In vitro insertional mutagenesis with a selectable DNA fragment

Prentki, Pierre; Krisch, Henry

doi:10.1016/0378-1119(84)90059-3

Cited by 1,644 publications

(1,000 citation statements)

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“…(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].) Plasmid pJES875 carries a deletion of nifLA that encodes only the first 113 amino acids of the N-terminal domain of NifL and the last 6 amino acids of the C terminus of NifA (2).…”

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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“…The fragment was inserted between the Hindlll and the Pstl sites of pUC19, generating pUC19-χ. A Hindlll fragment (2014 bp) that contains the Spc′ gene of R100.1 (Ω fragment; Prentki and Krisch, 1984) was then inserted, after treatment with Klenow enzyme, into the Hincll site of pUC19-χ, generating pUC19-χΩ. The Ω fragment does not contain χ.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Asai

Bates

Kogoma

1994

Cell

113

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SummaryHomologous recombination-dependent DNA replication (RDR) of a λ cos site-carrying plasmid is demonstrated in E. coli cells when the cells express λ terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot χ. χ is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the θ mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (or/Ms) on the chromosome.

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“…M12987). The aadA gene encodes resistance to streptomycin and spectinomycin (10). Partition functions are encoded by the sopABC genes.…”

mentioning

confidence: 99%

A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex.

Biek

Shi

1994

Proc. Natl. Acad. Sci. U.S.A.

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Stable maintenance of the low-copy-number mini-F plasmid in Escherichia coil is dependent on a functional partition system. The sop partition region encodes proteins SopA and SopB and a cis-acting element sopC, which contains multiple sites to which SopB binds. We have found that SopB protein acting at sopC in vivo is associated with a marked effect on plasmid DNA supercolling, which may reflect the formation of a wrapped nucleoprotein complex. In this study, we demonstrate that a functional partition complex can form with a single 43-bp SopB binding site. Our experiments suggest that SopB bound at a single site nucleates the binding of additional SopB protein and wrapping of adjacent DNA sequences, such that approximately equal numbers of supercoils are restrained regardless of the number of tandem sopC repeats present. It is likely that this unusual nucleoprotein complex allows interaction of the plasmid with the partition apparatus.

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In vitro insertional mutagenesis with a selectable DNA fragment

Cited by 1,644 publications

References 17 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

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex.

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