Spermidine acetyltransferase is required to prevent spermidine toxicity at low temperatures in Escherichia coli

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In Escherichia coli, the speG gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of E. coli at low temperature was examined using a speG mutant. At 37 degrees C, growth of the speG mutant was normal in the presence of 0. 5 or 1 mM spermidine. However, following a shift to 7 degrees C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7 degrees C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg(2+) restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg(2+), resulting in ribosome inactivation at low temperatures.     

Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis

A specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N -acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli .     

Studies of the induction of spermidine/spermine N1-acetyltransferase using a specific antiserum

A specific antiserum to rat liver spermidine/spermine N1-acetyltransferase was used to study the induction of this protein. The antiserum had no effect on the spermidine acetylating capacity of crude nuclear extracts and very little effect on the activity present in crude cytosolic extracts from control rat tissues indicating that most of this activity is not due to spermidine/spermine N1-acetyltransferase. Treatment of rats with carbon tetrachloride, spermidine, thioacetamide, or methylglyoxal bis(guanylhydrazone) produced a substantial increase in the spermidine acetylating capacity of rat liver cytosolic extracts which was exclusively due to an increase in the immunoprecipitable spermidine/spermine N1-acetyltransferase protein. Exact measurement of the extent of this increase was not possible because the basal amount was too low to determine precisely but the amount of this enzyme increased about 250-fold with 6 h of treatment with carbon tetrachloride, about 25-fold at 6 h after spermidine, about 23-fold at 24 h after thioacetamide and up to 300-fold at 24 h after methylglyoxal bis(guanylhydrazone). Treatment of rats with spermidine also increased spermidine/spermine N1-acetyltransferase in other tissues including lung, kidney, and pancreas. The spermidine/spermine N1-acetyltransferase protein was found to turn over very rapidly with a half-life of about 15 min in thioacetamide-treated rats and 180 min after carbon tetrachloride.     

Kinetics of induction and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus

Plasmid-mediated chloramphenicol resistance in Staphylococcus aureus has been shown to involve acetylation of chloramphenicol by an enzyme induced by growth in the presence of the antibiotic and certain analogues. Analysis of the kinetics of induction has been complicated by (i) the intrinsic inhibitory effects of chloramphenicol on induced enzyme synthesis and (ii) the rapid disappearance of inducer after synthesis of the acetylating enzyme. The compound related to d-threo chloramphenicol which lacks a C(3) hydroxyl substituent (3-deoxychloramphenicol) is a potent inducer of chloramphenicol acetyltransferase but is ineffective as an antibiotic and is not a substrate for the enzyme. The availability of such a "gratuitous" inducer has simplified an analysis of the kinetics of induction of chloramphenicol acetyltransferase. The enzyme from induced bacteria has been purified to homogeneity and has been compared with the analogous enzyme present in E. coli which harbors a resistance transfer factor with the chloramphenicol resistance determinant.     

Identification of N1-acetylnorspermidine in Vibrio parahaemolyticus and an enzyme activity responsible for its formation

N1-Acetylnorspermidine [CH3CONH(CH2)3 NH(CH2)3NH3] was identified in Vibrio parahaemolyticus, which contains norspermidine as a major polyamine. This is the first example for the natural occurrence of monoacetylated unusual polyamine. The N1-acetylnorspermidine content was the highest 4 h after inoculation. Incubation of norspermidine and acetyl CoA with a cell extract from V. parahaemolyticus produced N1-acetylnorspermidine. A remarkable increase in specific activity of the acetyltransferase was observed at the exponential phase of growth. Spermidine also served as a substrate for the enzyme, with the formation of two isomers of the acetylspermidines (N1-acetylspermidine was predominant), but the reaction rate was less than 50% of that with norspermidine. These results suggest that norspermidine in V. parahaemolyticus may be associated with the cell growth and its role may be controlled through acetylation, as reported for spermidine in Escherichia coli.     

Temperature-sensitive Chloramphenicol Acetyltransferase from Escherichia coli Carrying Mutant R Factors

Bacteria carrying temperature-sensitive mutant R factors for chloramphenicol resistance were isolated. In the presence of chloramphenicol, these bacteria grew at 34 C but not at 43 C. The mutations in the chloramphenicol resistance gene of the R factors affected neither the resistance of the bacteria to dihydrostreptomycin and tetracycline nor the stability of the R factors at 43 C. The chloramphenicol acetyltransferase obtained from Escherichia coli K-12 carrying the mutant R factors was heat-labile as compared with that from a strain carrying the wild-type R factor. We could not find chloramphenicol acetyltransferase activity in 17 chloramphenicol-sensitive and 5 -resistant strains (selected in vitro) of E. coli examined. The results strongly suggest that the chloramphenicol resistance gene of the R factors is the structural gene of the chloramphenicol acetyltransferase rather than the genome controlling the expression of a chromosomal determinant for the enzyme. Furthermore, the studies confirm that the existence of the chloramphenicol acetyltransferase is the primary cause of chloramphenicol resistance of bacteria carrying the R factor. Both the enzyme activity producing the monoacetyl derivative from chloramphenicol and the subsequent formation of the diacetate from the monoacetyl product were heat-labile to the same degree. The results suggest that only one enzyme participates in two steps of chloramphenicol acetylation.     

Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine.     

Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.     

Identification of N1-acetylnorspermidine in Vibrio parahaemolyticus and an enzyme activity responsible for its formation

Abstract N ¹-Acetylnorspermidine [CH₃CONH(CH₂)₃NH(CH₂)₃NH₃] was identified in Vibrio parahaemolyticus , which contains norspermidine as a major polyamine. This is the first example for the natural occurence of monoacetylated unusual polyamine. The N ¹-acetylnorspermidine content was the highest 4 h after inoculation. Incubation of norspermidine and acetyl CoA with a cell extract from V. parahaemolyticus produced N ¹-acetylnorspermidine. A remarkable increase in specific activity of the acetyltransferase was observed at the exponential phase of growth. Spermidine also served as a substrate for the enzyme, with the formation of two isomers of the acetylspermidines ( N ¹-acetylspermidine was predominant), but the reaction rate was less than 50% of that with norspermidine. These results suggest that norspermidine in V. parahaemolyticus may be associated with the cell growth and its role may be controlled through acetylation, as reported for spermidine in Escherichia coli .     

Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C).     

Acetylation of polyamines in chicken brain and retina

Both spermidine and spermine are acetylated in chicken brain and retina. From spermidine, more N1-acetylspermidine than N8-acetylspermidine is formed by both the brain and the retinal cytosol. Km for spermidine is similar with the enzyme preparation of the two tissues, but that for spermine is lower with the retinal preparation. Both tissues contain an activity able to reduce spermidine acetyltransferase activity. Both alkaline phosphatase and cAMP-dependent protein kinase (catalytic subunit) are able to inactivate the spermidine acetyltransferase activity of both tissues. Spermidine acetyltransferase activity and polyamine levels have been measured in both brain and retina during embryonic life. Only in the last part of the development can enzyme activity be correlated with the retina spermidine and spermine concentration.     

Growth-phase-dependent expression of cspD, encoding a member of the CspA family in Escherichia coli.

The cspD gene of Escherichia coli encodes a protein of high sequence similarity with the cold shock protein CspA, but cspD expression is not induced by cold shock. In this study, we analyzed the regulation of cspD gene expression. By using a cspD-lacZ fusion and primer extension analysis, the expression of cspD was found to be dramatically induced by stationary-phase growth. However, this induction does not depend on the stationary-phase sigma factor sigmaS. Moreover, the expression of cspD is inversely dependent on growth rates and induced upon glucose starvation. Using a (p)ppGpp-depleted strain, we found that (p)ppGpp is one of the positive factors for the regulation of cspD expression.     

大肠杆菌N~α-乙酰基转移酶RimI对人胸腺素α原N末端乙酰化修饰的影响

目的:研究大肠杆菌Nα-乙酰基转移酶RimI对人胸腺素α原(ProTα)N末端乙酰化修饰的影响。方法:在大肠杆菌中共表达ProTα与大肠杆菌Nα-乙酰基转移酶RimI,同时设置只表达ProTα的对照菌,分别提取纯化ProTα,利用HPLC检测其发生乙酰化修饰的程度。结果:ProTα的乙酰化修饰发生在翻译后水平,有无RimI的过表达、诱导时间的长短以及这两个因素之间的交叉效应均对ProTα的乙酰化程度有影响,其F值分别为53.8、89.22及16.28,P值均小于0.0001;无论有无RimI过表达,乙酰化的ProTα所占比例在诱导6 h后逐渐升高(P<0.0001);在过表达Ri-mI的菌株中,乙酰化的ProTα在诱导12 h后占37.4%,而在无RimI过表达的菌株中占64.3%;RimI对ProTα乙酰化的抑制作用从诱导6 h后开始显现(P=0.0007)。结论:在大肠杆菌中,过量的Nα-乙酰基转移酶RimI可抑制人胸腺素α原的乙酰化修饰。     

Regulation of spermidine/spermine N1-acetyltransferase in L6 cells by polyamines and related compounds.

Exposure of rat L6 cells in culture to exogenous polyamines led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase. Spermine was more potent than spermidine in bringing about this increase, but in both cases the elevated acetyltransferase activity increased the cellular conversion of spermidine into putrescine. The N1-acetyltransferase turned over very rapidly in the L6 cells, with a half-life of 9 min after spermidine and 18 min after spermine. A wide variety of synthetic polyamine analogues also brought about a substantial induction of spermidine/spermine N1-acetyltransferase activity. These included sym-norspermidine, sym-norspermine, sym-homospermidine, N4-substituted spermidine derivatives, 1,3,6-triaminohexane, 1,4,7-triaminoheptane and deoxyspergualin, which were comparable with spermidine in their potency, and N1N8-bis(ethyl)spermidine, N1N9-bis(ethyl)homospermidine, methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-amino-guanidine ), which were even more active than spermidine. It is suggested that these polyamine analogues may bring about a decrease in cellular polyamines not only by inhibiting biosynthesis but by stimulating the degradation of spermidine into putrescine.     

Spermidine nuclear acetylation in rat hepatocytes and in logarithmically growing rat hepatoma cells: comparison with histone acetylation.

Spermidine acetylation has been studied in nuclear homogenates and in entire nuclei from rat hepatocytes and rat hepatoma tissue culture (HTC) cells, isolated at different stages of logarithmic growth, and compared to histone acetylation. Under all experimental conditions, N8-acetylspermidine was the predominant product of the reaction (90%). Unlike histone, spermidine acetylation in HTC cell and hepatocyte entire nuclei was almost absent or strikingly reduced relative to acetylation using nuclear homogenates as the enzyme sources. This was due to the lack of a free minor pool of spermidine, most likely lost during the purification of entire nuclei. Thus, preincubation of intact nuclei in the presence of spermidine restored activities to values observed using nuclear sonicates. Spermidine acetylation in HTC cell nuclei fluctuated moderately during cell growth, being stimulated immediately after initiation of proliferation and decreasing progressively as cultures reached high cell density. This pattern corroborated that of N8-acetylspermidine intracellular accumulation induced by culturing cells in the presence of 1 mM 7-amino-2-heptanone, a competitive inhibitor of N8-acetylspermidine deacetylase. Histone acetylation during HTC cell growth was not markedly different qualitatively from that of spermidine. Moreover, spermidine and histone acetylations in hepatocyte nuclei were of the same order of magnitude as those seen in rat hepatoma cell nuclei. Finally, inhibition of deacetylation of N8-acetylspermidine had no apparent deleterious effects on cell and growth. It remains to be determined whether the acetylation step is of higher physiological importance, in particular, and as discussed in nuclear spermidine turnover.     

Loss of expression of cspC, a cold shock family gene, confers a gain of fitness in Escherichia coli K-12 strains

The CspA family of cold shock genes in Escherichia coli K-12 includes nine paralogs, cspA to cspI. Some of them have been implicated in cold stress adaptation. Screening for mutations among common laboratory E. coli strains showed a high degree of genetic diversity in cspC but not in cspA and cspE. This diversity in cspC was due to a wide spectrum of variations including insertions of IS elements, deletion, and point mutation. Northern analysis of these mutants showed loss of cspC expression in all but one case. Further analysis of the loss-of-function cspC mutants showed that they have a fitness advantage in broth culture after 24 h over their isogenic wild-type derivatives. Conversely, introduction of mutated cspC alleles conferred a competitive fitness advantage to AB1157, a commonly used laboratory strain. This provides the evidence that loss of cspC expression is both necessary and sufficient to confer a gain of fitness as seen in broth culture over 24 h. Together, these results ascribe a novel role in cellular growth at 37 degrees C for CspC, a member of the cold shock domain-containing protein family.     

Polyamines and the Accumulation of Ribonucleic Acid in Some Polyauxotrophic Strains of Escherichia coli

The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA.Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.