Avirulent isolates of Corynebacterium fascians that are unable to utilize agmatine and proline.

Growth of a highly virulent strain of the phytopathogen Corynebacterium fascians on rich media at 37 degrees C resulted in a loss of virulence in a majority of the population within 10 generations. Strains retained virulence during cultivation at 30 degrees C on a minimal medium with ammonia as a nitrogen source. Populations of avirulent strains on the surfaces of pea seedlings decreased, whereas the number of cells of the virulent strain increased 1,000-fold during a 3-week period. All avirulent mutants isolated by growth on rich media at 37 degrees C were unable to grow on media containing agmatine or proline as sole sources of nitrogen. The ability of the mutants to grow on pea seedlings and cause fasciation disease appeared to be related to their ability to utilize nitrogen sources available on plant surfaces.     

Role of glycoprotein gIII of pseudorabies virus in virulence.

Deletion mutants of pseudorabies virus unable to express glycoprotein gIII, gI, or gp63 or double and triple mutants defective in these glycoproteins were constructed, and their virulence for day-old chickens inoculated intracerebrally was determined. Mutants of wild-type pseudorabies virus defective in glycoprotein gIII, gI, or gp63 were only slightly less virulent (at most, fivefold) for chickens than was the wild-type virus. However, mutants defective in both gIII and gI or gIII and gp63 were avirulent for chickens, despite their ability to grow in cell culture in vitro to about the same extent as mutants defective in gIII alone (which were virulent). These results show that gIII plays a role in virulence and does so in conjunction with gI or gp63. The effect of gIII on virulence was also shown when the resident gIII gene of variants of the Bartha vaccine strain (which codes for gIIIB) was replaced with a gIII gene derived from a virulent wild-type strain (which codes for gIIIKa); gIIIKa significantly enhanced the virulence of a variant of the Bartha strain to which partial virulence had been previously restored by marker rescue. Our results show that viral functions that play a role in the virulence of the virus (as measured by intracerebral inoculation of chickens) may act synergistically to affect the expression of virulence and that the ability of the virus to grow in cell culture is not necessarily correlated with virulence.     

Capsulation and virulence in Erwinia amylovora

Evidence is presented that capsulation may be one virulence determinant for Erwinia amylovora, the fireblight pathogen. When 15 virulent and seven avirulent strains were grown on a medium containing asparagine as the only source of carbon and nitrogen, or yeast peptone agar, or on a sugar medium containing an inorganic source of nitrogen, capsule production and virulence were not correlated. However, if a sugar or sugar alcohol was added to the asparagine medium or to yeast peptone agar all the virulent strains produced some or many capsulated cells whereas six of the avirulent ones did not. Capsules were also produced by all the virulent strains during infection. The existence of a seventh avirulent strain which was capsulated on all media except unsupplemented asparagine agar, suggested that capsule production was not the only virulence determinant.     

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.     

Differences in aerosol survival between pathogenic and non-pathogenic strains of Legionella pneumophila serogroup 1

A strain of Legionella pneumophila serogroup 1 known to be virulent for guinea-pigs was found to be least stable at a relative humidity (r.h.) of 60% when stored as a small particle aerosol. Three L. pneumophila serogroup 1 strains of different virulence for guinea-pigs were then tested at a r.h. of 60% at 20 degrees C. The most virulent strain was found to have the best survival and the avirulent strain was least stable. The strain of intermediate virulence did not survive as well as the virulent strain but was more stable than the avirulent strain. Strains of L. pneumophila serogroup epidemiologically associated with legionnaires' disease had better survival in small particle aerosols than strains which were not associated with disease. Subtyping with monoclonal antibodies also showed that the type more commonly associated with disease survived longer in aerosols than the other subtypes.     

Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection

The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5' end and a chitin synthase (class V)-encoding region at its 3' end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5' upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25 degrees C but have severe growth and cellular abnormalities at 37 degrees C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Delta mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Delta mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37 degrees C and is of critical importance to its virulence.     

Comparative growth of Porphyromonas gingivalis strains in a defined basal medium

Porphyromonas gingivalis is an asaccharolytic bacterium whose metabolism is dependent on the uptake of small peptides and amino acids. The aim of this work was to study the growth of P. gingivalis in a defined basal medium (DBM) supplemented with various sources of proteins. The strain 49417 as well as other virulent isolates could grow in DBM containing 1% bovine serum albumin (BSA). Cells cultivated under this condition showed a slightly modified protein profile, and expressed hemagglutinating as well as proteolytic activities. Other natural proteins under investigation could not support the growth in the DBM. On the other hand, the strain 33277 as well as other avirulent strains of P. gingivalis could not use BSA as a substrate. The ability of P. gingivalis to grow in DBM-BSA is not entirely dependent on its ability to degrade the protein substrate as strain 33277 was able to extensively hydrolyse the molecule. Differences in either metabolic enzymes or peptide transport mechanisms may explain the distinctive behavior between virulent and avirulent strains. Data from this work suggest a relationship between nutritional requirements and virulence of P. gingivalis in an animal model. The DBM-BSA may represent a more appropriate medium for studies on the physiology of P. gingivalis.     

Isolation and characterization of hemolysin mutants of Vibrio vulnificus

Abstract The importance of the cytolysin/hemolysin in the virulence of Vibrio vulnificus was investigated using both the naturally occuring virulent and avirulent colony variants and ethylmethane-sulfonate generated mutants. Both virulent and avirulent isogenic morphotypes produced similar amounts of hemolysin. Two mutants deficient in the production of hemolysin and negative for CHO cell activity were characterized and their virulence for mice was examined. Non-hemolytic mutants were found to be as virulent as their parent strain. It is concluded that the hemolysin produced by V. vulnificus is not required for the full virulence of this pathogen.     

Experimental adaptation of an RNA virus mimics natural evolution

Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.     

A 78-megadalton plasmid occurs in avirulent strains as well as virulent strains ofCorynebacterium fascians

Ten wild-type strains ofCorynebacterium fascians, which differed in degree of virulence as measured by ability to cause hyperplasias (multiple stems; fasciation) in pea seedlings, were examined for the presence of plasmids. Four strains were highly virulent, three were avirulent, and three were intermediate in virulence. All of these wild-type strains harbored one plasmid each of approximately 78 megadaltons, as estimated from electrophoretic mobilities in agarose gels capable of resolving reference plasmids ranging from 8.8 to 350 Mdal. Restriction endonuclease (EcoRI andBamHI) cleavage patterns of these nominally 78-MdalC. fascians plasmids suggest that the plasmids are not uniformly of high homology, although similar or identical in electrophoretic mobility in the system used. The relationship of the 78-Mdal plasmids to the phytopathogenicity ofC. fascians remains uncertain, although the restriction endonuclease cleavage patterns do indicate that the plasmids from the highly virulent strains are more closely related to the plasmids from the strains intermediate in virulence than they are to the plasmids from the avirulent strains.     

Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes

Agrobacterium rhizogenes strain 15834, which incites hairy root disease in plants, harbors three large plasmids: pAr15834a (107 x 10(6) daltons), pAr15834b (154 x 10(6) daltons), and pAr15834c (258 x 10(6) daltons). Kanamycin-resistant transconjugants were selected in a cross of kanamycin-resistant derivate of strain 15834 and an avirulent recipient. The transconjugants belonging to one class were virulent and contained all three donor plasmids. These transconjugants also acquired sensitivity to the bacteriocin agrocin 84. The loss of plasmids from virulent transconjugants during growth at 37 degrees C indicated that virulence genes reside on pAr15834b, whereas agrocin 84 sensitivity genes reside on pAr15834a. The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain of A. rhizogenes. Restriction endonuclease fragment analysis of plasmids from the transconjugants and the donor revealed that pAr15834c is a cointegrate of pAr15834a and pAr15834b. Kanamycin-resistant transconjugants belonging to a second class were avirulent and contained an altered form of pAr15834b. Strain 15834 can utilize octopine. However, this trait was not detected in any of the transconjugants. Octopine is not synthesized by infected plant tissue.     

Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.     

Metabolic characterization of the genus Brucella. VI. Growth stimulation by i-erythritol compared with strain virulence for guinea pigs

Strains of Brucella abortus isolated 20 to 38 years ago and strains recently isolated were assessed for their virulence to guinea pigs and for their ability to grow in half-strength Tryptose Broth with and without i-erythritol. The recently isolated strains were virulent and i-erythritol enhanced their growth. The aged strains were avirulent and grew equally well in both media. Three of the recently isolated strains were subcultured serially every 24 hr alternately on Tryptose Agar slants and in half-strength Tryptose Broth without i-erythritol. After 8 to 13 such transfers, the growth of each strain was equivalent in both media. The subculture on which growth equivalence occurred was retested for virulence. None of the three strains had decreased in its virulence for guinea pigs. The conclusion was drawn that strain virulence for guinea pigs and growth enhancement by i-erythritol are independent characteristics.     

Comparative characteristics of cell-free filtrates of Shigella flexneri of different virulence]

It was shown for the first time that the virulent Sh. flexneri strain grown on Luria broth differed from the avirulent one by the yield of readily released surface-located complexes--lipopolysaccharide (determined by rhamnose) and protein into the filtrate. There was no distinct correlation between the strain virulence and the content of rhamnose-determined lipopolysaccharide in the filtrate; growing bacteria in the presence of Ca and Mg ions had no significant influence on the lipopolysaccharide release into the filtrate. Protein release into the cell-free filtrate was thrice that in the virulent shigella strain than in the avirulent one. When bacteria were grown in the presence of Ca ions protein release from the virulent strain increased 1.5-fold and changed but little in the avirulent culture. Cell-free filtrates of the virulent strain produced toxic action on L tissue culture cells; in conjunctival infection of guinea pigs they caused some reduction of the LD50 of the virulent strain and sharply aggravated the course of the infectious process. Heating of the filtrate at 100 degrees C for 15 min decreased their toxic action on L cells. The data obtained indicated that the active biological factor revealed in the virulent strain of Sh. flexneri was protein or its derivative.     

Petite-negative mutants of Saccharomyces cerevisiae

Summary A series of yeast mutants has been isolated with the inability to grow on fermentable carbon sources whilst growing normally on ethanol media. One of the mutants, namely MC16/206 lacks pyruvate decarboxylase activity and does not grow on glucose at 37°C but grows on both ethanol and glucose at 27°C. In this strain rho ^- petites are non-viable.     

Clonal structure of Yersinia pestis populations in experimental soil ecosystems

Two basic tendencies--formation of latent (uncultivable) form (LF) and hemin storage variability--has been revealed during study of clonal structure dynamics of Y. pestis populations in artificial soil ecosystems in long-term incubation conditions. Y. pestis populations disappeared within 3 - 6 months at 18 - 22 degrees C, whereas at 4 - 8 degrees C a subsequent replacement of vegetative cells on LF, which are capable to prolonged survival (up to 22 months) in soil with ability to reversion in the presence of abundance of nutrients, has been observed. Bacteria of virulent strain retained all determinants of pathogenicity when reverted to LF, whereas bacteria of avirulent strain (defective on plasmid of Ca-dependence), on the contrary, undergo further degradation that resulted in loss of a pgm locus and gradual disappearance of population. LF revertants of highly virulent strain restored properties of initial population and were highly virulent.     

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.