Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems.

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

A novel pleiotropic mutation in Escherichia coli K12 which affects transduction, transformation and rates of mutation.

A mutant strain of Escherichia coli K12, R2721, has been shown to differ from its parent strain, S491, in four associated phenotypic characters as a result of a single mutation. This strain did not give recombinants with DNA transduced by bacteriophage PI or bacteriophage Mu, nor transformats after exposure to R factor DNA: lysates of bacteriophage PI grown on this strain did not appear to contain any transducing particles when tested on normal recipients. Moreover, the reversion rates, both spontaneous and ultraviolet-induced, for two auxotrophic markers were reduced. The frequency of revertants was at least two orders of magnitude lower in cultures of R2721 than in cultures of S491I. Many of the rare revertants for one or other of the auxotrophic markers were found to have regained normal reversion frequencies for the other marker and for the capacity to be transduced. In all other respects, recombination in R2721 appeared normal, the frequency of chromosomal mobilization by and F' factor was unaffected and normal yields of recombinants were obtained from matings with Hfr strains. The only circumstance in which transduction of R2721 was observed was when the capacity to ferment galactose was selected and PI had been grown on a strain carrying lambdadgal when, presumably, integration was effected by the phage-coded gene products. The mutation has been located on the E. coli chromosone map between tonA and pro and has been given the symbol tdi (transduction inhibition). Double mutants, (tdi recA) and (tdi recB), have been isolated and show no unexpected properties.     

Male-Specific Bacteriophage MS2 Propagation in Fluorophenylalanine-Resistant Escherichia coli K12

Mutation of Escherichia coli K12 HfrH to resistance to fluorophenylalanine resulted in changes in the plaque morphology of bacteriophage MS2 on this strain and led to an increased efficiency of propagation of the phage in liquid cultures. Evidence was obtained that the mutation resulted in inhibition of early lysis in infected cells and that lysis involved the production of a lysozyme. Genetic studies suggested that the observed pleiotropy of the resistance mutation was due to informational suppression.     

Synthesis of Soluble Ribonucleates in Escherichia coli Infected with Bacteriophage R17

Measurements were made on the level of synthesis of soluble ribonucleates (sRNA) in Escherichia coli K-12 infected with the ribonucleic acid (RNA)-containing bacteriophage R17. Little or no decrease in sRNA synthesis was found to occur under conditions of infection which result in a 70 to 80% inhibition of ribosomal RNA synthesis.     

Streptomycin Action: Greater Inhibition of Escherichia coli Ribosome Function with Exogenous than with Endogenous Messenger Ribonucleic Acid

Inhibition of protein synthesis by streptomycin was tested in extracts from a strain of Escherichia coli sensitive to streptomycin. Three kinds of messenger ribonucleic acid (RNA) were employed: endogenous cellular RNA, extracted cellular RNA, and phage R17 RNA. Protein synthesis directed by extracted cellular RNA was inhibited three- to fourfold more than protein synthesis directed by endogenous RNA. With R17 RNA as messenger, nearly total inhibition of protein synthesis at initiation was again observed. The greater inhibition of function of extracted RNA, which must initiate new polypeptide chains in vitro, is in accord with the observation that in whole cells streptomycin blocks ribosomes at an early stage in protein synthesis. When streptomycin was added at successively later times during protein synthesis, the subsequent inhibition was progressively less. This was observed with either extracted cellular RNA or phage R17 RNA. A model is presented that can explain the less drastic inhibition by streptomycin of messenger RNA that is already functioning on ribosomes.     

Effect of Infection with Ribonucleic Acid Bacteriophage R23 on the Inducible Synthesis of β-Galactosidase in Escherichia coli

Infection by ribonucleic acid (RNA) bacteriophage R23 inhibited the synthesis of beta-galactosidase in Escherichia coli. The inhibition, although not complete, was apparent shortly after infection and was maximal after the first 20 min of infection. R23 diminished the beta-galactosidase-synthesizing capacity when inducer was added after phage infection, but not when infection followed inducer removal. These findings suggested that the primary effect of R23 on enzyme-forming capacity was limitation of synthesis of enzyme-specific messenger RNA. Studies with ultraviolet irradiated phage and amber mutants of R23 indicated that the inhibitory process could be separated into two phases. Early inhibition did not require the expression of the viral genome, whereas late inhibition required the expression of the viral RNA synthetase cistron.     

High resolution autoradiography of Escherichia coli cells infected with bacteriophage R17

Granboulan, Nicole (Institute de Recherches sur le Cancer, Villejuif, Seine, France), and Richard M. Franklin. High-resolution autoradiography of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:849-857. 1966.-The ultrastructural alterations in Escherichia coli infected with the RNA bacteriophage R17 were further investigated by means of the technique of high-resolution autoradiography. Tritiated precursors to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein were employed in separate experiments. A striking inhibition of cellular RNA, DNA, and protein synthesis was noted. Whereas normal RNA synthesis occurs in the nucleoid, in infected cells RNA synthesis is predominantly cytoplasmic, but later in the latent period, and during the stage of active viral growth, the label is localized in a polar region. In the late stages of viral growth, RNA synthesis occurs only around the crystals. Protein synthesis also becomes localized in a polar region, but DNA synthesis remains confined to the nucleoid. Under conditions of chloramphenicol inhibition of viral-coat protein synthesis, RNA label is localized in the paranuclear lesion, providing further indication that RNA forms this fibrillar structure.     

Growth suppression in early-stationary-phase nutrient broth cultures of Salmonella typhimurium and Escherichia coli is genus specific and not regulated by sigma S.

We have studied the growth suppression seen in early-stationary-phase LB broth cultures of Salmonella typhimurium. Multiplication of small numbers of an antibiotic-resistant S. typhimurium mutant was prevented when the mutant was added to 24-h cultures of the antibiotic-sensitive parent strain, whereas an antibiotic-resistant mutant of an Escherichia coli strain added to the same culture grew well. A 24-h E. coli culture produced a similar specific bacteriostatic inhibition against E. coli. In older cultures, a specific bactericidal effect similar to that observed by M. M. Zambrano and R. Kolter (J. Bacteriol. 175:5642-5647, 1993) was also observed. Whether incubated statically or shaken, sufficient nutrients were present in the filtered supernatants of 24-h cultures for small inocula of the same strain to multiply to ca. 10(9) CFU/ml after reincubation. Introduction of the rpoS mutation had no effect on the specific bacteriostatic inhibition. Similar specific inhibition was also observed in strains of Citrobacter freundii, Klebsiella pneumoniae, Enterobacter agglomerans, and Shigella spp. Experiments in which the 24-h culture was physically separated from the antibiotic-resistant mutant by using a dialysis membrane were carried out. These results indicated that the inhibition might be mediated by a diffusible but labile chemical mediator.     

Galactose-specific messenger ribonucleic acid contents in Escherichia coli

A method is described for measuring the proportion of galactose-specific mRNA (gal-mRNA) in the total RNA extracted from pulse-labelled cells of Escherichia coli K12, by DNA-RNA hybridization with DNA prepared from bacteriophage lambdadg. RNA from wild-type E. coli was compared with RNA from a homogenote carrying the gal operon both in the chromosome and in a substituted sex-factor, and with RNA from a deletion strain that carried the galactose operon only in the exogenote. In each case the cultures were induced with fucose. Under these conditions the amount of gal-mRNA was found to be proportional to the content of galactokinase in the different cultures, and to the gene frequency. The amounts of gal-mRNA in an O(c) mutant and an R(-) mutant were also proportional to the observed contents of galactokinase. In cultures repressed for the enzymes of the galactose operon with thiomethylgalactoside, the content of gal-mRNA was higher than expected from the content of galactokinase. Possible explanations of this finding are discussed.     

Mutants of Escherichia coli variably resistant to bacteriophage T1

Carta, Guy R. (Rutgers, The State University, New Brunswick, N.J.), and Vernon Bryson. Mutants of Escherichia coli variably resistant to bacteriophage T1. J. Bacteriol. 92:1055-1061. 1966.-Mutants resistant to bacteriophage T1 were isolated from ultraviolet (UV)-irradiated cultures of Escherichia coli B/r, a UV-resistant variant. Bacterial populations derived from some of these mutants were partially but not completely resistant to the bacteriophage. Such mutants, designated variably resistant (B/r/1v), could not be obtained from E. coli B. Phage-free mutant populations taken from different stages in growth consisted of significantly different proportions of T1-resistant and T1-sensitive cells. The growth stage-dependent range of variation exceeded 1,000-fold. In broth cultures, the highest proportion of resistant cells consistently appeared at mid-log phase, and the highest proportion of sensitive cells at lag and stationary phases. Comparable evidence for environmentally dependent changes in host-cell phenotype was obtained by efficiency of plating and cloning efficiency analysis tests. Micromanipulation showed that, in clones growing in the presence of phage T1, sensitive bacteria appeared with high frequency and underwent lysis.     

In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium.

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20 degrees C and 40 degrees C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A TnphoA insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.     

Ultrastructure of Escherichia coli cells infected with bacteriophage R17

Franklin, Richard M. (Institut de Recherches sur le Cancer, Villejuif, Seine, France), and Nicole Granboulan. Ultrastructure of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:834-848. 1966-Ultrastructural changes in Escherichia coli cells infected with ribonucleic acid (RNA) bacteriophage R17 were studied under conditions of one-step growth. No morphological alterations were seen during the latent period. During the period of rapid viral synthesis, a fibrillar lesion surrounded by ribonucleoprotein particles was observed in a polar region. Late in infection, paracrystalline arrays of virions were found in over 90% of the cells. When protein synthesis was blocked by in over 90% of the cells. When protein synthesis was blocked by chloramphenicol at 20 min postinfection, allowing continued viral RNA synthesis without production of coat protein, a dense fibrillar area appeared in a paranuclear region. Cytochemical studies were done on cells embedded in hydroxypropyl methacrylate, a water-miscible embedding agent. The paracrystalline arrays of virions were digested after extensive treatment with either pepsin or ribonuclease. Shorter digestion with the pepsin resulted in better definition of the crystal regions. The fibrillar area found in chloramphenicol-treated cells was digested by ribonuclease but not by pepsin, and was also resistant to lead extraction. This region probably represents a pool of virus-specific RNA.     

Synthesis of viral and rRNA in bacteriophage R17 infection of a stringent strain of Escherichia coli.

A previous paper (1973) indicated that infection with bacteriophage R17 permits the synthesis of RNA and spermidine in Escherichia coli (CP78 in the absence of the exogenous essential amino acid, arginine. We have now isolated RNA formed under such conditions and analyzed the newly synthesized species by agarose-acrylamide electrophoresis. It has been shown that infection of the stringent cells in the absence of exogenous arginine resulted in a marked incorporation of uracil into rRNA, as well as into R17 RNA. It was shown that, although the organism was nonauxotrophic for uracil, addition of [-14C]uracil resulted in the rapid formation of TUP, the specific radioactivity of which approached that of the exogenous uracil. This indicated that the incorporation of exogenous uracil into rRNA in R17 infection of the stringent strain reflected a true stimulated synthesis of this nucleic acid. Infection of the essentially isogenic relaxed strain, CP79, under the same conditions inhibited the RNA synthesis to a much less extent than the inhibition caused during the normal infection. These observations provide another example of the close correlation between synthesis of spermidine and of host RNA, even in cells infected by an RNA bacteriophage.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.     

Host interference with viral gene expression: mode of action of bacterial factor i

The mechanism of interference with R17 viral RNA expression by a host protein, factor i, was studied. Formation of initiation complexes on native bacteriophage R17 RNA molecules, as well as translation of R17 RNA in vitro, is blocked almost quantitatively by factor i. This inhibition is readily overcome by the addition of excess R17 RNA. Extensive complex formation between factor i and R17 RNA occurs during inhibition of initiation complex formation. Moreover, the extent of inhibition of R17 RNA translation correlates closely with the extent of complex formation between factor i and R17 RNA, and exhibits the same sigmoid concentration dependence on factor i.Although initiation complex formation is totally dependent upon initiation factor IF-3, neither this function of IF-3, nor its ability to prevent the association of 30 S and 50 S ribosomal subunits into single ribosomes, is affected by factor i. IF-3, even when present in tenfold molar excess over factor i, fails to relieve the inhibition of initiation on R17 RNA.It is concluded that factor i is a translational represser acting directly on messenger RNA. It is suggested that this repression is cistron-specific, affecting only viral coat protein synthesis. Messenger RNA discrimination by factor i does not involve initiation factor IF-3.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.