Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K12

Reiss, Jochen; Kleinhofs, A.; Klingmüller, Walter

doi:10.1007/bf00333594

Cited by 30 publications

(24 citation statements)

References 26 publications

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“…Mu is a polar mutagen which strongly decreases transcription from genes which lie downstream of the gene into which it has inserted (4). The (13). The cloned DNA which complemented all seven mutants, plasmid pJR1, was subcloned, and the smaller plasmid, pJR12, complemented only RK5203, RK5245, RK5216, and RK5221.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, we present data which support the hypothesis that chlN has both converting factor proteins but lacks a function needed to activate the smaller converting factor protein. Stewart (13,15) flbB5201 ayrA219 non-9 ptsF25 relAl rpsL150 RK5200 As RK4353 but chIA200::Mu cts V. Stewart (13,15) RK5203 As RK4353 but chlA203::Mu cts V. Stewart (13) RK5204 As RK4353 but chIA204::Mu cts V. Stewart RK5211 As RK4353 but chIA208::Mu cts V. Stewart RK5213 As RK4353 but chlA2J0::Mu cts V. Stewart RK5216 As RK4353 but chIA213::Mu cts V. Stewart (13) RK5217 As RK4353 but chIA2J4::Mu cts V. Stewart (13) RK5221 As RK4353 but chIA2J8::Mu cts V. Stewart ( (2,3).…”

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Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Pitterle

Rajagopalan

1989

J Bacteriol

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Molybdopterin (MPT) is not produced by the Escherichia coli mutants chlA1, chlM, or chlN or by the Neurospora crassa mutant nit-1. Extracts of E. coli chlA1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of N. crassa nit-1 into molybdopterin in vitro. In this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons [kDa]) which can be separated by using either anion-exchange or gel filtration chromatography. Neither protein is able to complement extracts of nit-1 by itself. Analysis of chlA Mu insertion mutants has shown that the two proteins are distinct gene products encoded at the chlA locus. Twelve chlA Mu insertion strains which lacked converting factor activity were deficient in one or both of the proteins. Converting factor activity could be generated by mixing extracts from strains having the 25-kDa protein with those having the 10-kDa protein but not those lacking both proteins. Finally, it was shown that the chlM mutant lacks the 10-kDa protein while the chlN mutant, which contains both the 10- and 25-kDa proteins, lacks a function required to activate the 10-kDa protein.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Pitterle

Rajagopalan

1989

J Bacteriol

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“…Biochemical analysis of some of these mutants confirmed that the mod mutation decreased the rate of molybdate transport and thus its accumulation by cells (9,16,38). By complementing these mutants, the wild-type genes coding for various components of the molybdate transport system have been isolated from E. coli (16,21,35). Johann and Hinton (21) determined the DNA sequence of an internal segment of the mod operon (modC gene) from E. coli.…”

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Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli

et al. 1995

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Although they are few in number, molybdoenzymes play an essential role in microbial metabolism. These enzymes (except dinitrogenase) contain a unique form of molybdopterin-nucleotide as the cofactor (33). In Escherichia coli, the main cofactor found in molybdoproteins (formate dehydrogenase, nitrate reductase, etc.) is molybdopterin guanine dinucleotide (33). The biosynthesis of molybdopterin guanine dinucleotide and thus active molybdoenzymes starts with the transport of molybdate into cells. Mutant strains which are defective in one molybdoenzyme, nitrate reductase activity, have been isolated from several microorganisms as chlorate-resistant strains (11,19,42). Pleiotropic molybdoenzyme-defective mutants whose phenotype can be suppressed by increasing the molybdate concentration in the growth medium were defined as transport-negative (mod; previously termed chlD [39]) mutants (11). By using this rationale, a fraction of chlorate-resistant mutants were identified as mod mutants (19,42). Biochemical analysis of some of these mutants confirmed that the mod mutation decreased the rate of molybdate transport and thus its accumulation by cells (9,16,38). By complementing these mutants, the wild-type genes coding for various components of the molybdate transport system have been isolated from E. coli (16,21,35). Johann and Hinton (21) determined the DNA sequence of an internal segment of the mod operon (modC gene) from E. coli. However, the complete DNA sequence of the E. coli mod operon is not available, although a large number of presumptive mod mutants of E. coli have been described. Besides E. coli, mod ϩ DNA was also isolated from Azotobacter vinelandii and Rhodobacter capsulatus and sequenced (27,43). Analysis of the mod DNA sequences from these organisms suggests that the transport of molybdate into cells is achieved by a typical periplasmic binding protein and an ATP-dependent transport system similar to the ones reported for other solutes, like sulfate, histidine, maltose, etc. (2,17,40,41).In this communication, the complete DNA sequence of the mod operon from E. coli is presented. By using cloned mod ϩ DNA, the mutations in a number of mod mutants were mapped within the mod operon. However, several chlorateresistant mutants initially identified as Mod Ϫ had unique phenotypic characteristics, and on the basis of complementation analysis, the mutation in these mutants was found to be outside the mod operon. MATERIALS AND METHODSBacterial strains. The bacterial strains used in this study are presented in Table 1 and are derivatives of E. coli K-12.Media and growth conditions. L broth which served as the rich medium was supplemented with glucose (0.3%; LBG) (25), formate, or molybdate as needed at the concentrations indicated for each experiment. Glucose-minimal medium and low-sulfur medium (LSM) have been described previously (26). For molybdate-free glucose-minimal medium, sodium molybdate (normally present at a final concentration of 40 M) was omitted. No attempt was made to remove contaminating molybdate ...

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“…This seems to be caused by disorder of the membrane when cloned genes are expressed and their gene products are inserted into the membrane. Although the chlD gene was cloned recently (18), all attempts to establish a cosmid with chlD and the adjacent genes have failed (30). In our experiments, we have used the cosmid vector pJE258 cop(ts).…”

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“…Cosmid pBK229 was introduced into several mutants carrying Mu cts integrations in genes chiA, chlB, chlE, and chlG (30). Only complementation of the chiA mutant was observed, since the chlA locus maps very close to chiD (approximate distance, 0.5 min).…”

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Isolation of Escherichia coli mutants defective in uptake of molybdate

et al. 1991

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For the study of molybdenum uptake by Escherichia coli, we generated TnSlac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants Al, A4, and M22 were finally selected for further analysis. Even in the presence of 100 ,uM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chiD mutant and the TnSlac-induced mutants. Certain subfragments of pBK229 which do not contain the chiD gene are still able to complement the TnSlac mutants. Mapping experiments showed that the TnSlac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the TnSlac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.In Escherichia coli, the trace element molybdenum is essential for a variety of bacterial enzymes including nitrate reductase (NR), formate dehydrogenase (FDH), and trimethylamine-N-oxide reductase (TOR).These enzymes are induced under anaerobic conditions and contain molybdenum bound to a low-molecular-weight cofactor (Mo-co) (19). The ICP-MS. Growth conditions. Bacterial cultures were grown at 37°C

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Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K12

Cited by 30 publications

References 26 publications

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli

Isolation of Escherichia coli mutants defective in uptake of molybdate

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