Partial characterization of a lysU mutant of Escherichia coli K-12

Hassani, Morad; Saluta, Maria V.; Bennett, George N.; Hirshfield, Irvin N.

doi:10.1128/jb.173.6.1965-1970.1991

Cited by 17 publications

(18 citation statements)

References 23 publications

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“…In Escherichia coli, a previously characterized lysine decarboxylase (EC 4.1.1.18) is encoded by cadA at 93.7 min and participates in the synthesis of cadaverine from lysine. This enzyme is inducible under anaerobic conditions at pH 5.5 and by adding lysine to the culture medium (7,19,23,27). Some evidence has been presented for the existence of a second, much less active, constitutive lysine decarboxylase in E. coli (6,30), but the data are still too incomplete to permit any definitive conclusions on the presence of this second enzyme.…”

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Characterization of a second lysine decarboxylase isolated from Escherichia coli

et al. 1997

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We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated ldc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.There are two types of bacterial amino acid decarboxylase, constitutive and inducible. The former type includes decarboxylases for L-ornithine, L-arginine, S-adenosyl-L-methionine, and diaminopimelic acid (27). In Escherichia coli, a previously characterized lysine decarboxylase (EC 4.1.1.18) is encoded by cadA at 93.7 min and participates in the synthesis of cadaverine from lysine. This enzyme is inducible under anaerobic conditions at pH 5.5 and by adding lysine to the culture medium (7,19,23,27). Some evidence has been presented for the existence of a second, much less active, constitutive lysine decarboxylase in E. coli (6, 30), but the data are still too incomplete to permit any definitive conclusions on the presence of this second enzyme. Igarashi et al. made several observations concurring with the suggestion that cadaverine is actually formed by ornithine decarboxylase, which would then account for the alleged constitutive lysine decarboxylase (8). The presence of constitutive lysine decarboxylase has not been detected in organisms, except for Selenomonas ruminantium, a strictly anaerobic gram-negative bacterium (12).We previously reported the overproduction of lysine by strain WC196, a lysine analog (S-aminoethyl-L-cysteine)-resistant strain of W3110 formed by N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenesis. During the construction of strains derived from this strain, we found that a cadA deletion mutant (WC196C) could still degrade lysine to cadaverine (15a). This finding suggested the existence of another lysine decarboxylase besides cadA in E. coli.Here, we report the existence of a new lysine decarboxylase gene, designated ldc, at 4.7 min on the E. coli chromosome, and we describe the lysine decarboxylase activities of strains containing cadA, ldc, or cadA ldc mutations. MATERIALS AND METHODSBacterial strains, bacteriophag...

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Characterization of a second lysine decarboxylase isolated from Escherichia coli

et al. 1997

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“…Additionally, LysRS was normally induced in ECOR strains EC10, EC16, EC40, and EC50, suggesting the presence of two LysRS genes in these strains as well. The two distinct fragment sizes for the two lysU homologs is consistent with the widely separated genomic locations of the lysS and lysU genes in E. coli K-12 (13,19,50 E. coli K-12 has been our paradigm for the study of the dual-gene LysRS system, and it is known that regulation of the lysU gene is complex and environmentally sensitive. Expression of lysU, but not that of lysS, is stimulated by nutritional or environmental factors such as L-leucine (23), L-leucine dipeptides (23,24), L-alanine (23,24), heat shock (21, 50), anaerobiosis (28), or growth to low pH in rich medium (18,20).…”

Section: Discussionmentioning

confidence: 79%

“…A critical tool for analyzing which restriction fragment from E. coli K-12 contained the lysU gene was strain GNB10181. There is no induction of the LysU protein in this strain, and it is suspected of harboring a deletion that includes the lysU gene (1,19). The chromosomal DNAs of GNB10181 and its isogenic parent, MC4100, were double digested with either BamHI-HindIII or BamHI-SalI, enzymes which do not cut into either the lysS or the lysU gene of E. coli K-12.…”

Section: Resultsmentioning

confidence: 99%

“…All of the bacterial strains used in this study are listed in Table 1. Strain GNB10181, an E. coli strain derived from MC4100 by Mud lac fusion, has been shown to be deficient in inducible lysine decarboxylase (the cadA gene product) (1), inducible LysRS activity (LysU), melibiose utilization (mel operon), and inducible arginine decarboxylase (adi) (19). Strain JA200/ pLC4-5 is a Clarke-Carbon colony bank strain (9) which has been shown to express LysU by maxicell analysis (50).…”

Section: Methodsmentioning

confidence: 99%

“…Studies on strains lacking the lysU gene indicate that it is not essential for growth of E. coli at temperatures in the range of 42 to 45ЊC (19,27). Examination of the duplicate LysRS gene system has been confined to E. coli K-12.…”

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The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes

Saluta

Hirshfield

1995

J Bacteriol

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The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS ؉ lysU ؉ ) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS ؉ ⌬lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginosa and B. megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.

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