Involvement of cyclic AMP and its receptor protein in the sensitivity of Escherichia coli K 12 toward serine: excretion of 2-ketobutyrate, a precursor of isoleucine.

Summary A relationship between serine-induced growth sensitivity and the cAMP-CAP complex is established. Mutants of Escherichia coli K 12 deficient either in the cya or crp gene function exhibit a resistant phenotype on serine media although they harbor a relA allele normally leading to sensitivity toward serine. The presence of a crp ^* allele in a cya rela background restores the sensitivity phenotype, while the analysis of serine resistant mutants selected from a crp ^* cya relA strains shows that the mutation leading to resistance is located at, or very near, the crp gene, giving a more or less Crp^- phenotype. In addition crp ^* cya relA strains excrete large quantities of 2-ketobutyrate when grown on glucose M63 medium. This excretion is unambiguously linked to the presence of the crp ^* allele and is correlated with an enhanced threonine deaminase activity. Besides, the complex regulation exerted on the acetolactate synthase activities is discussed.     

Control of minicell producing cell division by cAMP-receptor protein complex in Escherichia coli.

Summary It has been established that the strain CA8000 of Escherichia coli K 12 produces minicells. This phenotype of CA8000 has been shown to be suppressed by additional mutations in cya or crp genes. Minicell production by cya ⁺ crp ⁺ min bacteria is probably a consequence of error, introduced by horizontal growth, in the selection of site on the envelope for initiation of hemispherical growth.     

Carbohydrate uptake genes inEscherichia coli are induced by carbon starvation

Escherichia coli produces several membrane-associated and periplasmic proteins in response to deprivation for a carbon source. The carbon starvation response involves a two- to fourfold, cAMP-dependent induction of operons involved in carbohydrate uptake and utilization, including thelac operon. Threecarbon-starvation-inducible (sci) gene fusions to aphoA reporter sequence were characterized. ThephoA-fusions werecya ⁺/crp ⁺-dependent and located in three previously characterized genes involved in high-affinity uptake of alternative carbon sources:mglB, encoding the periplasmic galactose binding protein;rbsB, encoding the periplasmic ribose binding protein; andlamB, encoding the maltodextrin-specific outer membrane porin.     

Resistance to mecillinam produced by the co-operative action of mutations affecting iipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium

Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high-level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of SpoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 μg ml^?1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K-12 is probably due to the natural LPS defectiveness of this strain.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

RecA, Tus protein and constitutive stable DNA replication inEscherichia coli rnhA mutants

Constitutive stable DNA replication (cSDR), which uniquely occurs inEscherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. TherecA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR inrnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations inrecB, recD, recJ, ruvA andruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR inrnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in theter region of the chromosome, was ruled out because introduction of thetus : :kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.     

Catabolite repression of the colonization factor antigen I (CFA/I) operon ofEscherichia coli

Experiments were performed to study whether the synthesis of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenicEscherichia coli is affected by glucose. The CFA/I-producing strain H-10407 (O78:H11:CFA/I) was grown in CFA medium containing various concentrations of glucose. Addition of 1% glucose into the medium resulted in a pronounced decrease in CFA/I production by H-10407 as assessed by ELISA, hemagglutination, and electron microscopy. The repressive effect of glucose was reversed by the addition of 10 mM cAMP to the medium. Examination of the promoter sequence of thecfaA gene of the CFA/I operon revealed a consensus binding site for the catabolite activator protein-cAMP complex. With a reporter plasmid containing a fusion of thecfaA promoter, a portion of thecfaA gene, and thelacZ gene, it was shown that the activity of this promoter was influenced by glucose. In a wild-typeE. coli strain, addition of 0.5% glucose to the growth medium diminished the promoter activity more than 70%. ThecfaA promoter also exhibited a lower level of activity incya (adenyl cyclase) andcrp (cAMP receptor protein) mutants than in the wild-type strain. The addition of 10 mM cAMP resulted in a marked increase in the expression from thecfaA promoter in thecya but not in thecrp mutant. These results suggest that the suppressive effect of glucose in the CFA/I system is mediated via the mechanism of catabolite repression through thecfaA promoter of the CFA/I operon.     

Cyclic AMP can replace the relA-dependent requirement for derepression of acetohydroxy acid synthase in E. coli K-12.

Derepression of the isoleucine and valine biosynthetic enzymes was strongly impaired in a relA strain of E. coli K-12 grown in an amino acid-glucose medium. The expression of the isoleucine and valine operons during leucine starvation was markedly defective in the relA mutant as compared to an isogenic rel⁺ strain. Downshift to a poor carbon and energy source or the addition of cyclic AMP to the glucose medium allowed normal derepression in the relA mutant of one of the isoleucine and valine enzymes, acetohydroxy acid synthase. The other isoleucine and valine enzymes failed to derepress under these conditions, in contrast to the high enzyme levels in the rel⁺ parent. No increase in acetohydroxy acid synthase was found in relA cya or relA crp strains during glycerol or pyruvate downshift. Cyclic AMP allowed derepression in the relA cya mutant but not in the relA crp strain. These data strongly suggest that the relA requirement for normal expression of acetohydroxy acid synthase can be replaced by cyclic AMP.     

Outer membrane proteins of Escherichia coli K-12: Isolation of a common receptor protein for bacteriophage T6 and colicin K

Summary tsx mutants, resistant to T6-like bacteriophages and colicin K, of Escherichia coli K-12 lack an outer membrane protein of 26,000 molecular weight. This protein is shown to have receptor activity for both bacteriophage T6 and colicin K. The protein has been purified and its amino acid composition determined. Some tsx mutants appear to have an altered receptor protein, as indicated by their ability to plate extended host-range mutants of bacteriophage T6. These mutants are also cotransducible with proC and can be arranged in an order of increasing resistance to the host range phages, which appear to have differing degrees of ability to propagate on the tsx mutants.The tsx protein was shown to be catabolite repressible both by use of varying growth conditions and cya and crp mutants.     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Pleiotropic mutations in appR reduce pH 2.5 acid phosphatase expression and restore succinate utilisation in CRP-deficient strains of Escherichia coli

Summary Several strains of Escherichia coli K12 were compared for activity of the periplasmic pH 2.5 acid phosphatase, an enzyme whose expression is regulated negatively by cyclic AMP. Two distinct enzyme levels differing by about four-fold were observed. This strain-dependent difference does not involve modifications in the structure of the enzyme, but results from a difference in its expression. We show that (1) strains with a high- or a low level of enzyme differ in the gene locus appR located in the 59 min region of the chromosome, a site remote from the structural gene appA; (2) the appR ⁺ versus appR enzyme ratio is 3–4 in wild-type strains, adenylate cyclase-deficient strains (cya) or cyclic AMP receptor protein-deficient strains (crp) grown in rich medium or in glucose minimal medium, but is close to 1 in cya strains in the presence of 0.1 mM cyclic AMP and in wild-type strains grown with succinate as carbon source; (3) in a crp genetic background, appR strains, contrary to appR ⁺ strains, are able to grow on minimal medium with succinate as the sole carbon source. The selection, from an appR ⁺ crp strain, of clones growing on succinateminimal medium. yielded mutations in the same region of the chromosome and showing the same phenotype as naturally-occurring appR strains.All appR strains analysed so far showed other similar deficiencies. The possibility that mutated appR gene products might function as weak substitutes for a functional cAMP-CRP complex is discussed.     

Genetic characterization of the secretory mutants ofParamecium caudatum

Summary Two trichocyst-nondischarge (TND) mutants ofParamecium caudatum, tndl andtnd2, are unable to discharge the trichocyst matrix (tmx) in response to chemical stimuli, although they contain many docked trichocysts at predetermined sites in the cortex. Freeze-fracture electron microscopy (FEM) of the plasma membrane showed thattndl possess two typical intramembrane particle arrays at the trichocyst docking site in the cortex, the outer ring and the inner rosette, as observed in wild-type (WT) cells, whereastnd2 possess the ring but not the rosette. The tmx of both TND mutants are able to expand when they are freed and exposed to an extracellular medium containing 1.5 mM Ca²⁺. When mutant cells were treated with ionophore A23187 and Ca²⁺, tmx-expansion took place intnd2, but not intndl cells. Thetnd2 mutant could be rescued by an injection of the WT cytoplasm and also by partial cell fusion during conjugation with the WT andtndl cells. However, the secretion capacity oftndl was not restored either by a microinjection of the WT cytoplasm or by the conjugating pair formation. Freeze-fracture electron microscopy on the double homozygote fortndl andtndl genes, revealed only the parenthesis instead of the ring and the rosette, indicating that trichocysts do not dock to the cortical site. Double mutation at thetndl andtndl loci caused a decrease in the number of the trichocysts at the cortical site. These results suggest that cooperative action of the two TND genes is necessary for stable docking of the trichocysts to the cortical sites.Abbreviations FEM freeze-fracture electron microscopy - IMP intramembrane particle - TD trichocyst discharge: tmx trichocyst matrix - TND trichocyst nondischarge - WT wild-type     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

Characterization of a hemin-storage locus ofYersinia pestis

Summary The pigmentation phenotype (Pgm⁺) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm^– phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm⁺ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms⁺) inY. pestis spontaneous Pgm^– strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms⁺ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm⁺, spontaneous Pgm^–, and Hms^– transposon strains, as well as a spontaneous Pgm^– strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms⁺ phenotype.     

Round-cell mutants of Salmonella typhimurium produced by transposition mutagenesis: Lethality of rodA and mre mutations

Summary Thirty-three insertions of transposon Tn10l6l7 into genes involved in the control of rod cell shape were isolated in Salmonella typhimurium by the characteristic glossy appearance of colonies composed of spherical cells. Genetic tests demonstrated that 25 (76%) were insertions in the rodA gene, 7 (21 %) were mre mutants, and 1 (3%) was a divD mutant. No insertion in the pbpA gene were found. Insertions in cell shape genes only appeared when strains displaying resistance to mecillinam (not caused by -lactamase production) were employed. Neither rodA nor mre insertions could be transduced to wild-type strains but they were normally accepted by mecillinam-resistant derivatives and by cya and crp mutants, which, unlike the corresponding Escherichia coli strains, did not display resistance to mecillinam. On the other hand, the divD insertion could be efficiently transduced to any strain. It is concluded that the rodA, mre, and divD genes are involved in the control of rod cell shape but, in addition, the RodA and Mre products perform some function(s) that is essential for wild-type cells but dispensable for some mecillinam-resistant strains, and for cya and crp mutants.