Nelson Guerreiro

Proteome Analysis of Differentially Displayed Proteins As a Tool for the Investigation of Symbiosis

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

PROTEOME ANALYSIS OF THE MODEL MICROSYMBIONT SINORHIZOBIUM MELILOTI : ISOLATION AND CHARACTERISATION OF NOVEL PROTEINS

Sinorhizobium meliloti is an agriculturally and ecologically important microbe due to its capacity to establish nitrogen‐fixing symbiosis with plant legumes. Two‐dimensional gel electrophoresis of total cellular protein was used to establish a proteome reference map for the model microsymbiont Sinorhizobium meliloti strain 1021. The extent of changes in the gene expression of cells grown in a defined medium at different growth phases was established. After examination of over 2000 resolved protein spots, a minimum of 52 reproducible changes in protein expression levels were detected when early exponential phase cells were compared to late exponential phase cells. In contrast, induction of nodulation gene expression by the addition of the flavonoid luteolin to cells did not result in detectable changes in protein expression at either early or late exponential phase. N‐terminal microsequencing of eighteen unknown constitutive proteins plus four proteins, induced or up‐regulated in late exponential phase cells, allowed the identification of proteins not previously described in rhizobia. These included an amide‐binding protein, a putative hydrolase of the glyoxalase II protein family, a nucleoside diphosphate kinase, and a 5′‐nucleotidase. N‐terminal microsequencing was also valuable in revealing N‐terminal post‐translational processing and assigning a subcellular location to the analysed protein. Proteome analysis will provide a powerful analytical tool to complement the sequencing of the genome of strain 1021.

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA.

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.

Probing for pH-Regulated Proteins in Sinorhizobium medicae Using Proteomic Analysis

To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we have identified acid-activated gene transcription and now complement this approach by using a proteomic analysis to identify the changes that occur following exposure to acidity. Protein profiles of persistently or transiently acid-stressed S. medicae cells were compared to those grown in pH neutral, buffered media. Fifty pH-regulated proteins were identified; N-terminal sequences for 15 of these were obtained using the Edman degradation. Transient acid exposure downregulated GlnA and GlnK and upregulated a hypothetical protein. Continuing acid exposure downregulated ClpP, an ABC transporter, a hypothetical protein, a lipoprotein, the Trp-like repressor WrbA1 and upregulated DegP, fructose bisphosphate aldolase, GroES, malate dehydrogenase and two hypothetical proteins. These findings implicate proteolytic, chaperone and transport processes as key components of pH response in S. medicae.

Pleiotrophic Alterations in the Cellular Protein Synthesis of Tn5-Induced Rhizobium Mutants as Revealed by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) is currently unequalled in its capacity to separate individual components of complex protein mixtures, making it the method of choice for monitoring global changes in gene expression in response to defined conditions (Guerreiro et al, 1997). Using 2-DE we have examined the global changes in Rhizobium gene expression which occur in response to specific Tn5-induced mutations that lead to defective symbiotic phenotypes. Exopolysaccharides (EPS) of Rhizobium leguminosarum have been implicated to be involved in a complex series of interactions which lead to the establishment of an effective Rhizobium -legume symbiosis, but the precise role of EPS in this interaction is not well defined. The mutant ANU437, a derivative of R. l. bv. trifolli strain ANU794, contains a Tn5 insertion in pssA (also called pss4), produces very low levels of acidic exopolysaccharide and induces delayed formation of small non-nitrogen-fixing nodules on various clover species (Rolfe et al, 1996). Using 2-DE we have shown ANU437 to exhibit multiple differences in gene expression when compared with its wild-type parent. Such pleiotrophies would have been difficult to show via other methodologies. Twenty-one proteins in ANU437 were either newly-induced or up-regulated in their level of synthesis. Six of these proteins have been characterised by N-terminal sequencing, all were novel proteins except for one which showed a high similarity to a phosphogluconate dehydrogenase. These results have shown the PSSA protein, in addition to being a glycosyltransferase, may be directly or indirectly involved in influencing the expression of a complex series of genes whose levels of expression may be the actual contributing factor to the ineffective nodulation observed with exo− mutants.