Single-Strand Breaks in Deoxyribonucleic Acid and Viability Loss During Deoxyribonucleic Acid Synthesis Inhibition in Escherichia coli

The effects of deoxyribonucleic acid (DNA) synthesis inhibition brought about in four different ways-thymidine starvation, nalidixic acid, hydroxyurea, and dnaB mutation-were examined in isogenic strains of Escherichia coli K-12. Three parameters were examined to determine whether there are strict correlations among them: (i) the extent of DNA synthesis inhibition; (ii) cell survival; and (iii) the rate of breakage of DNA molecules. There was no significant correlation between the extent of DNA synthesis inhibition and the rate of viability loss caused by the four DNA synthesis inhibitors, nor was there a strict correlation between the rate of occurrence of single-strand breaks in DNA and loss of viability. During treatment with hydroxyurea (0.1 M), no viability loss was observed and little, if any, single-strand breakage of DNA occurred. Both thymidine starvation and nalidixic-acid (20 mug/ml) treatment resulted in viability loss and breakage of DNA. For these latter two inhibitors, the two events appeared to be associated because greater rates of both viability loss and DNA breakage were observed for nalidixic acid compared with thymidine starvation. However, viability loss need not be associated with extensive breakage of DNA as demonstrated with a temperature-sensitive DNA synthesis mutant; at 39 C, viability loss occurred at a high rate without significant DNA breakage. With the other agents, the amount of DNA breakage accumulated when a cell population has sustained an average of one lethal hit was estimated to be about 30 single-strand breaks per genome. Differences in chromosomal and episomal breakage rates were observed.     

Quantification and Comparison of Soil Elements in the Tibetan Plateau Kaschin-Beck Disease Area

The prevalence rate of Kaschin-Beck disease (KBD) in most parts of China is currently decreasing, but the disease is still active and severe on the Tibetan Plateau. Soil samples including the surface layer (0–20 cm) and the subsurface layer (20–40 cm) in the cultivated and natural soil profiles were collected, and the mechanical composite and total concentration of arsenic, cobalt, copper, iron, mercury, manganese, molybdenum, selenium, and zinc were determined. Concentrations of arsenic, iron, manganese, copper, and selenium in the surface soil were lower than those in the subsurface soil. The same was true of physical clay in the soil profiles. However, there were no significant differences between the different soil layers. The concentrations of selenium, molybdenum, and mercury were somewhat lower compared with the average concentrations of soils in China. Deficiencies of molybdenum and selenium both play a critical role in occurrence of KBD, but whether or not soil mercury at a low level contributes to KBD is still unclear. A correlation analysis of soil elements showed that there is a positive correlation between iron, cobalt, and manganese due to their similar chemical characteristics. Selenium concentrations in soil as well as the physical clay and iron descend with the deterioration of KBD, but mercury concentrations in soil ascend with the aggravation of the disease. The results show that selenium deficiencies greatly influence the disease, and a deficiency of molybdenum is likely another important factor in inducing KBD. Moreover, determining whether low levels of soil mercury contribute to KBD should be investigated in the future.     

Tungstate can substitute for molybdate in sustaining growth of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to grow on media supplemented with tungstate rather than with molybdate. The Archaeon then synthesized a tungsten iron-sulfur isoenzyme of formylmethanofuran dehydrogenase. The isoenzyme was purified to apparent homogeneity and shown to be composed of four different subunits of apparent molecular masses 65 kDa, 53 kDa, 31 kDa, and 15 kDa and to contain per mol 0.4 mol tungsten, <0.05 mol molybdenum, 8 mol non-heme iron, 8 mol acid-labile sulfur and molybdopterin guanine dinucleotide. Its molecular and catalytic properties were significantly different from those of the molybdenum isoenzyme characterized previously. The two isoenzymes also differed in their metal specificity: the active molybdenum isoenzyme was only synthesized when molybdenum was available during growth whereas the active tungsten isoenzyme was also generated during growth of the cells on molybdate medium. Under the latter conditions the tungsten isoenzyme was synthesized containing molybdenum rather than tungsten.Abbreviations MFR methanofuran - CHO-MFR N-formylmethanofuran - MGD molybdopterin guanine dinucleotide - MAD molybdopterin adenine dinucleotide - MHD molybdopterin hypoxanthine dinucleotide - FPLC fast protein liquid chromatography - SDS/PAGE sodium dodecylsulfate/polyacrylamide gel electrophoresis - ICP-MS inductively coupled plasma mass spectrometry     

Effects of Iron Starvation on the Ultrastructure of the Cyanobacterium Agmenellum quadruplicatum

The effects of iron starvation on the ultrastructure of the unicellular cyanobacterium Agmenellum quadruplicatum were studied by using thin sectioning and transmission electron microscopy. Intracellular polysaccharide began to accumulate at the onset of iron limitation. This was followed by degradation of ribosomes and (later) degradation of the thylakoid membranes, both of which were virtually absent by 200 h. The thylakoids underwent structural modifications and rearrangements before they actually began to break down. Iron starvation did not appear to affect carboxysomes or the extracellular glyocalyx. On the other hand, polyphosphate bodies may have been partially degraded, and an electrontransparent gap eventually appeared between the cell wall and the cytoplasmic membrane. All of these changes were reversed when iron was added back to 200-h starved cultures. The sequence of ultrastructural changes observed during iron starvation clearly differed from those previously reported to occur during nitrogen, phosphorous, or carbon limitation.     

Iron starvation leads to oxidative stress in Anabaena sp. strain PCC 7120

We establish here that iron deficiency causes oxidative stress in the cyanobacterium Anabaena sp. strain PCC 7120. Iron starvation leads to a significant increase in reactive oxygen species, whose effect can be abolished by treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl). Oxidative stress induced by iron starvation could be a common feature of photosynthetic bacteria.     

AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency

Metal homeostasis is critical for the survival of living organisms, and metal transporters play central roles in maintaining metal homeostasis in the living cells. We have investigated the function of a metal transporter of the NRAMP family, AtNRAMP3, in Arabidopsis thaliana. A previous study showed that AtNRAMP3 expression is upregulated by iron (Fe) starvation and that AtNRAMP3 protein can transport Fe. In the present study, we used AtNRAMP3 promoter beta-glucoronidase (GUS) fusions to show that AtNRAMP3 is expressed in the vascular bundles of roots, stems, and leaves under Fe-sufficient conditions. This suggests a function in long-distance metal transport within the plant. Under Fe-starvation conditions, the GUS activity driven by the AtNRAMP3 promoter is upregulated without any change in the expression pattern. We analyze the impact of AtNRAMP3 disruption and overexpression on metal accumulation in plants. Under Fe-sufficient conditions, AtNRAMP3 overexpression or disruption does not lead to any change in the plant metal content. Upon Fe starvation, AtNRAMP3 disruption leads to increased accumulation of manganese (Mn) and zinc (Zn) in the roots, whereas AtNRAMP3 overexpression downregulates Mn accumulation. In addition, overexpression of AtNRAMP3 downregulates the expression of the primary Fe uptake transporter IRT1 and of the root ferric chelate reductase FRO2. Expression of AtNRAMP3::GFP fusion protein in onion cells or Arabidopsis protoplasts shows that AtNRAMP3 protein localizes to the vacuolar membrane. To account for the results presented, we propose that AtNRAMP3 influences metal accumulation and IRT1 and FRO2 gene expression by mobilizing vacuolar metal pools to the cytosol.     

SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly

Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.     

Iron Overload and the Predisposition of Cells to Antioxidant Consumption and Peroxidative Damage

We have investigated the effects of iron overload in vivo on the tocopherol levels and the extent of lipid peroxidation in rat liver microsomes and their response to subsequent oxidative stress in vitro. The results demonstrate a direct correlation between consumption of antioxidant defences and the induction and extent of malondialdehyde production in microsomes prepared from iron-loaded rats. The data are consistent with the requirement for iron (II)/iron (III) ratios in lipid peroxidation in control microsomes.     

Citrate as a siderophore in Bradyrhizobium japonicum.

Under iron-limiting conditions, many bacteria secrete ferric iron-specific ligands, generically termed siderophores, to aid in the sequestering and transport of iron. One strain of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, 61A152, was shown to produce a siderophore when 20 B. japonicum strains were screened with all six chemical assays commonly used to detect such production. Production by strain 61A152 was detected via the chrome azurol S assay, a general test for siderophores which is independent of siderophore structure. The iron-chelating compound was neither a catechol nor a hydroxamate and was ninhydrin negative. It was determined to be citric acid via a combination of thin-layer chromatography and high-voltage paper electrophoresis; this identification was verified by a specific enzymatic assay for citric acid. The inverse correlation which was observed between citric acid release and the iron content of the medium suggested that ferric citrate could serve as an iron source. This was confirmed via growth and transport assays. Exogenously added ferric citrate could be used to overcome iron starvation, and iron-deficient cells actively transported radiolabeled ferric citrate. These results, taken together, indicate a role for ferric citrate in the iron nutrition of this strain, which has been shown to be an efficient nitrogen-fixing strain on a variety of soybean cultivars.     

Globus pallidus: a target brain region for divalent metal accumulation associated with dietary iron deficiency

Recently, iron deficiency has been connected with a heterogeneous accumulation of manganese in the rat brain. The striatum is particularly vulnerable, for there is a significant negative correlation between accumulated manganese and gamma-aminobutyric acid levels. The effect of dietary iron deficiency on the distribution of zinc and copper, two other divalent metals with essential neurobiological roles, is relatively unexplored. Thus, the primary goal of this study was to examine the effect of manipulating dietary iron and manganese levels on the concentrations of copper, iron, manganese and zinc in five rat brain regions as determined with inductively coupled plasma mass spectrometry analysis. Because divalent metal transporter has been implicated as a transporter of brain iron, manganese, and to a lesser extent zinc and copper, another goal of the study was to measure brain regional changes in transporter levels using Western blot analysis. As expected, there was a significant effect of iron deficiency (P < 0.05) on decreasing iron concentrations in the cerebellum and caudate putamen; and increasing manganese concentrations in caudate putamen, globus pallidus and substantia nigra. Furthermore, there was a significant effect of iron deficiency (P < 0.05) on increasing zinc concentration and a statistical trend (P = 0.08) toward iron deficiency-induced copper accumulation in the globus pallidus. Transporter protein in all five regions increased due to iron deficiency compared to control levels (P < 0.05); however, the globus pallidus and substantia nigra revealed the greatest increase. Therefore, the globus pallidus appears to be a target for divalent metal accumulation that is associated with dietary iron deficiency, potentially caused by increased transporter protein levels.     

Physiological impact of acute molybdenum exposure in juvenile kokanee salmon (Oncorhynchus nerka)

A series of experiments were conducted to determine the physiological impact of acute sublethal molybdenum exposure to juvenile kokanee salmon (Oncorhynchus nerka Kennerlyi). Molybdenum was found to be relatively non-toxic to kokanee as the 96 h LC(50) was greater than 2,000 mg Mo l(-1). Exposure to either 25 or 250 mg Mo x l(-1) for 7 days was found to stimulate a significant 1.6- to 1.7-fold increase in ventilation which was later characterized to be dose-dependent between 5 and 250 mg Mo l(-1). Acute sublethal molybdenum exposure was found to have little or no impact on kokanee oxygen consumption at rest or immediately following a bout of forced activity or on physiological indicators of stress such as plasma lactate, sodium and cortisol. Despite these findings, prior exposure to 25 or 250 mg Mo l(-1) resulted in post-exercise loss of equilibrium and exercise-induced delayed mortality that were not observed in controls. Molybdenum accumulation in gill and liver of kokanee was also characterized. The findings of this study suggest that despite the non-toxic nature of molybdenum, acute sublethal exposure to this metal has physiological consequences to those fish exposed even for only a brief period. Further studies are needed to more fully elucidate the metabolism and mode of action of this metal in fish.