The functional organization of the glnB-A cluster ofAzospiriUum brasilense, which codes for the PI, protein and glutamine synthetase, respectively, was studied with the aid of lacZ fusions, deletion mapping, site-directed mutagenesis, and complementation. It was shown previously by mRNA mapping that the cluster contains two tandemly organized promoters, glnBp1 and glnBp2, of the a7o and cr-4 types, respectively, upstream of glnB and a third unidentified promoter upstream of ginA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription ofglnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from ginBpp2 and a low level from ginAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, PI, polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that PI, plays an important role under these conditions. Null mutant strains of ginB, ntrB-ntrC, nifA, and point mutant strains in ginA were analyzed. NtrB and NtrC are not involved in the regulation of ginBA expression, in contrast to PII and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and PI, acts by modifying the properties of an unidentified regulator which might be a functional homolog of NtrC. In addition, a Nif-null mutant strain of glnB was characterized further. A Nif+ phenotype was restored to the strain by nifA from Klebsiella pneumoniae but not by nWfA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions inA. brasilense. It is therefore most likely that PI, is required for NiWA activation under conditions of nitrogen fixation. Deletion mapping and site-directed mutagenesis showed that glnAp was located within a 45-bp DNA fragment upstream of the mRNA start site, dissimilar to previously described consensus sites for cr factors.The nitrogen fixation (nif) genes are largely conserved among diazotrophs, and the requirement of a nifA product for their activation seems to be a general rule in proteobacteria (for recent reviews, see references 10 and 25). However, different regulatory pathways for the overall regulation of nitrogen metabolism have evolved in different groups of nitrogen-fixing bacteria.In the model system of Kiebsiella pneumoniae, ntrBC and ginA, which code for the two-component regulatory proteins NtrB and NtrC and glutamine synthetase (GS), respectively, are clustered. The PI1 protein, encoded by glnB, that maps elsewhere on the chromosome plays a central role by coordinating the response of the Ntr system to the intracellular nitrogen status (3,14,28,33

Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in Azospirillum brasilense Sp7, designated flcA. The deduced amino acid sequence of flcA shared high similarity with a family of transcriptional activators of the LuxR-UphA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in Pseudomonas aeruginosa. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the flcA coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that flcA controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by Azospirillum.

SummaryNumerous bacteria are able to use free and haemoprotein-bound haem as iron sources because of the action of small secreted proteins called haemophores. Haemophores have very high affinity for haem, and can therefore extract haem from the haemcarrier proteins and deliver it to the cells by means of specific cell surface receptors. Haem is then taken up and the empty haemophores are recycled. Here, we report a study of the regulation of the Serratia marcescens has operon which is involved in haemophoredependent haem acquisition. We characterized two genes encoding proteins homologous to specific ECF sigma and antisigma factors. We showed that they regulate the synthesis of the haemophore-specific outer membrane receptor, HasR, by a signal transduction mechanism similar to the siderophore surfacesignalling systems. We also showed the essential role of HasR itself in this process. Using haem-loaded and haem-free haemophore, we identified the stimulus for the HasR-mediated signal transduction as being the binding of the haem-loaded haemophore to HasR. Thus, unlike siderophore-uptake systems, in which the signalling molecule is the transported substrate itself, in the haemophore-dependent haem uptake system the inducer and the transported substrate are different compounds.

The coexistence of two different P II proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding P II ) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [ 32 P]phosphate or [ 3 H]uracil labeling or by cross-reaction with an anti-A. brasilense P II -antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second P II protein, named P Z , was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of P II . Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that P II and P Z are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.Azospirillum brasilense is a plant growth-promoting rhizobacterium that associates with the roots of grasses and fixes nitrogen under microaerobic conditions in the free-living state (33). As in all proteobacteria, the transcription of nif (nitrogen fixation) genes proceeds from 54 (NtrA or RpoN)-dependent promoters with upstream activator sequences that are the binding target of the transcriptional activator NifA (reviewed in reference 10). In contrast, the regulation by nitrogen of both the expression and activity of NifA differs considerably from that described for other free-living diazotrophs. In A. brasilense, NifA is synthesized in an inactive form under conditions incompatible with nitrogen fixation (24), and NtrC is not essential for the regulation of nifA expression (23).A. brasilense possesses a glutamine synthetase (GS) encoded by glnA (5). GS activity is modulated by reversible adenylylation as in enteric bacteria (8,19). However, the regulation of glnA expression has some unusual features. The glnA gene is adjacent to glnB, the structural gene for the P II protein. P II plays an essential role in regulation of nitrogen metabolism in Escherichia coli by controlling the level and activity of GS (reviewed in references 27 and 32). The transcription of the glnB-glnA cluster in A. brasilense depends on three different, selectively used and nitrogen-regulated promoters (11) and does not involve the two-component regulatory system NtrBNtrC (12). A glnB null mutant strain does not have impaired ammonia assimilation but is strictly Nif Ϫ (1...

SummaryIn Gram-negative bacteria, the TonB -ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonB SM , and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonB SM and HasB are significantly similar and can replace each other for haem acquisition. However, TonB SM , but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonB SM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonB EC for haem acquisition. As the change affects a domain involved in specific TonB EC -ExbB EC interactions, HasB may be unable to interact with ExbB EC , and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonB EC -dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.

The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.

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