Hauke Hennecke

Potential Symbiosis-Specific Genes Uncovered by Sequencing a 410-Kilobase DNA Region of the Bradyrhizobium japonicum Chromosome

The physical and genetic map of the Bradyrhizobium japonicum chromosome revealed that nitrogen fixation and nodulation genes are clustered. Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis. Therefore, we determined the nucleotide sequence of a 410-kb region. The overall G+C nucleotide content was 59.1%. Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions. Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia. Sixteen percent were similar only to proteins of other bacteria. No database match was found for 29%. Repetitive DNA sequence-derived ORFs accounted for the rest. The sequenced region contained all nitrogen fixation genes and, apart from nodM, all nodulation genes that were known to exist in B. japonicum. We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources. Some of them are preceded by -24/-12 promoter elements. A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host.

One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes.

This report is concerned with the structural characterization and genetic regulation of new bacterial groES and groEL chaperonin genes, and presents two novelties. The first is the discovery that the nitrogen fixing soybean root nodule bacterium, Bradyrhizobium japonicum, unlike all other prokaryotes investigated so far, possesses a multigene family consisting of five very similar, though not identical, groESL‐like genes. The second novelty relates to the finding that these five homologues are expressed to different degrees and, in particular, that one family member (namely groESL3) is induced by a mechanism that does not involve the well‐known heat shock response. By contrast, the groESL3 genes are co‐regulated together with symbiotic nitrogen fixation genes, in that they are activated by the nitrogen fixation regulatory protein NifA at low oxygen conditions and transcribed from a −24/−12 promoter by the sigma 54 RNA polymerase. Two other members of the groESL gene family are apparently expressed constitutively at different levels, and yet another one is strongly induced by high temperature. As an attractive hypothesis it follows that B. japonicum may modulate its cellular contents of GroES‐ and GroEL‐like chaperonins in response to specific environmental conditions and physiological needs.

Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria

It was known that nitrogenase genes and proteins are well conserved even though they are present in a large variety of phylogenetically diverse nitrogen fixing bacteria. This has lead to the speculation, among others, that nitrogen fixation (nif) genes were spread by lateral gene transfer relatively late in evolution. Here we report an attempt to test this hypothesis.We had previously established the complete nucleotide sequences of the three nitrogenase genes from Bradyrhizobium japonicum, and have now analyzed their homologies (or the amino acid sequence homologies of their gene products) with corresponding genes (and proteins) from other nitrogen fixing bacteria. There was a considerable sequence conservation which certainly reflects the strict structural requirements of the nitrogenase iron-sulfur proteins for catalytic functioning. Despite this, the sequences were divergent enough to classify them into an evolutionary scheme that was conceptually not different from the phylogenetic positions, based on 16S rRNA homology, of the species or genera harboring these genes. Only the relation of nif genes of slow-growing rhizobia (to which B. japonicum belongs) and fast-growing rhizobia was unexpectedly distant. We have, therefore, performed oligonucleotide cataloguing of their 16S rRNA, and found that there was indeed only a similarity of SAB=0.53 between fast- and slowgrowing rhizobia.In conclusion, the results suggest that nif genes may have evolved to a large degree in a similar fashion as the bacteria which carry them. This interpretation would speak against the idea of a recent lateral distribution of nif genes among microorganisms.

Genome-Wide Transcript Analysis of Bradyrhizobium japonicum Bacteroids in Soybean Root Nodules

The transcriptome of endosymbiotic Bradyrhizobium japonicum bacteroids was assessed, using RNA extracted from determinate soybean root nodules. Results were compared with the transcript profiles of B. japonicum cells grown in either aerobic or microaerobic culture. Microoxia is a known trigger for the induction of symbiotically relevant genes. In fact, one third of the genes induced in bacteroids at day 21 after inoculation are congruent with those up-regulated in culture by a decreased oxygen concentration. The other induced genes, however, may be regulated by cues other than oxygen limitation. Both groups of genes provide a rich source for the possible discovery of novel functions related to symbiosis. Samples taken at different timepoints in nodule development have led to the distinction of genes expressed early and late in bacteroids. The experimental approach applied here is also useful for B. japonicum mutant analyses. As an example, we compared the transcriptome of wild-type bacteroids with that of bacteroids formed by a mutant defective in the RNA polymerase transcription factor sigma54. This led to a collection of hitherto unrecognized B. japonicum genes potentially transcribed in planta in a sigma54-dependent manner.

Localized Mutagenesis in Rhizobium japonicum

SummaryThe slow-growing soybean symbiont, Rhizobium japonicum, has not readily been accessible so far to classical mutational analysis of genes responsible for symbiotic nitrogen fixation. We have overcome part of this problem by the successful application of a site-directed mutagenesis technique to this organism. The following steps are involved: (i) local Tn5 mutagenesis, in E. coli, of cloned R. japonicum DNA (e.g. the nifDK operon); (ii) conjugational transfer of the mutated DNA into R. japonicum using vectors which are unable to replicate there; (iii) selection of R. japonicum exconjugants which have exchanged their wild-type genomic DNA region for the Tn5-containing fragment by homologous recombination. While using this technique it appeared mandatory to distinguish double-crossover-events (true replacements) from single-crossover events (replicon fusions or cointegrations). Only the true replacement mutants were genetically stable; their phenotypes were determined with respect to nodulation (Nod) and nitrogen fixation (Fix) by plant infection tests. Tn5 mutations within nifD and nifK caused a Nod+ Fix- phenotype, whereas mutants with insertions in the immediate vicinity on either side of nifDK were found to be Nod+ Fix+, suggesting that genes flanking nifDK may not be involved in the nitrogen fixing symbiosis. Nodule reisolates were found to carry Tn5 at their original locations.

Repressor properties of the nifL gene product in Klebsiella pneumoniae

SummaryCertain mutations in the nifL gene of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster resulted in altered nif regulaiton such that nitrogenase synthesis was no longer repressed by low levels of exogenous fixed nitrogen, by oxygen or by high temperature. Introduction of a plasmid with a nifL+ allele restored fixed nitrogen and oxygen repression. We therefore conclude that the nifL product acts as a nif-specific repressor in response to these effectors.Hence nif-specific regulation is controlled by the products of two regulatory genes, nifLA, which comprise a single operon. As previously reported (Dixon et al. 1980; Buchanan-Wollaston et al. 1981), the nifA product is necessary for transcription from other nif promoters but not from that of its own operon. We find that the nifL gene product also acts at other nif promoters but does not repress its own synthesis. Transcription of nifLA is in turn controlled by a general nitrogen-regulatory system in the cell, mediated by the products of the ntrA (glnF), ntrB (glnL) and ntrC (glnG) genes.

Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum.

Heat‐shock regulation was detected for three out of the five members of the groESL multigene family in Bradyrhizobium japonicum. The results uncovered the simultaneous presence of two distinct heat‐shock control systems which so far have not been reported to co‐exist in a single prokaryotic organism. The first system concerns groESL1 whose transcription is controlled in a σ32‐dependent manner similar to that known from work done with Escherichia coli. Heat‐shock control of groESL4 is mediated by the second system, which is characterized by an inverted‐repeat DNA structure originally described as a heat‐shock regulatory element (CIRCE) in Bacillus subtilis. This element represses expression of groESL4 under non‐stress conditions, as inferred from the increased expression of a groESL4′–′lacZ fusion suffering a 4 bp deletion within the CIRCE element. The two control systems clearly differ with respect to the temperature dependence and the kinetics of the heat‐shock response, and they also respond differently to the stress signal elicited by incorporation of the amino acid analogue p‐F‐phenylalanine into cellular protein. Knock‐out mutations in groEL4 resulted in an increased expression of groESL4, suggesting that repression via CIRCE depends, itself, upon the cellular level of GroEL4 protein.

Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids

It has previously been demonstrated that the unnatural amino acid p‐Cl‐phenylalanine can be attached to tRNAPhe by a modified phenylalanyl‐tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p‐Cl‐ or p‐Br‐phenylalanine into full‐length luciferase in vitro. The incorporation of p‐Cl‐phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery.

The Bradyrhizobium japonicum fixGHIS genes are required for the formation of the high-affinity cbb3-type cytochrome oxidase

Abstract We report structural and functional analyses of the Bradyrhizobium japonicumfixGHIS genes, which map immediately downstream of the fixNOQP operon for the symbiotically essential cbb3-type heme-copper oxidase complex. Expression of fixGHIS, like that of fixNOQP, is strongly induced in cells grown microaerobically or anaerobically. A fixGHI deletion led to the same prominent phenotypes as those known from a fixNOQP deletion: defective symbiotic nitrogen fixation (Fix–) and decreased cytochrome oxidase activity in cells grown under oxygen deprivation. Only traces, if any, of cytochrome cbb3 subunits were present in membranes isolated from the ΔfixGHI strain, as revealed by Western blot analysis with subunit-specific antibodies. This effect was not due to lack of fixNOQP transcription. The results suggested a critical involvement of the fixGHIS gene products in the assembly and/or stability of the cbb3-type heme-copper oxidase. On the basis of sequence similarities between the FixI protein and a Cu-transporting P-type ATPase (CopA) of Enterococcus hirae, and between FixG and a membrane-bound oxidoreductase (RdxA) of Rhodobacter sphaeroides, we postulate that a membrane-bound FixGHIS complex might play a role in uptake and metabolism of copper required for the cbb3-type heme-copper oxidase.

Assembly and Function of the Cytochrome cbb Oxidase Subunits in Bradyrhizobium japonicum

The Bradyrhizobium japonicum cbb-type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme.