Scanning Electron Microscope Study of Neisseria gonorrhoeae

Morphological studies utilizing various microscopy techniques have aided in our understanding of the gonococcus and gonorrhea. In this study scanning electron microscopy was used to study differences in virulent (colony types 1 and 2) and avirulent (colony types 3 and 4) gonococci relative to colony appearance, patterns of growth in liquid media, and surface features of individual cocci. Colony types of virulent gonococci are smaller in diameter but have a higher evaluation than those of avirulent mutants. Colony type 2 has a convex undersurface that is associated with surface pitting of solid media. When the colonies are grown in liquid media, various degrees of autoagglutination are observed; this is most pronounced with type 2 and least evident with type 4. Although pili may be involved in this phenomena, other mechanisms must be employed, since type 3 gonococci that lack pili autoagglutinate. Pili are seen on types 1 and 2 and are absent from types 3 and 4. They appear as individual threads radiating from the bacteria or as bundles of pili attaching adjacent cocci. Another extracellular structure consists of small spherical bodies that can coat the bacteria surface, attach to pili, or exist free from other bacterial components. These spheres are least evident with type 4. The gonococcal surface is pebbly with multiple sulci.     

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25°C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17⁻ mutants at 25°C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17⁻ mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.     

Selection from gonococci grown in vitro of a colony type with some virulence properties of organisms adapted in vivo.

Gonococci from subcutaneously implanted chambers in guinea pigs produced, on agar, more than 95% small colonies showing a "double highlight" (DH) effect in oblique reflected light combined with transmitted light. Laboratory strains of gonococci produced some DH colonies, but other showed a single highlight (SH) or no highlight (NH). Selection of DH colonies and comparison of their organisms with gonococci grown in vivo and with those from SH colonies, showed that the DH character was associated with high infectivity for guinea-pig chambers, resistance to killing by human phagocytes and heavy pilation. Furthermore, DH colonies were found in the first culture of three fresh samples of urethral pus. Thus, the DH colony characteristic may be a more reliable criterion of pathogenicity of gonococcal isolates than systems used previously. There were, however, some differences between the gonococci grown in vivo and the DH colony types. The gonococci grown in vivo and cultured once on solid medium possessed one or two antigens which differed from those of DH (or SH) colonies. They also formed smooth suspensions (which separated slowly) in saline, compared with the rough suspensions (which separated quickly) formed by gonococci from DH (or SH) colonies. Finally, the organisms grown in vivo were resistant to killing by human serum whereas the DH (and SH) colony types were susceptible; the resistance of the organisms grown in vivo was lost during one subculture on agar suggesting that the property is a phenotypic characteristic. Hence, in addition to selecting DH colony types the conditions in vivo produce organisms which differ, probably phenotypically, from cultured organisms.     

Genetic transformation of pilation and virulence into Neisseria gonorrhoeae T4.

Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.     

Screening for Spontaneous Virulent Mutants of Erysiphe graminis D.C. f.sp. hordei on Barley lines with Resistance Genes Ml-a1, Ml-a6, Ml-a12 and Ml-g

Seedlings of 4 barley lines with powdery mildew resistance genes Ml-a1, Ml-a6 Ml-a12 and Ml-g were inoculated with powdery mildew culture CR3 which is avirulent to the 4 host lines. The inoculation density was 1.2 infectious conidia per mm², and in total 50 million conidia were screened for the occurrence of virulent mutans. During 30 cycles of screening, 43 putative virulent mutants were selected, multiplied and tested. They could be grouped in 5 different genotypes according to virulence spectrum. Based on the virulence spectre, mating type, biochemical tests and analyses of test crosses, 3 of the types were rejected as being of mutational origin, and the verification of the remaining 2 were not consistent with the expectations deduced from a gene-for-gene interaction. Provided that none of the genotypes found were of mutational origin, the spontaneous mutation frequency from avirulence to virulence in barley powdery mildew is therefore below 2 × 10^–8. A reconstructation experiment showed that the density of avirulent inoculum did not reduce the survival rate of rate virulent genotypes     

Transformation of virulence factor among bacterial blight ofXanthomonas campestris pv.malvacearum in plants

Transformation of an avirulent strain of bacterial blight of the cotton pathogenXanthomonas campestris pv.malvacearum (Xcm) to a virulent strain was achieved in plants by using total (i. e. chromosomal and plasmid DNA) as well as chromosomal DNA, but not the plasmid DNA, of a virulent Xcm strain, thereby indicating that virulence was located in the chromosomal replica. The process of transformation began 1 d after inoculation of the DNA preparation with the avirulent strain in plant tissues and was completed within 2 d with a recombination frequency of 10⁻²−10⁻⁴.     

Virulence of Escherichia coli in acute pyelonephritis,acute cystitis and asymptomatic bacteriuria

E. coli strains were isolated from urine specimens of hospitalised patients with acute pyelonephritis, acute cystitis or asymptomatic bacteriuria (ABU), and tested for virulence in an experimental mouse model. Of 12 pyelonephritisstrains 11 were shown to be virulent and 1 avirulent; of 12 cystitis-strains 4 were virulent and 8 avirulent; of 12 ABU-strains 5 were virulent and 7 avirulent. It is concluded that, while no difference in virulence was found between cystitis-and ABU-strains, pyelonephritis-strains were more often virulent than cystitis-and ABU-strains.No associations could be shown between virulence of the isolated strains and the presence of antibody-coated bacteria in the urine. Common urinary O types were not more often virulent than other O types. No relationship was seen between virulence and the presence of K antigen or the presence of particular K types.     

Dominance and recessiveness at loci for virulence against potato and tomato in Phytophthora infestans

Summary In this study we investigated the genetic control of virulence in the diploid fungal pathogen, Phytophthora infestans, against host resistance genes R1, R2, R3, and R4 (potato) and Ph1 (tomato). For four of these virulence traits, the presence or absence of segregation indicated conclusively which phenotype was dominant. We observed a 31 (virulentavirulent) segregation on R2 in the progeny of parents which were both virulent, suggesting that virulence is dominant and both parents are heterozygous. In a cross in which one parent was virulent and the other avirulent on potato gene R3, all progeny tested were avirulent, so avirulence against R3 is dominant. The same virulent parent crossed with a different avirulent parent produced virulent and avirulent progeny in a 13 ratio, indicating that a second locus may be involved. The progeny of two parents virulent on R4 segregated for virulence and avirulence, so virulence against R4 is dominant. For Ph1, a 13 segregation in the progeny of two avirulent parents showed that the avirulent phenotype is dominant, and a 31 ration in a second cross suggested the involvement of a second locus. The segregations for virulence against R1 did not indicate which phenotype was dominant, but did suggest singlelocus control.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Adaptation to resistant hosts increases fitness on susceptible hosts in the plant parasitic nematode Globodera pallida

Trade‐offs between virulence (defined as the ability to infect a resistant host) and life‐history traits are of particular interest in plant pathogens for durable management of plant resistances. Adaptation to plant resistances (i.e., virulence acquisition) is indeed expected to be associated with a fitness cost on susceptible hosts. Here, we investigated whether life‐history traits involved in the fitness of the potato cyst nematode Globodera pallida are affected in a virulent lineage compared to an avirulent one. Both lineages were obtained from the same natural population through experimental evolution on resistant and susceptible hosts, respectively. Unexpectedly, we found that virulent lineages were more fit than avirulent lineages on susceptible hosts: they produced bigger cysts, containing more larvae and hatching faster. We thus discuss possible reasons explaining why virulence did not spread into natural G. pallida populations.     

Neisseria gonorrhoeae II. Colonial Variation and Pathogenicity During 35 Months In Vitro

During 35 months of selective in vitro cultivation, Neisseria gonorrhoeae cells retained their virulence for humans and were shown to be closely related to a particular colonial morphology. Saline-autoagglutinability was the only other characteristic distinguishing virulent from avirulent cells. Human responses to challenge with cells of the different colonial types were studied for their relationships to virulence or avirulence.     

Agglutination of Erwinia stewartii Strains with a Corn Agglutinin: Correlation with Extracellular Polysaccharide Production and Pathogenicity

A bacterial agglutinin was extracted from ground corn (WI hybrid 64A × W117) seed with phosphate-buffered saline (pH 6.0) and precipitated with (NH₄)₂SO₄ at 70% saturation. The activities of this agglutinin against 22 strains of Erwinia stewartii (agent of bacterial wilt of corn) that varied in virulence were determined. Specific agglutination (agglutination titer per milligram of protein per milliliter) values were correlated negatively with virulence ratings. Strains with high specific agglutination values (15 or higher) were avirulent or weakly virulent; strains with low specific agglutination values (10 or lower) were highly virulent, with two exceptions. Avirulent strains produced butyrous colonies and released only small amounts of extracellular polysaccharide (EPS) into the medium, and the cells lacked capsules; virulent strains produced fluidal colonies and released large amounts of EPS, and the cells were capsulated. There was a strong correlation between the amount of EPS produced by each strain (as determined by increase in viscosity of the medium) and the specific agglutination value; in contrast, lipopolysaccharide compositions were similar in all strains. When cells of six fluidal strains were washed by repeatedly centrifuging and resuspending them in buffer, they were agglutinated more strongly by corn agglutinin than were unwashed cells. When avirulent cells were washed, their specific agglutination values did not increase significantly. Eight EPS-deficient mutants of E. stewartii, selected for resistance to the capsule-dependent bacteriophage K9, had lower virulence but higher specific agglutination than did their corresponding wild-type parents. Production of EPS appears to be essential for virulence; EPS may prevent agglutination of bacteria in the host, thus allowing their multiplication.     

Changes in properties of phytopathogenic bacteria effected by plasmid pRD1

Transfer of plasmid pRD1 from Escherichia coli K12J62-1 to phytopathogenic bacteria, Agrobacterium tumefaciens, Xanthomonas beticola and Erwinia carotovora subsp. carotovora caused changes in conjugant properties not determined by the plasmid and the emergence of the properties not present in the parent strains. Clones have been obtained with intermediate properties between donor and recipient, including those with altered or lost virulence. In transconjugants of A. tumefaciens virulence increased. In transconjugants of X. beticola and E. carotovora subsp. carotovora highly virulent as well as avirulent forms have been observed. The loss of virulence in X. beticola correlated with the Nif^* phenotype. Plasmid pRD1 also affected the biochemical properties of the new hosts.     

Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation

Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.     

Type and Strain Variation in Induction of L Forms of Neisseria gonorrhoeae

Strains of Neisseria gonorrhoeae were inoculated onto brain heart infusion (Difco) agar supplemented with penicillin and polyvinylpyrrolidone (PVP) as a stabilizer and cultivated in a candle extinction jar. L-form colonies became visible after a few days. They continued to grow and were viable for up to 38 days. The extent of inducibility of L forms of N. gonorrhoeae was examined semiquantitatively. It was found to be constant for each type and strain and to depend only slightly on the amount of penicillin added to the medium. None of the types of one strain produced L-form colonies. In another strain, avirulent types(T₃, T₄) showed more ability to produce L forms than virulent types (T₁, T₂) and no L forms were produced by the subtypes of T₁ and T₂–T_1a and T_2a. In a third strain, only T₄ produced L forms. Electron microscopic studies showed that the L forms consisted of a number of membranous vesicles and a variety of cell types such as those completely lacking cell walls and those with only remnants of cell walls. The results indicate the existence of subtypes of T₁ and T₂ of gonococci and the intrinsic inducibility of gonococcal types and strains to produce L forms.     

Gain of virulence by Soybean mosaic virus on Rsv4‐genotype soybeans is associated with a relative fitness loss in a susceptible host

‘Gene‐for‐gene’ theory predicts that gain of virulence by an avirulent pathogen on plants expressing resistance (R) genes is associated with fitness loss in susceptible hosts. However, the validity of this prediction has been studied in only a few plant viral pathosystems. In this study, the Soybean mosaic virus (SMV)–Rsv4 pathosystem was exploited to test this prediction. In Rsv4‐genotype soybeans, P3 of avirulent SMV strains provokes an as yet uncharacterized resistance mechanism that restricts the invading virus to the inoculated leaves. A single amino acid substitution in P3 functionally converts an avirulent to a virulent strain, suggesting that the genetic composition of P3 plays a crucial role in virulence on Rsv4‐genotype soybeans. In this study, we examined the impact of gain of virulence mutation(s) on the fitness of virulent variants derived from three avirulent SMV strains in a soybean genotype lacking the Rsv4 gene. Our data demonstrate that gain of virulence mutation(s) by all avirulent viruses on Rsv4‐genotype soybean is associated with a relative fitness loss in a susceptible host. The implications of this finding on the durable deployment of the Rsv4 gene in soybean are discussed.     

Interrelation of virulent and avirulent (vaccinal) strains of Shigella flexneri 2A in long-term bacterial carriage in gnotobiotic rats

The ability of S. flexneri 2a virulent and avirulent (vaccine) strains No. 516 and No. 516 m to displace one another during long-term carrier state in germ-free rats has been studied. The long-term persistence of the vaccine strain in the intestine of the rats has been shown to produce colonization resistance to subsequent infection with the virulent culture of these bacteria. During carrier state in rats progressive S--R dissociation of the bacteria occurs, type II antigen is lost with simultaneous retention of group 3, 4 antigens and the resulting transformation of S. flexneri, serotype 2a, into variant y; the virulence of Shigella S-forms is also lost.     

Quantitative Studies of Resistance Induced By Avirulent Cultures of Erysiphe graminis f. sp. hordei in Barley

Quantification of resistance induced by avirulent cultures of Erysiphe graminis f. sp. hordei against virulent cultures in barley was attempted. Four mildew cultures and 4 barley varieties with known genes of virulence and resistance respectively were used. Pre, post and simultaneous inoculation of leaves was done with avirulent and virulent cultures. Pre-inoculation with avirulent cultures induced resistance in the host such that the pustule number and spore production by later inoculation of virulent cultures was reduced significantly. Once induced, such resistance was active up to 8 days. There was some indication of induced susceptibility if the inducing culture was characterized by medium virulence. Increase of inceulum density of the avirulent (inducer) culture increased the amount of induced resistance Further studies of the phenomenon of induced resistance are needed in relation to possible applications for disease control through inoculations. varietal mixtures and multilines.     

Independent Inheritance of Genes Governing Virulence and the Production of a Polypeptide in Ustilago hordei*

Detergent soluble polypeptides from teliospores of Ustilago hordei were examined by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis. A relatively prominent polypeptide (M.W. 28,000, P¹ 6.8) was found in extracts of teliospores from two isolates that were avirulent on the barley cultivar ‘Plush’ but was not detected in extracts from two isolates that were virulent on this cultivar. The polypeptide was also detected in extracts from teliospores of an F₁, hybrid between one of the virulent and one of the avirulent isolates. Haploid cultures from five meiotic tetrads, isolated from the F₁ hybrid, were backcrossed to haploids from the virulent parent to determine the pathogenicity of each strain. The polypeptide segregated among the 20 progeny as if under the control of a single, dominant allele. The gene governing virulence on ‘Plush’ was not linked to the gene governing the segregating polypeptide.     

Mating frequency, genetic structure, and sex ratio in the intermorphic female producing ant species Myrmecina nipponica

1. Myrmecina nipponica has two types of colonies: a queen colony type, in which the reproductive females are queens and new colonies are made by independent founding, and an intermorphic female colony type, in which reproductive females belong to a wingless intermediate morphology between queen and worker, and where colonies multiply through colonial budding. 2. The mating frequencies of reproductive females in both types indicate monoandry. The relatedness among nestmates in both types was almost 0.75, however relatedness between mother and daughter in intermorphic female colonies was slightly higher than that of queen colonies. 3. The sex ratio (corrected investment female ratio) was 0.70 at the population level, suggesting that the sex ratio is controlled by workers in this species, however the ratio differed greatly between the two types of colonies. Queen colonies (n = 37) had a female‐biased sex ratio of 0.77 while intermorphic female colonies (n = 33) had a ratio of 0.56. 4. Each reproductive intermorphic female was accompanied by an average of 2.9 workers (including virgin intermorphic females) in the colonial budding, and when the investment to those workers was added to the female investment, the sex ratio reached 0.81. 5. The frequency distribution of sex ratio was bimodal, with many colonies producing exclusively males or females, however mean estimated relatedness within colonies was almost 0.75. These data are inconsistent with the genetic variation hypothesis, which is one of the predominant hypotheses accounting for the between‐colony variation in sex ratio.