Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

Method of selecting mutations for the plasmid genes of streptomycin resistance in Escherichia coli K-12 cells

Evidence is presented that streptomycin-dependent mutants of Escherichia coli K-12 are incompatible with the functional activity of streptomycin-resistance genes coding for aminoglycoside adenilyl transferase. On the basis of this phenomenon, a method for selection of streptomycin-sensitive mutations in these genes is proposed, and the possibility of the direct selection of hybrid DNA molecules carrying inserts inactivating streptomycin-resistance genes is discussed. The effectiveness of the method for isolation of streptomycin-sensitive R100.1 plasmid derivatives was demonstrated.     

Prevalence of recombinational versus mutational events in damaged plasmid DNA containing regions of homology with the chromosome.

Plasmid DNA modified by in vitro treatments was transformed in E. coli bacterial cells. A streptomycin-resistant strain, carrying the peculiar rpsL421 mutation, was used as a recipient for the cloning vector pNO1523, which carries the wild-type (streptomycin-sensitive) rpsL allele. Transformants were streptomycin-sensitive unless a change in plasmid sequence had occurred. The analysis of the MaeI restriction pattern of plasmids isolated from streptomycin-resistant transformants, together with the detection of the phenotype that they conferred to a streptomycin-dependent strain, allowed us to identify plasmids that had undergone recombination with the host chromosome. The number of these plasmids exceeded by far that of plasmids resulting from mutational events.     

Development of Auxotrophy by Streptomycin-Resistant Mutation

Several streptomycin-resistant mutants of Escherichia coli have been isolated which require exogenous isoleucine for growth. The majority of these strains were of streptomycin-dependent phenotype. If grown in the absence of streptomycin, these streptomycin-dependent auxotrophs (Sm(d-aux)) strains were unable to produce beta-galactosidase and aldolase activities and also failed to exhibit donor properties in conjugation. Genetic analysis indicated that the isoleucine requirement of these strains could be caused by a mutation at the strA locus.     

New approach to the genetics of Streptomyces coelicolor

Sermonti, G. (Istituto Superiore di Sanità, Rome, Italy), Milena Bandiera, and Isabella Spada-Sermonti. New approach to the genetics of Streptomyces coelicolor. J. Bacteriol. 91:384-392. 1966.-Mixed cultures of complementary auxotrophic strains of Streptomyces coelicolor A3(2) were preincubated on discs of cellophane on complete medium and were then transferred onto selective media. When one strain was streptomycin-resistant and the other was streptomycin-sensitive, and the transfer medium contained streptomycin, distinct minute tufts of aerial mycelium appeared on the background growth of the mixed culture. They turned out to be heterozygous clones (heteroclones) in which the streptomycin-sensitive allele was, as a rule, missing. The pattern of marker contribution of the streptomycin-sensitive parent to the zygotes was indicative of a continuous structure carrying the hereditary material. A gradual transfer of the donor genome during conjugation was suggested by the progressive completion of the zygotes obtained by increasing preincubation time.     

Effect of Streptomycin Deprivation on Enzyme Synthesis in Streptomycin-Dependent Escherichia coli

During growth of streptomycin-dependent strains of Escherichia coli in the absence of streptomycin (deprived growth), both constitutive and inducible synthesis of beta-galactosidase were preferentially inhibited. A similar preferential inhibition of constitutive and derepressed synthesis of alkaline phosphatase was observed. Catabolite repression accounted for part, but not all, of the inhibition of the inducible beta-galactosidase synthesis. Serological experiments indicated that that part of the inhibition specificially associated with streptomycin deprivation was not a result of the production of altered beta-galactosidase. It is suggested that during deprived growth the ribosomes of streptomycin-dependent bacteria become impaired in their ability to translate certain messages.     

The 503nm pigment of Escherichia coli

The yield of cell protein was one-third less for streptomycin-dependent Escherichia coli B than for the wild-type parent strain when both were grown aerobically on a medium with limiting glucose, but anaerobically the yield of protein was similar for both strains. The transient pigment absorbing at 503nm that is known to be present in E. coli and other organisms was not detectable in streptomycin-dependent mutants nor in a non-dependent (energy-deficient) revertant. When wild-type E. coli B was grown on limiting glucose–salts medium containing 2,4 dinitrophenol, the yield of cell protein was decreased and formation of the 503nm pigment was inhibited. Fumarase, aconitase and glucose 6-phosphate dehydrogenase were de-repressed in E. coli B cells grown with excess of glucose in a medium containing 2,4-dinitrophenol. In air-oxidized, wild-type E. coli B cells, the 503nm pigment appeared before reduced cytochromes when gluconate was the substrate but failed to appear when succinate was the substrate. The results provide evidence for a role of the 503nm pigment in aerobic energy metabolism, possibly as an electron acceptor from NADPH.     

Localization of a non-Mendelian gene in Chlamydomonas reinhardii

Summary Transfer of a non-Mendelian neamine-dependent (nd) mutant to an antibioticfree medium results in neamine-sensitive and neamine-resistent revertants. These reversions are caused by extranuclear mutations.The neamine-sensitive revertants are no more able to split offnd-cells after back-donation to neamine containing medium. Therefore they are different from the streptomycin-sensitive revertants of a streptomycin-dependent (sd) mutant. These mutants were capable ofsd-segregation though their potence ofsd-segregation diminished on antibiotic-free medium with increasing time of cultivation.The different behaviour can be explained by the fact that manysd-genes are present which have to be appointed to the mitochondria. On the other side, thend-gene exists only in few copies and is located therefore in the chloroplast.Several experiments with differing methods are discussed to localize the extranuclear genes.

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Vorgelegt durch G. Melchers     

Functional interactions between mutated forms of ribosomal proteins S4, S5 and S12

Here we show that ram mutations, either in ribosomal protein S4 or S5, decrease the proofreading flows for both cognate and noncognate ternary complexes bound by streptomycin-dependent (SmD) ribosomes. This effect is accompanied by a slight increase in the overall error frequency. More important, however, is the decreased proofreading of the cognate species which is almost reduced to wild-type levels. The data suggest that it may be the reduction of the proofreading of the cognate substrate that is important for suppressing streptomycin dependence. Furthermore, we show that rpsE mutants, selected from streptomycin-dependent strains, behave kinetically very similarly to the previously described rpsD mutants.     

Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus

We have isolated spontaneous streptomycin-resistant, streptomycin-dependent and streptomycin-pseudo-dependent mutants of the thermophilic bacterium Thermus thermophilus IB-21. All mutant phenotypes were found to result from single amino acid substitutions located in the rpsL gene encoding ribosomal protein S12. Spontaneous suppressors of streptomycin dependence were also readily isolated. Thermus rpsL mutations were found to be very similar to rpsL mutations identified in mesophilic organisms. This similarity affords greater confidence in the utility of the crystal structures of Thermus ribosomes to interpret biochemical and genetic data obtained with Escherichia coli ribosomes. In the X-ray crystal structure of the T. thermophilus HB8 30 S subunit, the mutated residues are located in close proximity to one another and to helices 18, 27 and 44 of 16 S rRNA. X-ray crystallographic analysis of ribosomes from streptomycin-resistant, streptomycin-pseudo-dependent and streptomycin-dependent mutants described here is expected to reveal fundamental insights into the mechanism of tRNA selection, translocation, and conformational dynamics of the ribosome.     

Overexpression of a Mutant B Subunit in Toxigenic Vibrio cholerae Diminishes Production of Active Cholera Toxin In Vivo

A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V. cholerae. The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium. The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo. Overexpression of B^G33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains. Received: 29 January 1998 / Accepted: 30 April 1998     

Freeze-drying of live oral streptomycin-dependent mutant Shigella vaccines: survival of streptomycin-dependent Shigella flexneri 2a after freeze-drying

Although the relatively successful freeze-drying of streptomycin-dependent mutant Shigella vaccines has been reported previously, further studies have been undertaken for the following reasons: (1) To improve the survival rate of the vaccine strains after freeze-drying. (2) To see whether monosodium glutamate can be eliminated as a stabilizer as it is reported to produce pharmacological effects in man.     

The use of the gene hybridization method for the identification of epidemiologically dangerous strains of cholera vibrios

Using the labeled DNA fragments containing the genes for cholera toxin the strains of cholera vibrios were studied for the presence of cholera toxin genes. Vibrio cholerae strains isolated from natural water reservoirs under the favourable epidemic situation do not contain the cholera toxin genes. The DNA hybridization method was compared with other methods used in research and practical work for estimation of epidemic importance of cholera vibrios.     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Die Suppression der außerkaryotisch bedingten Streptomycin-Abhängigkeit bei Chlamydomonas reinhardii

Zusammenfassung Die Streptomycin-Abhängigkeit der Mutante sd₃ wird außerkaryotisch vererbt. Nach Übertragung auf streptomycin-freies Medium entstehen in einer Häufigkeit von 10^-7 bis 10^-6 streptomycin-empfindliche Zellen. Diese Revertanten unterscheiden sich in verschiedenen Merkmalen von dem ebenfalls streptomycin-sensiblen Wildtyp. Die Reversion kommt also nicht durch Rückmutation zustande. Durch Kreuzungsexperimente konnte gezeigt werden, daß die Mutation, die zur Suppression der Streptomycin-Abhängigkeit führt, auf der extranucleären DNS stattgefunden hat.

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Suppression of the extranuclear streptomycin dependence in Chlamydomonas reinhardii

Summary The streptomycin-dependent mutant sd₃ shows uniparental inheritance. From their transfer on streptomycin-free medium streptomycin-sensitive cells result in a frequency of 10^-7 to 10^-6. These revertants differ in several marks from likewise sensitive wild-type cells. Therefore they were formed not by backmutation. Crossings showed, that also this suppression took place on the extranuclear DNA.

     

Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin

Orias, E. (University of California, Santa Barbara), and T. K. Gartner. Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin. J. Bacteriol. 91:2210-2215. 1966.-Streptomycin-induced suppression of amber and ochre rII mutants of phage T4 was studied in a streptomycin-sensitive strain of Escherichia coli and four nearly isogenic streptomycin-resistant derivatives of this strain, in the presence and in the absence of an ochre suppressor. Most of the 12 rII mutants tested were suppressed by streptomycin in the streptomycin-sensitive su(-) strain. This streptomycin-induced suppression in the su(-) strain was eliminated by the independent action of at least two of the four nonidentical mutations to streptomycin resistance. In two of the su(+)str-r strains, streptomycin markedly augmented the suppression caused by the ochre suppressor. In those su(-)str-r hosts in which significant streptomycin-induced suppression could be measured, the amber mutants were more suppressible than the ochre mutants.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.