Characterization of the lysogenic bacteriophage MAV1 from Mycoplasma arthritidis

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of Mycoplasma arthritidis, was characterized. Several strains of M. arthritidis were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA (attP) and bacterial DNA (attB) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the attB sequence. A model for MAV1 integration-excision is proposed.     

Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTXPhi: molecular basis for origination of new strains with epidemic potential

Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXPhi. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.     

The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V. cholerae O139

We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V. cholerae strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection. Susceptibility is restored by mshA complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the mshA pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.     

Production of phage-resistant mutants of E. coli, producers of penicillin acylase

Full resistance to the virulent phage C1 in E. coli N68 is usually accompanied by decreased capacity for penicillinacylase production. For rapid selection of phage resistant mutants possessing penicillinacylase activity comparable with that of the initial bacteria a method was proposed. The method provided comparison of the penicillinacylase activity of the bacterial colonies grown on solid media with addition of phenylacetic acid as the enzyme inductor. A great number of mutants forming colonies on the solid medium in the presence of phage C1 was compared with the use of the above method and a mutant of E. coli N68-PR-I resistant to the phage with penicillinacylase activity equal to 68 per cent of that of the bacteria of the wild type was selected. Mutant N68-R-5 with increased resistance to phage CI was selected among the mutants of E. coli N68 resistant to rifampicin. The penicillinacylase activity of this mutant was not less than of E. coli N68. Phage CI can lyse the cells of strain N68-R-5. Still these bacteria possess a markedly decreased capacity for the phage reproduction.     

Interaction of Pseudomonas aeruginosa plasmids and mu-like phages: the suppression of the early stages of cell infection by phage D3112 in the presence of plasmid RPL11

The growth of Mu-like, D3112, B39 and B3 bacteriophages of Pseudomonas aeruginosa on bacterial strains containing R plasmids was studied. Plasmids RPL11, Rms148 and Rms163 were shown to interfere with phage growth: 1) D3112 and B39 phages do not produce plaques on a lawn of PAO1 (Rms148) giving e.o.p. less than 10(-9); 2) RPL11 plasmid restricts phage D3112 growth (e.o.p. less than 10(-9), the growth of phage B3 being also restricted by this plasmid, though in considerably less extent; 3) phage B39 makes small and very turbid plaques on a lawn of PAO1 (Rms163) with e.o.p. 0,13, while c mutants form clear plaques on this lawn and grow with e.o.p. 1,0. The interference of plasmid RPL11 with phage D3112 growth was examined in detail. The plasmid did not affect phage D3112 adsorption and no restriction of phage DNA in R+ cells was found. However, phage genes controlling establishment of lysogeny and the lytic cycle were not expressed after infection. It was observed though, that if a cell contains both prophage D3112 and plasmid RPL11, no interference with repressor synthesis or phage development takes place after induction of prophage. The results obtained allow to conclude that: 1) RPL11 plasmid interference with phage D3112 growth is caused by the plasmid effect on one of the early stages in the development preceding phage DNA integration; 2) the process of primary integration after infection and that of reintegration of DNA after prophage induction are likely to differ.     

Isolation and biological characterization of Legionella pneumophila mutants resistant to aminoglycoside antibotics and their protective action]

Preliminary estimation of virulence in some antibiotic resistant mutants of Legionella pneumophila, Philadelphia 1 in various models of infection revealed its decrease in the mutants resistant to azlocillin, cefotaxime, fluoroquinolone LIB-80, neamine and streptomycin. Detailed investigation of the neamine resistant mutants showed that in relation to streptomycin susceptibility such mutants could be divided into 3 classes: susceptible to streptomycin, resistant to high concentrations of streptomycin and resistant to moderate concentrations of streptomycin. Part of mutants Nea(r)Strr and Nea(r)Strr500 and all mutants Nea(r)Strr100 proved to be less virulent with respect to guinea pigs and chick embryos. The study of the spectinomycin resistant mutants of Legionella spp. did not reveal any changes in the virulence which made it possible to suggest that the influence of the mutations in the ribosomal protein genes determining resistance to streptomycin and neamine on virulence of L. pneumophila was based on the interdependence of the mutant effect on the suppression and the influence on the virulence detected by us in S. flexneri, Y. pseudotuberculosis, L. monocytogenes and F. tularensis. The Legionella mutants Nea(r)Strr100 were characterized by significant protective activity and protected immunized guinea pigs when tested in a model of their aerogenic infection.     

Selection and properties of totally phage-resistant mutant Pseudomonas putida PpG1

The efficiency of using bacteria in open systems to degrade different anthropogenic toxic pollutants can depend strongly on the interaction between these bacteria and natural bacteriophages. The possibility of selecting bacterial Pseudomonas putida mutants resistant to all bacteriophages of this species known so far was tested (in our work, these mutants were designated totally phage-resistant mutants). In a model experiment, changes in the composition of a population upon prolonged growth of bacteria in the presence of one of the virulent phages were examined. On the basis of the results obtained, it is postulated that: (1) Mutants differing in resistance to various phages accumulate in a population; relative numbers of different mutants can undergo alterations over the course of time; mutants selected in the presence of a given virulent phage do not often manifest complete resistance to this phage. (2) It is possible to isolate totally phage-resistant mutants of P. putida PpG1. These mutants carry up to three different mutations simultaneously; however, these mutants regain sensitivity to many phages upon pseudoreversion occurrence.     

The action of toxins derived from scorpion venom on the ileal smooth muscle preparation

The properties of the 37400 oncogenic strain of Agrobacterium tumefaciens are described. This strain was derived from the VI lysogenic strain originally isolated by Hamilton from a Zinnia elegans tumour. Strain 37400 has a number of properties which render it suitable for quantitative and genetic studies. It is cured of prophages and can serve as a universal sensitive indicator for a number of phages isolated from various lysogenic strains of Agrobacterium tumefaciens. Its good growth properties in synthetic media and at elevated temperatues enable the isolation of auxotrophic mutants and temperature sensitive phage mutants. Preliminary experiments show that strain 37400 will serve as suitable starting material for conjugation experiments under defined conditions.     

The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence

Melioidosis, an infection caused by the gram-negative bacterial pathogen Burkholderia pseudomallei, is endemic in south-east Asia and northern Australia. Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north-east Thailand. B. pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B. pseudomallei 1026b multiplies in 10-30% NHS. We developed a simple screen for the identification of serum-sensitive mutants based on this novel phenotype. Approximately 1200 Tn5-OT182 mutants were screened, and three serum-sensitive mutants were identified. The type II O-antigenic polysaccharide (O-PS) moiety of lipopolysaccharide was not present in the serum-sensitive mutants. A representative serum-sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis. The Tn5-OT182 integrations in the serum-sensitive mutants were physically linked on the B. pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O-PS production. The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis. The results presented here demonstrate that type II O-PS is essential for B. pseudomallei serum resistance and virulence.     

Antimicrobial-resistant Salmonella spp isolated from healthy broiler chickens after slaughter

Of 105 Salmonella organisms of any serotype selected from a sample of 1,824 serotyped salmonellae isolated during a nationwide bacteriologic survey of healthy broiler chickens after slaughter, 60 (57%) were resistant to 1 or more antimicrobial agents and 47 (45%) were resistant to 2 or more agents. Highest resistance was to tetracycline (45%), streptomycin (41%), sulfisoxazole (19%), gentamicin (10%), and trimethoprim/sulfamethoxazole (8%). Additional isolates of S typhimurium, heidelberg, agona, and enteritidis were selected from the sample of 1,824 isolates for testing because of the high frequency with which these 4 serotypes are isolated from human patients. The highest frequency of resistance among 104 isolates of S heidelberg, 92 isolates of S typhimurium, and 30 isolates of S agona was to streptomycin (33 to 57%), sulfisoxazole (33 to 50%), tetracycline (26 to 50%), and gentamicin (13 to 40%); 51 to 63% of these isolates were resistant to 1 or more agents and 37 to 59% were resistant to 2 or more agents. Resistance to ampicillin among these 3 serotypes was uncommon (0 to 4%). In contrast, 15 of 19 tested isolates (79%) of S enteritidis were resistant to ampicillin and 13 of the 19 isolates (68%) were resistant only to ampicillin. This pattern of resistance was associated with a specific bacteriophage type and indicated the potential role of bacterial clones in determining the frequency and patterns of antimicrobial resistance in populations of broiler chickens. Resistance to gentamicin and trimethoprim/sulfamethoxazole was higher than that previously reported and is of public health concern because of the frequency with which these drugs are used to treat bacterial infections in human patients.     

Pyoverdin is essential for virulence of Pseudomonas aeruginosa

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.     

Oxidant inhibition of alphaLbeta2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage

Dithiothreitol (DTT) and other dithiol antioxidants with closely spaced thiol pairs strongly activate leukocyte function antigen-1 (LFA-1, alphaLbeta2 integrin) to bind intercellular adhesion molecule-1 (ICAM-1). Because direct biochemical modification of LFA-1 by DTT is not apparently involved, we investigated the possible role of a reduction-oxidation (redox)-sensitive adhesion-regulatory pathway. Phenylarsine oxide (PAO), an oxidant selectively reactive with closely spaced pairs of thiol groups, inhibited LFA-1-dependent adhesion of human natural killer and HSB2 T leukemia cells to murine cells expressing human ICAM-1. PAO also induced disappearance of a conformation-sensitive LFA-1 epitope recognized by KIM127 antibodies and promoted an increase in total apparent cytoskeleton-linked LFA-1 in which a novel cytochalasin D-resistant linkage was involved. Exposure of PAO-pretreated cells to DTT caused a decline in LFA-1/cytoskeleton linkages in conjunction with rapid restoration of KIM127 epitope expression and LFA-1 adhesive function. Implicating an intracellular site of action were findings that (1) an epitope-tagged PAO probe bound predominantly to intracellular proteins but not detectably to immunoprecipitation-purified LFA-1 chains, and (2) membrane permeant but not impermeant dithiol antioxidants reversed PAO adhesion-inhibitory effects. These results support the concept of a reversible redox-sensitive linkage between LFA-1 and cytoskeleton by which oxidants and antioxidants may exert profound opposing effects on LFA-1 conformation and adhesive function.     

Mu gem2ts DNA integration is not necessary for induction of synchrony of cell division in Escherichia coli K12

The gem2ts mutant of bacteriophage Mu induced synchrony of cell division on bacteria surviving infection. Induction of synchronous growth could also be observed as a response to the entire infected bacterial population, as in the case of infection of hic mutants, a peculiar class of gyrB alleles. After Mu wild-type or Mu gem2ts infection of hic mutants, there was a lack of viral DNA integration and replication, while phage gene expression (including that of A gene, coding for the transposase) seemed to be quite normal. These data indicate that the mechanism of bacterial synchronization induced by Mu gem2ts does not require integration nor replication of the phage DNA.     

Acriflavine-resistant mutant of Streptococcus cremoris

Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40 degrees C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures.     

Cognition in children does not suffer from very low lead exposure

It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.     

Characterization of host-range mutants of cyanophage N-1

Fifteen host-range (h) mutants of cyanophage N-1 were characterized with reference to their efficiency of plating, time of appearance, morphology and size of plaques on Nostoc muscorum and its three phage-resistant (Nm 1/N-1, Nm 2/N-1 and Nm 8/N-1) mutants. While phage N-1 did not adsorb to the three phage-resistant mutants, the h mutants differed one from the other in having lower or higher adsorption rate constants on N. muscorum or the phage-resistant mutants. The inability of majority of h mutants isolated on Nm 1/N-1 to grow in Nm 8/N-1 was shown to be due to a failure of adsorption. The h mutants also differed one from the other in their reversion (back mutation) frequencies. The lethal doses (LD37) required to kill 37% of free phage particles after UV-irradiation, heating and ethylenediamine tetraacetate (EDTA) treatment greatly varied. Most of the h mutants were found to be considerably more sensitive to UV and thermic inactivation than N-1 while they were resistant to EDTA. The h mutants except five of them were unable to multiply at 40 degrees C. The significance of these features is discussed.     

Biological properties of Streptococcus bovis bacteriophages isolated from lysogenic cultures and sheep rumen

Biological characteristics of eleven phages for Streptococcus bovis were investigated; seven phage were isolated from ovine rumen and four were virulent mutants of temperate phages of lysogenic cultures. The phages had many properties in common: similar morphology of negative colonies, the identical spectrum of lytic action, related antigens, absolute or high requirement of calcium ions, thermolability, and inactivation by the content of the rumen. Their susceptibility to the inactivating action of acetic acid, urea and temperature was however different. Chloroform and phenol may be used during purification and conservation of the phages.