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.     

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.     

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.     

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.     

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.     

Catabolite-Like Repression of Extracellular Enzyme Production in Vibrio parahaemolyticus

Production of extracellular amylase and protease in Vibrio parahaemolyticus was repressed by various carbohydrates present in the medium. In addition, the protease production was repressed very strongly by peptones or casamino acids. Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) added exogenously could reverse the repression of amylase production, but not that of protease production irrespective of the “repressors” used. Mutants of V. parahaemolyticus, which resembled the reported cya (adenylate cyclase) and crp (cyclic AMP receptor protein) mutants of Escherichia coli and related organisms, were examined for the exoenzyme production. Amylase production in the mutants was defective, while their protease production was not defective, but rather accentuated as compared with that in the parental strain. These findings strongly suggest that amylase production is subject to catabolite repression mediated by cyclic AMP, whereas protease production is controlled by a repression mechanism which mimics in part, but may be distinct from catabolite repression.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.     

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     

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.     

The Effect of galE Gene Inactivation on Lipopolysaccharide Profile of Helicobacter pylori

The galE gene product, UDP-galactose 4-epimerase, mediates the incorporation of galactose in extracellular polysaccharide materials such as the O-side chain of lipopolysaccharide (LPS). The O-side chain in H. pylori LPS has been shown to cross-react with Lewis x and/or y blood group antigens, suggesting its potential involvement in H. pylori-linked autoimmune disease. To study its role in H. pylori LPS biosynthesis, the galE gene was cloned, sequenced, and a galE-knockout H. pylori strain was constructed. The H. pylori galE gene encoded a protein of 344 amino acids with a molecular weight of 39K. The LPS profile from the galE-knockout H. pylori strain showed a lower molecular weight than that of the parental strain, indicating the involvement of the galE gene in LPS biosynthesis of H. pylori. Received: 15 December 1997 / Accepted: 10 March 1998     

The Multifunctional Protein YdiV Represses P Fimbria-Mediated Adherence in Uropathogenic Escherichia coli

YdiV, a degenerate EAL domain protein, represses motility by interacting with FlhD to abolish FlhDC interaction with DNA. Here, we demonstrate that deletion of ydiV dysregulates coordinate control of motility and adherence by increasing adherence of Escherichia coli CFT073 to a bladder epithelial cell line by specifically increasing production of P fimbriae. Interestingly, only one of the two P fimbrial operons, pap_2, present in the genome of E. coli CFT073 was upregulated. This derepression of the pap_2 operon is abolished following deletion of either cya or crp, demonstrating cyclic AMP (cAMP)-dependent activation of the P fimbrial operon. However, the absence of YdiV does not affect the gene expression of cya and crp, and loss of SdiA in the ydiV mutant does not affect the derepression of the pap_2 operon, suggesting that YdiV control of adherence acts in response to cAMP levels. Deletion of ydiV increases motility by increasing expression of fliA, suggesting that in E. coli CFT073, YdiV regulates motility by the same mechanism as that described previously for commensal E. coli strains. Furthermore, analysis of site-directed mutations found two putative Mg²⁺-binding residues of four conserved YdiV residues (E29 and Q219) that were involved in regulation of motility and FliC production, while two conserved c-di-GMP-binding residues (D156 and D165) only affected motility. None of the four conserved YdiV residues appeared to affect regulation of adherence. Therefore, we propose a model in which a degenerate EAL, YdiV, utilizes different domains to regulate motility through interaction with FlhD and adherence to epithelial cells through cAMP-dependent effects on the pap_2 promoter.     

Catabolite repression in Escherichia coli mutants lacking cyclic AMP.

Summary The regulation of catabolite repression of -galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (cya ), and thus unable to synthesize cyclic AMP. It has been found that, provided a second mutation occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression. If the catabolite repression seen in the mutant strains corresponds to the mechanism operating in wild-type cells, the results would suggest that the intracellular concentration of cyclic AMP cannot be the unique regulator of catabolite repression.Jacques Monod was still with us when most of the work described in this and the following paper was accomplished. His constant interest, his unfailing advice, his warm support, were invaluable. It will be difficult for us to ever enjoy a successful experiment without regretting that he cannot share this pleasure with us.     

Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.     

Unaffected nodulation and nitrogen fixation in carbohydrate pleiotropic mutants ofRhizobium meliloti

Pleiotropic mutants were isolated fromRhizobium meliloti M5N1 with the resistance transposon Tn5. They failed to use carbohydrates as carbon source, but not dicarboxylic acids. Diauxic growth properties of the wild type and uptake assays in both M5N1 and a mutant strain 2.10 showed that the mutant was affected in the active transport of sugars. Exogenous cyclic AMP failed to affect the phenotype of the mutant, and a relatively high frequency of reversion was observed. This suggests that the mutant 2.10 was probably altered in acrp-like gene. However, its symbiotic properties remained unaffected. Thus, symbiosis does not depend on sugar utilization.     

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.     

Identification of a novel structural motif in the lipopolysaccharide of the galE/galK double mutant of Haemophilus influenzae strain Eagan

Defined mutants of the galactose biosynthetic (Leloir) pathway were employed to investigate lipopolysaccharide (LPS) oligosaccharide expression in Haemophilus influenzae type b strain Eagan. The structures of the low-molecular-mass LPS glycoforms from strains with mutations in the genes that encode galactose epimerase (galE) and galactose kinase (galK) were determined by NMR spectroscopy on O- and N-deacylated and dephosphorylated LPS-backbone, and O-deacylated oligosaccharide samples in conjunction with electrospray mass spectrometric, glycose and methylation analyses. The structural profile of LPS glycoforms from the galK mutant was found to be identical to that of the galactose and glucose-containing Hex5 glycoform previously identified in the parent strain [Masoud, H.; Moxon, E. R.; Martin, A.; Krajcarski, D.; Richards, J. C. Biochemistry1997, 36, 2091-2103]. LPS from the H. influenzae strain bearing mutations in both galK and galE (galE/galK double mutant) was devoid of galactose. In the double mutant, Hex3 and Hex4 glycoforms containing di- and tri-glucan side chains from the central heptose of the triheptosyl inner-core unit were identified as the major glycoforms. The triglucoside chain extension, β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, identified in the Hex4 glycoform has not been previously reported as a structural element of H. influenzae LPS. In the parent strain, it is the galactose-containing trisaccharide, β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, and further extended analogues thereof, that substitute the central heptose. When grown in galactose deficient media, the galE single mutant was found to expresses the same population of LPS glycoforms as the double mutant.