA) when grown in dilute cultures or in the presence of trypsin. Development of competence in both mutant and wild strains is temperature dependent, being 10-fold greater at 30 C than at 37 C. Induction of competence on a shift from 37 to 30 C requires protein synthesis and the presence of Mg(2+) and Ca(2+); uptake of DNA does not require protein synthesis. Competence decays exponentially at higher temperatures. As well as taking up DNA, competent cells release oligonucleotide fragments of donor DNA in the medium external to the cells. Normal strains release fragments comparable in amount to the DNA taken up; but, in a mutant selected for inability to degrade DNA in agar, the amount of fragments formed external to the cells is only 40% of DNA uptake. Requirements for external deoxyribonuclease action are identical to those for DNA uptake: prior development of competence and the presence during treatment with DNA of Mg(2+) ions and a source of energy.     

Spent culture supernatant of Mycobacterium tuberculosis H37Ra improves viability of aged cultures of this strain and allows small inocula to initiate growth

Spent culture supernatant from early-stationary-phase Mycobacterium tuberculosis H37Ra cultures increased the viability of bacilli from aged cultures of this strain and allowed small inocula to initiate growth in liquid culture. The resuscitation factor was acid labile and heat stable, with a mass of less than 1,375 Da.     

Phage formation in Staphylococcus muscae cultures; nucleic acid synthesis during virus formation

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse.