Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia

Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO₃⁻-inducible. NH₄⁺ was the initial repressor signal for the uptake process which was involved in the control of the NO₃⁻inducible reductase system.     

Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.     

Nitrite metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrite uptake and nitrite reductase by ammonia

Abstract Nitrogen regulation of nitrite uptake and nitrite reductase was studied in the cyanobacterium Anabaena cycadeae and its glutamine-auxotrophic mutant. The development of the nitrite-uptake system preceded, and was independent of, the development of nitrate reductase. The levels of both of the systems were higher in the glutamine auxotroph lacking glutamine synthetase (GS) than in the wild-type strain having normal GS activity. The nitrite-uptake system was found to be constitutive and ammonia-repressible whereas the nitrite-reductase system was ammonia-repressible and nitrite-inducible. Ammonia did not inhibit the nitrite-uptake and nitrite reductase activities in the glutamine auxotroph whereas glutamine did so, suggesting that repression of nitrite-uptake and nitrite reductase systems by ammonia requires the operation of GS and probably involves the participation of some organic nitrogen metabolites like glutamine.     

Regulation of urea uptake by ammonia in the cyanobacterium Anabaena doliolum

Abstract The urea uptake system was studied with regard to its repression and derepression in the cyanobacterium, Anabaena doliolum . The uptake of urea was energy-dependent and was repressed in ammonia grown cells. Repression of the urea uptake by ammonium did not require ammonium assimilation or de novo protein synthesis, suggesting that ammonium itself was the repressor signal. The derepression of the urea uptake system, however, required de novo protein synthesis and glutamine synthetase activity.     

Studies on the ribosomal RNA operons of Listeria monocytogenes

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165(2). Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.     

Role of glutamine synthetase activity in the uptake and metabolism of arginine and proline in the cyanobacterium Anabaena cycadeae

Abstract The uptake of arginine and proline and their assimilation as nitrogen source have been studied in the cyanobacterium Anabaena cycadeae and its glutamine auxotropic mutant lacking glutamine synthetase activity. The uptake pattern of arginine and proline was found to be biphasic in both wild-type and mutant strains, consisting of an initial fast phase lasting up to 60 s followed by a slower second phase. The uptake activities of both the amino acids were also found to be similar in both the strains. The wild-type strain, having normal glutamine synthetase activity, utilized arginine and proline as sole nitrogen source, whereas the mutant strain lacking glutamine synthetase activity could not do so. These results suggest that: (1) glutamine synthetase activity is necessarily required for the assimilation of arginine and proline as nitrogen source, but it is not required for the uptake of these amino acids; and (2) glutamine synthetase serves as the sole ammonia-assimilating enzyme as well as glutamine-forming route in heterocystous cyanobacteria.     

Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae

Growth and regulation of heterocyst and nitrogenase by fixed nitrogen sources were studied comparatively in parent and glutamine auxotrophic mutant of Anabaena cycadeae. The parent strain grew well on N2, NH+4 or glutamine while the mutant strain grew on glutamine but not on N2 or NH+4. The total lack of active glutamine synthetase in the mutant strain thus appears to be the reason for its observed lack of growth in N2 or NH+4, which explains why it is a glutamine auxotroph and at the same time shows glutamine synthetase to be the sole primary ammonia assimilating enzyme. NH+4 repression of heterocyst and nitrogenase in the mutant and the parental strains and their derepression by L-methionine-DL-sulfoximine suggest that NH+4 per se and not glutamine synthetase mediated pathway of ammonia assimilation is the initial repressor signal of heterocyst and nitrogenase in A. cycadeae.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup

Abstract An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37°C but not at 28°C. Cellular adherence properties of DS92, which belonged to enteropathogeci serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid. The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E. coli strains including the reference EAggEC strain 17-2. Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086.     

Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal

Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.     

Nitrogen regulation of urease synthesis in Saccharopolyspora erythraea ATCC 11365

Abstract Era is an essential GTP-binding protein of an unknown function in Escherichia coli . On the basis of its sequence similarities to other GTP-binding proteins such as E. coli EF-Tu, EF-G, IF2 and eukaryotic Ras proteins, it has been suggested that the Era function is activated by GTP binding, and that subsequent conversion of bound GTP to GDP by the intrinsic GTPase activity modulates its function. Two Era mutants, one dominant negative mutant (dE), which has a deletion mutation from Ala40 to Gly49, and the other non-functional mutant (T42A/T43A), which has two substitution mutations, Thr42 to Ala and Thr43 to Ala, were analyzed for their abilities of GTP-binding and GTPase activity. It was found that the dE mutant lost the GTP-binding ability, while it still retained the GTPase activity. On the other hand, the T42A/T43A mutant retained both the GTP-crosslinking and GTPase activities. However, the K _m values for GTPase activity increased 5-and 12-fold for dE and T42A/T43A mutants, respectively. These results indicate that both the GTP-binding and GTPase activities are important for the Era function.     

Evidence for direct repression of nitrogenase by ammonia in the cyanobacterium Anabaena cylindrica

The nitrogenase activity of the cyanobacterium Anabaena cylindrica was repressed upon addition of ammonium salts after preincubation in the presence of a concentration of L-methionine-DL-sulfoximine sufficient to totally inhibit glutamine synthetase. Repression was also observed when urea was added to cells in the presence of the glutamine synthetase inhibitor. Measurements of ammonia concentrations were made in each case and provided evidence that ammonia itself is a primary regulator of nitrogenase levels in A. cylindrica.     

Aerobactin production and dulcitol fermentation in clonal groups of Escherichia coli O1: K1 strains from urinary tract infections

Abstract 70 urinary Escherichia coli O1:K1 strains were characterized for O1 antigen factors, mannose-resistant hemagglutination of human erythrocytes, flagellar and fimbrial antigens, dulcitol fermentation and aerobactin production. On the basis of their O1 and H antigens the strains could be assigned to 6 distinct groups. The most prevalent groups were: O1abcd: H⁻ :F9 (33 strains; pattern II), O1abc: H⁻ :F11 (9 strains; pattern IV), and O1abc: H7: F11 (19 strains; pattern V). Strains with patterns IV and V, both expressing fimbrial antigen F11, fermented dulcitol and produced aerobactin, whereas strains with pattern II were negative for both characteristics.     

Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子

从江苏、江西、安徽等7个省疑似黄、白痢直肠棉拭及病死猪的十二指肠和肠系膜淋巴结中分离鉴定出339株病原性大肠杆菌。经O血清型鉴定,除77株未能定型、41株自凝外,测定出221个分离株的O血清型,这些分离株覆盖了64个血清型,以O107、O101、O20、O93、O11和O149为主,共99株,占定型菌株的44.80%。这些血清型与已报道的常见血清型间存在一定差异。运用黏附素单抗对以上菌株进行F4、F5、F6、F18、F41　5种黏附素检测,共97个分离株表达黏附素(28061%),而表达两种和3种黏附素的菌株分别有22株和8株,它们分别占表达黏附素菌株的22.68%和8.25%,其中单独表达F4、F6、F5+F41黏附素菌株分别有18、30、15株,分别占表达黏附素菌株的18.56%、30.93%和15.46%;同时运用多重PCR对其中145个分离株进行毒素基因(Sta、STb、LT、SLT2e)的检测,拥有Sta和STb毒素基因的菌株分别占检测菌株的51.72%和3724%。F6、F4、F5+F41和Sta、STb为该地区致初生仔猪腹泻大肠杆菌常见的毒力因子。     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.     

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.