Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen.

For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule. In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region. The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively. The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene. This mutation resulted in an Inf- phenotype in bean plants.

Identification and characterization of hupT, a gene involved in negative regulation of hydrogen oxidation in Bradyrhizobium japonicum.

The Bradyrhizobium japonicum hupT gene was sequenced, and its gene product was found to be homologous to NtrB-like histidine kinases. A hupT mutant expresses higher levels of hydrogenase activity than the wild-type strain under hydrogenase-inducing conditions (i.e., microaerobiosis plus hydrogen, or symbiosis), whereas in noninduced hupT cells, hupSL expression is derepressed but does not lead to hydrogenase activity. We conclude that HupT is involved in the repression of HupSL synthesis at the transcriptional level but that enzymatic activation requires inducing conditions.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative s54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense.

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum.

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion. The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.

Nucleotide sequence analysis of four genes, hupC, hupD, hupF and hupG, downstream of the hydrogenase structural genes in Bradyrhizobium japonicum.

The nucleotide sequence of a 2.2 kb region downstream of the hydrogenase structural genes in Bradyrhizobium japonicum was determined. Four genes encoding predicted polypeptides of 27.8 (HupC), 21.4 (HupD), 10.6 (HupF) and 15.8 (HupG) kDa were identified, of which the first three probably belong to the same operon as the hup structural genes, hupS and hupL. HupC is homologous to the hydrophobic polypeptides with four potential transmembrane regions that are encoded by open reading frames following the hydrogenase structural genes in Rhodobacter capsulatus, Escherichia coli, Azotobacter vinelandii, Wolinella succinogenes, Rhizobium leguminosarum and Alcaligenes eutrophus. Also HupD, HupF and HupG are homologous to genes involved in processing, maturation, functioning and regulation of hydrogenase activity in various hydrogen-oxidizing bacteria.

Identification of a potential transcriptional regulator of hydrogenase activity in free-living Bradyrhizobium japonicum strains.

In Bradyrhizobium japonicum, Tn5 insertions in a particular chromosomal DNA fragment result in a Hup- phenotype in free-living conditions without affecting hydrogenase (Hup) activity in the symbiotic state. By determination of the nucleotide sequence of this region, we were able to identify the nature of the inactivated genes. The fragment is located 9 kb downstream of the hydrogenase structural genes and contains one incomplete and three complete open reading frames. They are designated hypD', hypE, hoxX and hoxA respectively, since the deduced amino acid sequences display very strong homology with genes involved in the regulation of hydrogenase activity in Escherichia coli, Rhodobacter capsulatus, Azotobacter vinelandii (hypD' and hypE) and Alcaligenes eutrophus (hoxX and hoxA). This is the first report on transcriptional activators of the hup genes in B. japonicum. Implications of these findings with respect to regulation of hydrogenase synthesis by hydrogen, oxygen and nickel in free-living B. japonicum are discussed.

Hydrogenase in Bradyrhizobium japonicum: genetics, regulation and effect on plant growth.

A region of 15 kbp DNA located on the chromosome of Bradyrhizobium japonicum is essential for a Hup(+) phenotype and contains the hydrogenase structural genes. During the last few years, other genes with various functions related to hydrogenase oxidation, such as Ni(2+) incorporation into the hydrogenase enzyme, electron transport from hydrogenase to O2, and regulation of hydrogenase expression in free-living conditions, have been identified in this region. A region in front of the hydrogenase structural genes is necessary for transcriptional regulation of hydrogenase expression by O2, H2 and Ni(2+). In addition, the N2 fixation system and the tertiary structure of the chromosome seem to be involved in the expression of the hydrogenase genes. The effect of legume inoculation with Hup(+) rhizobia has been evaluated. The possible benefits of a Hup(+) phenotype, such as the regeneration of chemical energy in the form of ATP or reductants, and the removal of O2 and H2 from the active site of the nitrogenase enzyme where they might inhibit the nitrogenase reaction, are discussed. The data indicate that a H2-uptake system is beneficial in soybean nodules, but host plant and environmental factors may interfere with the effects of H2 cycling in the plant.

Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37.

We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A. tumefaciens T37. Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition.