Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

Fellay, R.; Frey, Joachim; Krisch, Henry

doi:10.1016/0378-1119(87)90041-2

Cited by 666 publications

(354 citation statements)

References 28 publications

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“…The EcoRV fragment containing most of nifLA was then replaced by a 2.1-kb HindIII cassette (made blunt ended with the Klenow fragment of DNA polymerase I) containing an ⍀ interposon fragment with a kanamycin resistance gene to yield plasmid pJES875. (The 2.1-kb HindIII cassette was derived from plasmid pHP45⍀-Km [13]. The ⍀ fragment is flanked in inverted orientation by transcriptional and translational termination signals [41].)…”

Section: Methodsmentioning

confidence: 99%

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Schmitz

Kustu

1996

J Bacteriol

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In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription by 54 -holoenzyme. In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen. In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 M) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogenlimiting conditions. Adding back Fe in only twofold molar excess over phenanthroline restores NifA activity, whereas adding other metals fails to do so. By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline. In the free-living diazotroph Klebsiella pneumoniae, expression of nitrogen fixation (nif) genes is regulated by the products of the nifLA operon (3,8,14,32). NifA activates transcription of all nif genes (except nifLA) by the alternative holoenzyme form of RNA polymerase 54 -holoenzyme. NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9). It catalyzes the ATP-dependent isomerization of closed complexes between 54 -holoenzyme and a nif promoter to transcriptionally productive open complexes (25,35). NifL, which is a negative regulator, inhibits NifA activity in vivo in response to molecular oxygen and/or combined nitrogen (22,32).NifL from K. pneumoniae is composed of two domains separated by a hydrophilic interdomain linker (Q-linker [12]). We showed recently that the C-terminal domain is sufficient to inhibit transcriptional activation by NifA in vitro and in vivo (37). Thus, the inhibitory function of NifL appears to lie in its C-terminal domain, which presumably interacts with NifA. The N-terminal domain of NifL contains a region of homology (ϳ30% amino acid identity over 130 residues) to the product of the bat gene from Halobacterium halobium (19,47,50). Since Bat is an oxygen-responsive activator of the synthesis of bacterio-opsin, it has been proposed that the region of homology may be involved in oxygen sensing. The mechanism(s) by which NifL senses oxygen and/or combined nitrogen is not understood.The N-terminal domain of NifL contains one CysXXCys motif (Cys-184-Ala-Asp-Cys-187). The similarity of this motif to sequences involved in binding metal clusters in proteins such as ferrodoxins and rubredoxins (1, 4, 24) suggested a possible metal-binding role of this region in NifL (21). On the basis of this prediction, Henderson et al. (21) examined the influence of metal ion deficiency on NifL activity. They concluded that iron (Fe) deficiency reduced the inhibitory activity of NifL expressed at high levels in Escherichia coli.To further study the function of a possible metal ion or iron-sulfur (Fe-S) cluster in oxygen-sensing by NifL, we examined the effect of Fe deficiency on NifL inhibition of NifA activity in vivo in a stra...

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Section: Methodsmentioning

confidence: 99%

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Schmitz

Kustu

1996

J Bacteriol

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“…coli plasmids used for cloning and vector construction were as follows: pBS8 (Spratt et al, 1986), pJRD16905 (this work), pSU19 and pSU20 (chloramphenicol resistance vectors derived from pSU27 19 and obtained from F. de la Cruz: Martinez et al, 1988); pT7-5 and pT7-6 were used to identify pCG 100-encoded polypeptides in E. coli (Tabor & Richardson, 1985). pHP45QCm (Fellay et al, 1987) was used as source of the f2 cartridge containing the chloramphenicol (Cm) resistance gene of pKT210. pJRD16905 and p13313 ( Fig.…”

Section: Escherichia Coli Nm522 (Supe Thi A(lac-proab) A(hsdms-mcrb)s F'mentioning

confidence: 99%

Structural organization of the Corynebacterium glutamicum plasmid pCG100

Trautwetter

Blanco

1991

Journal of General Microbiology

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pCG100, a 3 kb cryptic plasmid of Corynebacteriumgfutumicum ATCC 13058, probably identical with pSRl from C. gfutumicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.9 kb BgfII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a truns-acting replication factor. Derivatives of pCGl00 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCGl00 is compatible with pBE1, a cryptic plasmid of Brevibacterium factofermentum. Shuttle plasmid vectors, containing the kanamycinresistance gene from Tn903 or from Streptococcus faecdis as selectable markers and the AmpR, TetR or facZa genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.

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“…Earlier also, several workers have transformed toluene or other hydrocarbon degrading genes [33][34][35][36] in suitable target organisms. In an unrelated report, Fellay et al [37] verified the properties of omega transposon to mutagenize a broad-host range plasmid which contains the entire metacleavage pathway of the toluene degradation plasmid pWWO of P. putida. They showed that when the plasmid containing an omega transposon was transferred by conjugal mobilization from E. coli to R. leguminosarum or in few other bacteria, the appropriate interposon drug resistance was expressed.…”

Section: Discussionmentioning

confidence: 99%

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Goel¹,

Pandey

Sood³

et al. 2011

Indian J Microbiol

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Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT T ) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 9 10 -6 . DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.

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Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

Cited by 666 publications

References 28 publications

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Structural organization of the Corynebacterium glutamicum plasmid pCG100

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

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