Defective guanine uptake in an 8-azaguanine-resistant mutant of Salmonella typhimurium

An 8-azaguanine-resistant mutant, azg-11, derived from a guanine auxotroph, gua-1, of Salmonella typhimurium was isolated. This mutant was resistant to the analogue when grown on 2,6-diaminopurine, but showed greater susceptibility than the parent on guanine. Studies with the uptake of radioactive purines revealed that the mutant was defective in a mechanism for incorporation of guanine as well as of xanthine. Initial rates of uptake were determined for guanine at concentrations which were sufficiently low to make permeases limiting. The affinity constant K(m) for the mutant was found to be 2.5 x 10(-4)m; that of the parent was 2.3 x 10(-5)m. Examination of cell-free extracts suggested that the purine nucleotide pyrophosphorylases, responsible for the conversion of free intracellular purines to the corresponding nucleotides, were present and unaltered. The results indicate that the mutant is defective in a mechanism for the active transport for guanine and possibly xanthine.     

Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced. Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 muM respectively, compared to 60 muM and 45 muM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib-PP synthetase activity was observed in both strains, although to a lesser extent in the mutant. Our data suggest that the mutant harbors a mutation in the structural gene for PRib-PP synthetase. The mutation responsible for the altered PRib-PP synthetase was located in the purB-hemA region at 26 min on the recalibrated linkage map.     

Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.     

Growth responses to nutrients of an auxotroph and a prototroph of a predatory fungus

The growth responses of a prototroph and an auxotroph of Arthrobotrys dactyloides were compared, on several nutrient media. The prototroph can utilize a variety of nitrogenous compounds almost equally, but the auxotroph has an exacting requirement for several amino acids together. Both strains utilize sucrose best among the three sugars tested as a C-source but the prototroph responds strongly to phospholipid, in addition. They differ in their needs for purines, pyrimidines and B vitamins.     

Development and application of an assay for uranyl complexation by fungal metabolites,including siderophores

An assay to detect UO(2)(2+) complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe(3+) dye and the other containing a CAS-UO(2)(2+) dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO(2)(2+) chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.     

Comparative pathogenicity of auxotrophic mutants of Candida albicans

An induced mutant of Candida albicans with greatly decreased virulence for mice is described. The mutant was one of five auxotrophic mutants obtained by ultraviolet irradiation of a clinical isolate (strain MY 1044). The five mutants included two methionine auxotrophs, one methionine-cysteine auxotroph, one temperature-sensitive serine auxotroph, and one auxotroph with unknown growth requirements. Each of the mutants produced normal mycelium and had a normal profile of susceptibility to four antifungal drugs. The virulence of each mutant was compared with the parent strain by LD50 determination in mice. Four of the five auxotrophs exhibited LD50's that were not significantly different from the parent strain (mean LD50 = 7.5 x 10(5) cells). However, the temperature-sensitive serine auxotroph was significantly less virulent than the parent strain (LD50 greater than 10(7) cells), even though it grew well in vivo and in mouse serum at 37 degrees C in vitro. Use of this mutant in conjunction with its "isogenic" parent should help to elucidate true virulence factors in C. albicans.     

Photobactin: a catechol siderophore produced by Photorhabdus luminescens,an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1 nematodes

The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.     

Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5'-phosphate biosynthesis

Bacillus subtilis synthesizes pyridoxal 5'-phosphate, the active form of vitamin B(6), by a poorly characterized pathway involving the yaaD and yaaE genes. The pdxS (yaaD) mutant was confirmed to be a strict B(6) auxotroph, but the pdxT (yaaE) mutant turned out to be a conditional auxotroph depending on the availability of ammonium in the growth medium. The PdxS and PdxT proteins copurified during affinity chromatography and apparently form a complex that has glutaminase activity. PdxS and PdxT appear to encode the synthase and glutaminase subunits, respectively, of a glutamine amidotransferase of as-yet-unknown specificity essential for B(6) biosynthesis.     

Isolation and characterization of guanine auxotrophs in Neurospora crassa

An ad-9 strain of Neurospora crassa was mutagenized with ethylmethane sulfonate (5%) and selected for guanine auxotrophy. The resultant double adenine plus guanine mutant was backcrossed with wild type and a single guanine auxotroph was isolated from the progeny. In vitro assays indicated that the mutant had GMP synthetase activity comparable with wild type, but was completely lacking of IMP dehydrogenase activity. The guanine requirement can therefore be explained by the mutant's inability to convert IMP to XMP. Another guanosine auxotroph was able to adapt and grow on minimal medium after 3 days. This mutant had GMP synthetase activity comparable with wild type but had only 10% of the IMP dehydrogenase activity of wild type, which may possibly explain its ability to grow on minimal medium after 3 days. It was confirmed that the two isolates are not allelic by crossing the two and recovering 25% wild-type progeny. Out isolate must therefore be designated gua-2.     

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.     

Photobactin: a Catechol Siderophore Produced by Photorhabdus luminescens, an Entomopathogen Mutually Associated with Heterorhabditis bacteriophora NC1 Nematodes

The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.     

Role of siderophore in iron uptake in cowpea Rhizobium GN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Abstract Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.     

Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity

Under conditions of iron limitation Pseudomonas fluorescens ATCC 17400 produces two siderophores, pyoverdine, and a second siderophore quinolobactin, which itself results from the hydrolysis of the unstable molecule 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Pseudomonas fluorescens ATCC 17400 also displays a strong in vitro antagonism against the Oomycete Pythium, which is repressed by iron, suggesting the involvement of a siderophore(s). While a pyoverdine-negative mutant retains most of its antagonism, a thioquinolobactin-negative mutant only slowed-down Pythium growth, and a double pyoverdine-, thioquinolobactin-negative mutant, which does not produce any siderophore, totally lost its antagonism against Pythium. The siderophore thioquinolobactin could be purified and identified from spent medium and showed anti-Pythium activity, but it was quickly hydrolysed to quinolobactin, which we showed has no antimicrobial activity. Analysis of antagonism-affected transposon mutants revealed that genes involved in haem biosynthesis and sulfur assimilation are important for the production of thioquinolobactin and the expression of antagonism.     

Isolation of auxotrophic mutants from acetobacter methanolicus MB 58

The lysine content of the biomass of the acidophilic facultatively methylotrophic bacterium Acetobacter methanolicus MB 58 was increased by genetic manipulations. A homoserine auxotroph, MB 58.196, and a threonine auxotroph, MB 58.195, were obtained from Acetobacter methanolicus MB 58 by N-methyl-N′-nitro-N-nitrosoguanidine treatment. Investigations of enzyme activities revealed that the homoserine auxotroph lacks homoserine dehydrogenase activity, and the threonine auxotroph lacks homoserine kinase activity. Concerning the lysine-producing ability, only the homoserine auxotrophic mutant accumulates lysine in the intracellular pool. The intracellular lysine content of this mutant was increased 40-fold. An excretion of amino acids into the medium was not detected. A homoserine resistant mutant, MB 58.196.10, isolated from MB 58.196 by UV-irradiation, was able to excrete lysine. About 95% of free lysine were found in the culture medium. Altogether, the free lysine concentration was increased 800-fold in comparison to the wild-type strain. By these genetic manipulations the total lysine concentration of MB 58.196 was increased to 2.7% and of MB 58.196.10 to 56% in comparison to the wild-type strain.     

Urea: An intermediate of aerobic and anaerobic purine degradation in Rhodopseudomonas capsulata

Abstract A mutant strain ( pur ^-) defective in utilization of purines was isolated from Rhodopseudomonas capsulata . In the mutant, the loss of purine utilization correlated with urease deficiency. In contrast to the wild-type strain, the mutant catalyzed release of urea from purines. The nitrogen of the purine ring was completely liberated as urea indicating that the latter compound is an intermediate of the purine degradation pathway in Rps. capsulata . The degradation pattern was identical under aerobic and anaerobic conditions.     

结合缺陷型菌株显色法采用离子束诱变筛选高产L-精氨酸突变株

为快速高效筛选L-精氨酸高产突变株,建立一种缺陷菌株平板显色法并采用低能N+离子束对L-精氨酸生产用菌株钝齿棒杆菌SYPA5-5进行诱变处理,通过上述平板显色法筛选获得高产突变株.对突变株进行摇瓶发酵实验,最终选育出一株L-精氨酸产量较高且产酸性能比较稳定的突变菌株钝齿棒杆菌SYPA5-5-36.该菌株摇瓶发酵L-精氨酸产量可达35.85 g/L,比出发菌株提高了19.5％.因此,缺陷型菌株平板显色法可以用于快速、高效筛选高产L-精氨酸突变株.     

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.