N4-hydroxycytidine-a new mutagen of a base analogue type

N⁴-hydroxycytidine (N⁴-OHcyd)¹ is incorporated into nucleic acids of a cytidine-requiring strain of S.typhimurium 1045 and can act mutagenically. The reversion frequency of pyrG^? → pyrG⁺ is 10–20 fold higher than the spontaneous background. N⁴OHcyd-induced revertants show a strong inhibitory effect in the presence of N⁴OHcyd. The influence of N⁴OHcyd on cytidine metabolism is discussed.     

The N4-hydroxycytidine reduction system in toluenized cells of Salmonella typhimurium.

1. Enzymatic reduction of N4-hydroxycytidine to cytidine in Salmonella typhimurium is highly specific. The reaction occurs only at the nucleoside level. Free base or its 1-methyl analogue is not reduced. 2. The pH optimum shows a broad plateau with a maximum at pH 7.0. The apparent Km value, estimated in the toluene-treated cells, is 4.8 mM and Vmax 1.4 nmoles/min/mg of wet bacterial weight. The reaction is NADH-dependent, although in toluenized bacterial cells it can occure without addition of any exogenous factor.     

Effect of 4-azaleucine upon leucine metabolism in Salmonella typhimurium

dl-4-Azaleucine (5 x 3(-3)m) added to exponentially growing cells of Salmonella typhimurium resulted in an abrupt cessation of growth lasting 4 to 8 hr followed by a resumption of division. The transitory nature of inhibition was not due to the instability or modification of the analogue or to a derepression of leucine-forming enzymes. Of many compounds tested, leucine served most efficiently to reverse 4-azaleucine-induced inhibition. Inhibition of growth can be explained by the fact that 4-azaleucine inhibits alpha-isopropylmalate synthase, the first enzyme unique to leucine biosynthesis. The analogue was a poor inhibitor of both the transamination of alpha-ketoisocaproate to leucine and the charging of leucine to transfer ribonucleic acid. With a leucine auxotroph starved for leucine, the analogue was incorporated into protein specifically in place of leucine. Such incorporation was accompanied by the death of almost all of the cells.     

Redox potential-dependent nitrite metabolism by Salmonella typhimurium.

Salmonellae are generally resistant to the inhibitory effects of NaNO2. Removal of the lipopolysaccharide of Salmonella typhimurium by ethylenediaminetetraacetic acid pretreatment did not result in subsequent inhibtion of growth by NaNO2, indicating that lipopolysaccharide does not function to exclude NaNO2 from the cell. NaNO2 disappeared from the medium while the cells were growing, but, after stationary phase was reached, no further losses were observed unless the pH was maintained above 7.0. Similar losses were observed in a cell-free system if the redox potential of the medium was between -250 and -175 mV. If the disrupted cell suspension was first heated in a boiling water bath for 15 to 18 min, no NaNO2 loss was observed regardless of the redox potential. S. typhimurium is capable of metabolizing NaNO2, possibly by means of a nitrite-reducing enzyme function which is redox controlled.     

Redox potential-dependent nitrite metabolism by Salmonella typhimurium.

Salmonellae are generally resistant to the inhibitory effects of NaNO2. Removal of the lipopolysaccharide of Salmonella typhimurium by ethylenediaminetetraacetic acid pretreatment did not result in subsequent inhibtion of growth by NaNO2, indicating that lipopolysaccharide does not function to exclude NaNO2 from the cell. NaNO2 disappeared from the medium while the cells were growing, but, after stationary phase was reached, no further losses were observed unless the pH was maintained above 7.0. Similar losses were observed in a cell-free system if the redox potential of the medium was between -250 and -175 mV. If the disrupted cell suspension was first heated in a boiling water bath for 15 to 18 min, no NaNO2 loss was observed regardless of the redox potential. S. typhimurium is capable of metabolizing NaNO2, possibly by means of a nitrite-reducing enzyme function which is redox controlled.     

A mutagen assay detecting forward mutations in an arabinose-sensitive strain of Salmonella typhimurium.

Strain SV3 of Salmonella typhimurium is sensitive to arabinose, that is, unable to grow in a medium containing arabinose plus glycerol as carbon source. Arabinose resistance is the consequence of the mutational inactivation of one of at least three different genes. The selection of arabinose-resistant mutants provides a simple and sensitive assay for the detection of weak mutagens and for refined quantitative studies of strong ones. The assay is not influenced by experimental artifacts derived from physiological or lethal effects or from differences in plating density. Such artifacts are common with other bacterial mutagen assays, including those using strains analogous to SV3. As practical examples, the assay was used with N-methyl-N'-nitro-N-nitrosoguanidine and the fungicide captafol.     

Sterol hydroperoxide metabolism by Salmonella typhimurium

In order to rationalize multiphasic dose-response data evincing mutagenicity towards Salmonella typhimurium TA1537 for sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide their metabolism by the bacterial test strain was investigated. The 5 alpha-hydroperoxide was isomerized to the 7 alpha-hydroperoxide and reduced to 5 alpha-cholest-6-ene-3 beta,5-diol; the 7 alpha-hydroperoxide was reduced to cholest-5-ene-3 beta,7 alpha-diol and transformed to 3 beta-hydroxycholest-5-en-7-one. The 3 beta,5 alpha-diol and 3 beta,7 alpha-diol were not interconverted nor was either transformed to the 7-ketone.     

Effect of cyclopentaneglycine on metabolism in Salmonella typhimurium

Cyclopentaneglycine (CPG) inhibited the growth of wild-type Salmonella typhimurium. The inhibition was overcome by isoleucine or any isoleucine precursor formed after threonine. CPG appeared to mimic isoleucine as a strong inhibitor of the activity of l-threonine deaminase. The analogue was a poor inhibitor of isoleucyl-transfer ribonucleic acid synthetase. CPG did not appear to be incorporated into protein nor did it replace isoleucine in repression. Cells that had recovered from growth inhibition by CPG had derepressed levels of the isoleucine-valine biosynthetic enzymes.     

Peroxidative metabolism of benzidine derivatives by Salmonella typhimurium

Benzidine and several derivatives are activated to mutagenic species in an H2O2-dependent Ames test system. Optical and electron paramagnetic resonance (EPR) spectroscopy are employed in studies of the H2O2-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, and provide direct evidence for peroxidase activity in Salmonella typhimurium. The acetylase-proficient Ames tester strain TA98 and its acetylase-deficient derivative TA98/1,8-DNP6 are equally responsive to H2O2-dependent mutagenicity; enzymatic acetylation appears not to be involved in activation of benzidine, in this system. The H2O2-dependent mutagenicity of benzidine and oxidation of TMB are observed when the assays are carried out in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethane sulfonic acid (MES) buffer, at the same pH. This difference is interpreted in terms of the effects of these buffers on the intracellular pH of the bacteria. The H2O2-dependent mutagenicity of several benzidine congeners is also described.     

The role of ntrA in the anaerobic metabolism of Salmonella typhimurium

The possible involvement of NtrA in the expression of several anaerobically induced genes in Salmonella typhimurium was investigated. Unlike Escherichia coli, where hydrogenase 3 is ntrA dependent, the introduction of a mutation in ntrA had virtually no effect on the hydrogenase activity, thought to be hydrogenase 3, of S. typhimurium LT7. Fumarate reductase and alcohol dehydrogenase activities were found to be diminished in ntrA mutant strains, but this may very well be indirect since fdhF mutant strains showed the same effect. These results suggest that in S. typhimurium NtrA is highly specific for the anaerobic expression of fdhF.     

Hydroperoxidase I catalyzes peroxidative activation of 3,3'-dichlorobenzidine to a mutagen in Salmonella typhimurium

Dichlorobenzidine can be peroxidatively activated in Salmonella typhimurium Ames tester strains. Mutagenicity is observed when an S. typhimurium strain which is sensitive to frame-shift mutagens is incubated with dichlorobenzidine and hydrogen peroxide. In this paper, we show that the bacterial enzyme, hydroperoxidase I, is responsible for much of this activation. We constructed isogenic tester strains which lack hydroperoxidase I or II, due to Tn10 insertions in the structural genes encoding these proteins. Hydrogen peroxide-dependent mutagenicity of dichlorobenzidine was measured in each strain. A tester strain lacking hydroperoxidase I activity was much less sensitive than was the parent strain. When hydroperoxidase I activity was restored in this strain, via a plasmid-borne copy of the gene encoding the Escherichia coli protein, sensitivity to peroxide-dependent dichlorobenzidine mutagenicity was enhanced.     

Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium

The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT). Little is known about the ArnT protein structure because it has not previously been purified. We report here the first expression and purification of 6 x His-tagged S. typhimurium ArnT in NovaBlue cells. The enzyme was purified using sequential Q-Sepharose anion exchange and HisLink nickel affinity column chromatography. The purified protein has an apparent molecular weight of 62 kDa on SDS-PAGE and the identity of the purified ArnT was confirmed by Western blot using a monoclonal antibody against the His-tag and by MALDI-TOF mass spectrometry. Purified ArnT protein was shown to be highly alpha-helical as determined by circular dichroism analysis. A chromosomal ArnT knockout strain of E. coli BL21(DE3) was developed to allow in vivo functional analysis of plasmid-encoded ArnT constructs, and a polymyxin assay was used to confirm that the cloned ArnT proteins retained full activity. These studies provide an essential foundation for further analysis of ArnT structure and function using mutagenesis and biophysical techniques.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.     

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.