The response of normal and polyoma virus-transformed BHK-21 cells to exogenous purines

Adenosine (10 μM) stimulates the initial growth rate of BHK/21 cells seeded at low but not high density in monolayer culture; it does not affect final cell density or permit growth in agar. In labelling experiments With tritiated thymidine, adenosine also increases the response of quiescent cells to low concentrations of serum. Dialysis of serum to remove oxypurines only marginally reduces its effect on quiescent cell labelling or growth, indicating that BHK/21 cells are able to synthesise purines. The response of quiescent cells to 5% serum is inhibited by high MW (2 × 10⁶) dextran sulphate at 2 μg per milliliter. Low MW dextran sulphate (30,000) and heparin at 20 μg per milliliter produce the same effect. Exogenous adenosine (10 μM) prevents this inhibition. Many other purine derivatives replace adenosine for all the above activities but xanthine is completely inactive in all. It, therefore, appears that nucleotide synthesis is a necessary function of these compounds. The growth of cells of a polyoma-virus-transformed BHK/21 line in monolayer is not stimulated by exogenous purine, though their colony-forming ability in agar is increased five-fold. The stimulating effects of exogenous purines on normal BHK/21 cells and the absolute requirement for them in the presence of polyanions is discussed in relation to possible mechanisms of growth control.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Persistence of Mycobacterium paratuberculosis during manufacture and ripening of cheddar cheese

Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 10(4) to 10(5) CFU/ml) and low (10(1) to 10(2) CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log(10)) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.     

Synchronous exocytosis in Paramecium cells. IV. Polyamino compounds as potent trigger agents for repeatable trigger-redocking cycles

We found that different polyamino compounds trigger the instantaneous and simultaneous release of trichocysts from Paramecium tetraurelia cells (monoxenically bacterized cultures), provided amino groups are spaced in intervals of approximately 1.0 nm; in this case even diamines or oligopeptides exert some trigger effect. The most potent trigger agent found was aminoethyldextran (AED, MW = 40 kDa) which was used mainly as a derivative with 40 -NH2/molecule. A maximal response (strain K401) was achieved at 1.38 X 10(-6) M, a half maximal response at 1.08 X 10(-6) M. AED acts by a dual effect, i.e., not only by statistically properly spaced amino groups but also by some additional effect of the dextran molecule, since this greatly enhances the effect of oligoamines, although it exerts no trigger effect per se. From a total of approximately 1120 or approximately 1230 trichocysts per cell (strain K401 or 7S) approximately 95% are releasable by AED. In these strains the number of non-releasable trichocysts corresponds closely to the number of undocked trichocysts floating in the cytoplasm, so that practically all trichocysts which are docked to the cell membrane, can be released. (We also analysed different mutant strains for their response to AED.) Massive trichocyst release does not impair cell viability or culture growth, and multiple release-redocking cycles can be performed; up to 5 trigger-docking cycles were tested with individual cells in 12 h intervals. AED-triggered exocytosis requires a free extracellular [Ca2+] of greater than or equal to 10(-5) M; it is inhibited by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate), by a short pH 5.5 shock or by neomycin at 10(-5) M concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium

Fiala, Milan (University of Washington, Seattle), and George E. Kenny. Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium. J. Bacteriol. 92:1710-1715. 1966.-A reproducible macroplaque assay for six M and three H strains of rhinoviruses has been developed in several human heteroploid cell lines. Plaques were produced only with suitable solidifying agents: purified agar (Ionagar, Agarose) or methylcellulose. Plaque development was greatly enhanced by increasing Mg(+2) to 30 to 40 mm. Diethylaminoethyl (DEAE) dextran also increased plaque sizes, and the effects of Mg(+2) and DEAE dextran were additive. In addition, Ca(+2) substituted for Mg(+2). The suitability of human heteroploid cell lines for rhinovirus plaque assay varied greatly, ranging from insensitivity through partial to complete sensitivity. This assay was six to seven times more sensitive than an end point tube assay. These results indicate that potentiation of plaque formation by Mg(+2) known for some enteroviruses can also be extended to the rhinovirus group of picornaviruses.     

α<Superscript>5</Superscript>β<Subscript>1</Subscript> integrins and fibronectin are involved in adherence of the<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ER97314 clinical strain to A549 cells

The role of fibronectin (Fn) and its natural receptors alpha5beta1 integrins in the interaction of P. aeruginosa with A549 epithelial cells was compared in the clinical isolate ER97314 and the reference PAK strain. Both strains expressed functional type IV pili, as shown by the results of the twitching motility assay. The ER97314 strain was highly adherent to immobilized Fn (640 000+/-20 000 CFU per well) while the PAK strain adhered less efficiently (70 000+/-10 000 CFU per well). Both strains adhered to A549 cells (33 400+/-1200 and 1200+/-100 CFU per well, for PAK and ER97314, respectively), only the PAK strain being significantly internalized (9430+/-2020 CFU per well). Cytochalasin D and genistein significantly decreased bacterial adherence of the 2 strains and caused also a significant decrease in PAK internalization. This inhibitory activity was not related to changes in the expression of alpha5beta1 integrins. Antibodies to Fn and alpha5beta1 integrins inhibited the adherence of the ER97314 strain but had no significant effect on PAK interaction with human cells. These findings suggest that only some P. aeruginosa strains can target Fn and their natural receptors alpha5beta1 integrins for adherence to A549 cells.     

A-Factor as a selective agent for isolation of the soil Gram-negative bacterium strain--the producent of an antibacterial antibiotic]

It was shown the stimulating role of A-factor on soil prokaryotes growth. Soil sample culturing on agar medium, containing A-factor, resulted in the colony forming units (CFU) increasing in comparison with culturing on the medium without this regulator. Gram-negative bacteria were the reason of CFU increasing; previously the effect of A-factor on bacteria of this group was not shown. Gram-negative rod bacterial strain No. 35 was isolated and shown that CFU number was approximately twice increased at A-factor concentration in agar medium 2 and 7 mcg/ml; high A-factor concentration up to 28 mcg/ml was not effective. Isolated strain No. 35 is the producer of antibiotic, effective against gram-positive bacteria.     

Persistence of Mycobacterium paratuberculosis during Manufacture and Ripening of Cheddar Cheese

Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 10⁴ to 10⁵ CFU/ml) and low (10¹ to 10² CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log₁₀) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis.

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Fate of Mycobacterium avium subsp. paratuberculosis in Swiss hard and semihard cheese manufactured from raw milk

Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 10(4) to 10(5) CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 10(3) to 10(4) cells of M. avium subsp. paratuberculosis per g will be inactivated.     

Morphological and phenotypical characterization of Bacillus sporothermodurans

AIMS: Enumeration of resistant bacteria in ultra-high temperature (UHT) treated milk; morphological characterization and phenotyping of resistant strains by traditional and nontraditional methods and their identification by molecular biology. METHODS AND RESULTS: Modified standard plate count agar (PCA) and modified brain-heart infusion (BHI) agar were used for colony counts. Physiological culture traits were determined as suggested by Bergey's Manual of Systematic Bacteriology or in modified J-broth or in modified BHI agar. Scanning electron microscope (SEM) was used for microscopic examination. Strain identification was carried out by polymerase chain reaction (PCR). A total of 125 (62.81% of 199) samples were positive and the bacterial load was higher than 10(5) CFU ml(-1) in 46 samples (28.80% of 125). The 16S rRNA sequence of bacterial cultures obtained from UHT-treated milk was similar to that of Bacillus sporothermodurans M215 type strain((T)) and different biotypes were found by analysis of colony appearance, cell morphology and physiological traits. CONCLUSIONS: Bacillus sporothermodurans was the predominant sporigenous micro-organisms in UHT milk. SIGNIFICANCE AND IMPACT OF THE STUDY: BHI agar is more suitable than PCA for quality control of milk after UHT treatment. Modified J-broth medium is useful to determine selected physiological traits of B. sporothermodurans. The strains characterized and identified as B. sporothermodurans were significantly different compared with the type strain.     

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.     

Development of a plating medium for selection of Helicobacter pylori from water samples

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10(6) CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37 degrees C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.     

Influence of Polycations on the Interaction Between Poliovirus Multistranded Ribonucleic Acid and HeLa Cells

In contrast to single-stranded viral ribonucleic acid (RNA), poliovirus multistranded RNA (RI-RNA) is poorly adsorbed to suspended HeLa cells in the absence of polycations. In the presence of 20 mug of diethylaminoethyl (DEAE) dextran per ml and other polycations, 90% or more of the RI-RNA is adsorbed to HeLa cells within 2 min at 37 C. However, only 30% of the RI-RNA label penetrates into the cells independent of the concentration of DEAE dextran applied. DEAE dextran is adsorbed almost quantitatively to HeLa cells within 3 min at 37 C. Most of the DEAE dextran remains bound to the cell membrane and available for binding of RI-RNA for 15 min at 37 C.     

Changes in virulence, M protein and IgG Fc receptor activity in a type 12 group A streptococcal strain during mouse passages

A type 12 group A strain (1800) was passaged serially through mice 25 times. The ability to servive in normal human blood dropped from a growth index of 52 after the first passage to 1 after four passages. After 14 passages the growth index increased again and stabilized above 30. The virulence for mice increased from a LD100 of 10(8) colony forming units (CFU) to 10-100 CFU after 7 passages and then remained constant. The Mqw antigen disappeared after 4 passages as tested by immunodiffusion, electroimmunoassay and indirect bactericidal tests. Three antisera, raised in rabbits against strains originally belonging to types M3, M12 and M46 but devoid of type antigens after mouse passages showed high bactericidal indices against the 1800 strain after 14 or more passages on mice. Anti-type M1 serum was also found bactericidal for the passaged strains. The IgG Fc-receptor activity of the strain isolated after each mouse passage was tested in hemagglutination experiments with human red blood cells coated with "incomplete" anti-Rh and hot hydrochloric acid extracts of the strains. The capacity to agglutinate "Ripley"-coated cells increased gradually during the first 12 passages and subsequently the titres of the extracts stabilized between 1:160 and 1:320. The HUN coat, useful for detection of the G3m (5) maraker gave titraes increasing with the number of passages while the titres for IgG1 coats kept at 1:4 or below. On background of these results, the possible role of the IgG Fc-receptor as a virulence factor is discussed.     

Autoluminescent Mycobacterium tuberculosis for rapid, real-time, non-invasive assessment of drug and vaccine efficacy

Preclinical efforts to discover and develop new drugs and vaccines for tuberculosis are hampered by the reliance on colony-forming unit (CFU) counts as primary outcomes for in vivo efficacy studies and the slow growth of Mycobacterium tuberculosis. The utility of bioluminescent M. tuberculosis reporter strains for real-time in vitro and ex vivo assessment of drug and vaccine activity has been demonstrated but a simple, non-invasive, real-time surrogate marker to replace CFU counts for real-time evaluation of drug and vaccine efficacy in vivo has not been described. We describe the development of a fully virulent and stable autoluminescent strain of M. tuberculosis and proof-of-concept experiments demonstrating its utility for in vivo bioluminescence imaging to assess the efficacy of new drugs and vaccines for tuberculosis in a mouse model. Relative light unit (RLU) counts paralleled CFU counts during the active phase of bacterial growth, with a lower limit of detection of approximately 10(6) CFU in live, anesthetized mice. Experiments distinguishing active from inactive anti-tuberculosis drugs and bacteriostatic drug effects from bactericidal effects were completed in less than 5 days. The ability of a recombinant BCG vaccine to limit bacterial growth was demonstrated within 3 weeks. Use of this autoluminescent reporter strain has the potential to drastically reduce the time, effort, animals and costs consumed in the evaluation of drug activity in vitro and the in vivo assessment of drug and vaccine efficacy.     

Carboxyl-terminal protease regulates Brucella suis morphology in culture and persistence in macrophages and mice

The putative carboxyl-terminal processing protease (CtpA) of Brucella suis 1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. The B. suis CtpA protein shared up to 77% homology with CtpA proteins of other bacteria. A CtpA-deficient Brucella strain (1330DeltactpA), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growth in salt-free enriched medium compared to the wild-type strain 1330 or the ctpA-complemented strain 1330DeltactpA[pBBctpA]. Electron microscopy revealed that in contrast to the native coccobacillus shape of wild-type strain 1330, strain 1330DeltactpA possessed a spherical shape, an increased cell diameter, and cell membranes partially dissociated from the cell envelope. In the J774 mouse macrophage cell line, 24 h after infection, the CFU of the strain 1330DeltactpA declined by approximately 3 log(10) CFU relative to wild-type strain 1330. Nine weeks after intraperitoneal inoculation of BALB/c mice, strain 1330DeltactpA had cleared from spleens but strain 1330 was still present. These observations suggest that the CtpA activity is necessary for the intracellular survival of B. suis. Relative to the saline-injected mice, strain 1330DeltactpA-vaccinated mice exhibited 4 to 5 log(10) CFU of protection against challenge with virulent B. abortus strain 2308 or B. suis strain 1330 but no protection against B. melitensis strain 16 M. This is the first report correlating a CtpA deficiency with cell morphology and attenuation of B. suis.     

Genetic exchange of transposon and integrative plasmid markers in Mycoplasma pulmonis.

Matings of genetically marked derivatives of Mycoplasma pulmonis resulted in the exchange of chromosomal DNA and the appearance of doubly marked transconjugants. Transposons Tn916 and Tn4001, and a series of integrative plasmids derived from their cloned antibiotic resistance genes, were used to construct antibiotic-resistant mycoplasmal derivatives to examine this phenomenon at the molecular level. Genetic exchange occurred on agar surfaces at frequencies ranging from 3.3 X 10(-4) to 6.4 X 10(-8) transconjugants per CFU. Examination of chromosomal DNA from transconjugants by hybridization revealed that the transposons or integrated plasmids were in the same chromosomal locations as in the parental strains, indicating that exchange involved the transfer of chromosomal DNA and homologous recombination. Transfer was not affected by DNase, polyethylene glycol, EDTA, or calcium chloride but was affected by treatment of either parent with trypsin. Mixing of mating strains before plating had no effect on mating frequencies, but mating did occur in liquid media. The ability to exchange chromosomal markers was limited to selected strains of M. pulmonis; mating did not occur with Acholeplasma laidlawii or M. gallisepticum. Heat and UV inactivation studies revealed that nonviable cells could act as donors in matings. The evidence presented supports a conjugationlike mechanism involving specific trypsin-sensitive membrane components.