Characterization of the nifA regulatory gene of Rhizonium leguminosarum PRE

P. Roelvink

Research output: Thesisinternal PhD, WU


<TT></TT><p><TT>This thesis describes the characterization of the <u>nif</u> A regulatory gene of the pea endosymbiont <u>Rhizobium</u><u>leguminosarum</u> PRE.</TT><p><TT>Chapter I gives a general overview on the regulation of nitrogen fixation in diazotrophs, with special focus on the regulatory NifA protein. The regulation of genes involved in nitrogen fixation in two bacteria is discussed in detail: the free living <u>Klebsiella</u><u>pneumoniae</u> and the endosymbiont of alfalfa <u>R</u> . <u>meliloti</u> . Major differences exist between these organisms where the onset of nitrogen fixation is concerned. <u>K</u> . <u>pneumoniae</u> has a general nitrogen regulatory circuitry which senses an internal biochemical signal i.e. the level of available ammonia as defined by the glutamine to 2-ketoglutarate ratio, a high ratio indicating a surplus, a low ratio a deficit. Sensing of a N-deficit results is translated, through a chain reaction of protein modifications, into activation of the regulatory NtrC product by phosphorylation. The resulting NtrC-P activates transcription of the regulatory <u>nif</u> LA operon, which encodes the inhibitor NifL and the activator NifA. The <u>Klebsiella</u> NifA thereupon activates transcription of the genes involved in nitrogen fixation. In a recently published paper David et al. (1988) suggest that the onset of nitrogen fixation in <u>R</u> . <u>meliloti</u> starts with the sensing of the external oxygen level. The FixL protein is hypothesized to sense a decrease in oxygen level. This protein is thus activated and in turn activates the FixJ protein, which directly or indirectly activates transcription of the <u>nif</u> A gene. The <u>Rhizobium</u> NifA protein activates transcription of the nitrogen fixation genes. In this overview we hypothesize that the oxygen sensing protein FNR instead of FixL senses the internal oxygen level. FNR then activates transcription of the <u>fix</u> LJ operon. The FixL protein may be a moderator of the activity of FixJ, comparable to the role of NtrB in activating NtrC. To date all rhizobial NifA proteins, in contrast to <u>Klebsiella</u> NifA, were shown to be oxygen sensitive. The structural analysis of the NifA protein is described and possible functions ascribed to domains identified in this protein are discussed. A model for NifA activity emerging from data presented for <u>K</u> . <u>pneumoniae</u> is</TT><br/>discussed. At present a complete model cannot be presented for <u>Rhizobiaceae</u> . The similarities and differences between the models for <u>K</u> . <u>pneumoniae</u> and Rhizobium species are discussed.<p>In Chapter 2 the DNA sequence and deduced amino acid sequence of <u>R</u> . <u>leguminosarum</u> PRE are presented. The amino acid sequence differs in 30 amino acids from that published for <u>R</u> . <u>leguminosarum</u> 3855 (Grönger et al., 1987). A possible explanation for this difference is discussed. The NifA Open Reading Frame (ORF) reveals two potential translation start sites, which in a heterologous <u>E.</u><u>coli</u> background appear to be used both. The second translation start, which leads to a 488 amino acids, 53 kD protein, is preferred over the first, which leads to a 519 amino acids, 56.1 kD protein. The <u>R</u> . <u>meliloti</u> (Weber et al., 1985, Buikema et al., 1985) and <u>B</u> . <u>japonicum</u><u>nif</u> A genes (Thöny et al., 1987) also have two translation start sites. It was shown for <u>R</u> . <u>meliloti</u> NifA (Beynon et al., 1988) the full length protein is the active form in an <u>E</u> . <u>coli</u> background. It is discussed that a translational preference for the second translational start site, leading to the inactive protein, as was found in pulse labeling experiments in <u>E</u> . <u>coli</u> may also exist in <u>Rhizobium</u> . We therefore suggest that the experiments presented by Beynon et al. (1988) are not conclusive as to the size of the functional protein in a <u>Rhizobium</u> background. Primer extension experiments and S <sub>1</sub> -nuclease protection were used to identify the putative <u>nif</u> A promoter. A transcription terminator was identified by S1-nuclease protection.<p>Chapter 3 deals with a phenomenon reported by Hawkins and Johnston (1988) and Roelvink et al. (1988). A <u>nif</u> A::Tn <u>5</u> mutant can not be complemented by a plasmid having only the <u>nif</u> A coding DNA fragment. A detailed analysis of the <u>nif</u> A- <u>nif</u> B intergenic region is presented. The <u>nif</u> A gene has a transcriptional terminator typical of bacterial genes (Brendel et al., 1986) consisting of a four GC basepairs stem and a nine base loop followed by a thymidine rich DNA stretch. This terminator was sapped by S1-nuclease protection. The <u>nif</u> B gene has a RpoN dependent promoter, having all nucleotides thought to be crucial to its activity. The <u>nif</u> A terminator was fused to the <u>Tet</u> - promoter and this fusion was cloned in a low copy<TT>transcriptional <u>lac</u> Z vector. The results show that the <u>nif</u> A terminator allows 85% readthrough. RNA::DNA hybridisation studies show that the <u>nif</u> A gene is transcribed at a level twice of that of <u>nif</u> B. By using a plasmid, which has a DNA region encompassing <u>nif</u> A, <u>nif</u> B and a ferredoxin like gene downstream of nifB (Grönger et al., 1988, Klipp et al., 1988) it was shown that <u>nif</u> A::Tn <u>5</u> mutants can be fully complemented. Taken together these findings suggest that the <u>nif</u> A and the nifB gene are in one operon. The failure of plasmids having the nifA encoding DNA fragment alone to complement a <u>nif</u> A::Tn <u>5</u> mutant results from a polar effect of the Tn <u>5</u> transposon on <u>nif</u> B transcription.</TT><p><TT>Chapter 4 deals with the <u>nif</u> H promoter region of <u>R</u> . <u>leguminosarum</u> PRE as one of the target sites of the NifA protein. We determined the nucleotide sequence of this region and identified a pseudo upstream activator sequence (UAS), a pseudo promoter, a consensus UAS and a consensus promoter. The promoter, mapped by primer extension experiments, differs from the consensus in one of the nucleotides thought to be invariant (see Gussin et al., 1986). The function of the <u>nif</u> H promoter elements was tested in a heterolo gous <u>E</u> . <u>coli</u> and a homologous <u>Rhizobium</u> background. Fusions of the <u>nif</u> H promoter region to <u>lac</u> Z, and fusions of deleted <u>nif</u> H promoter regions to <u>lac</u> Z, were used in activation studies by</TT><u>E</u> .<TT><u>pneumoniae</u> NifA in <u>E</u> . <u>coli</u> . Both high and low copy (deletion) <u>nif</u> H:: <u>lac</u> Z fusions were conjugated to <u>Rhizobium</u> . The activation study in an <u>E</u> . <u>coli</u> background showed that the pseudo UAS and the pseudo promoter are not involved in the function of the promoter. A different result was obtained with low copy <u>nif</u> H:: <u>lac</u> Z constructs in a <u>Rhizobium</u> background. The construct having both pseudo and consensus UAS, when compared with a construct having the consensus UAS only, seems to delay the onset of nitrogen fixation by three days. We suggest that this indicates that the presence of one or more UAS's modulates the expression of <u>nif</u> and <u>fix</u> genes, as was suggested for UAS's of <u>B</u> . <u>Japonicum</u><u>nif</u> and <u>fix</u> genes (Gubler and Hennecke, 1988). A <u>nif</u> promoter region holding a UAS, when cloned in a multi copy vector, can inhibit nitrogen fixation by capturing the NifA activator needed for expression of <u>nif</u> and <u>fix</u> genes. A multicopy Inhibition study with (deleted) <u>nif</u> H:: <u>lac</u> Z</TT><br/><TT>fusions led to a surprising finding: deletion of part of the consensus UAS on the multicopy plasmid did not result in inhibition of nitrogen fixation. The relevance of this finding is discussed. We conclude that <u>R</u> . <u>leguminosarum</u><u>nif</u> H can function without an UAS as was found for</TT><u>R</u> .<TT>meliloti nifH <u>in</u><u>planta</u> (Better et al., 1985). We suggest that NifA way form a complex with</TT>RpoN-RNAP<TT>that can bind directly at the promoter to activate transcription.</TT><p><TT></TT>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van Kammen, A., Promotor, External person
  • van den Bos, R.C., Promotor, External person
Award date30 May 1989
Place of PublicationS.l.
Publication statusPublished - 1989


  • nitrogen fixing bacteria
  • symbiosis
  • rhizobium
  • microorganisms
  • genetics
  • heritability
  • rhizobium leguminosarum


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