María del Carmen Vargas

AniA Regulates Reserve Polymer Accumulation and Global Protein Expression in Rhizobium etli

Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::OmegaSm(r)/Sp(r) mutant CAR1, which is unable to synthesize poly-beta-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes beta-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.

Effect of aniA (Carbon Flux Regulator) and phaC (Poly-β-Hydroxybutyrate Synthase) Mutations on Pyruvate Metabolism in Rhizobium etli

The Rhizobium etli poly-beta-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source. The inactivation of aniA, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate. Pyruvate carboxylase (PYC) activity, pyc gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58. The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate. The possible relation between AniA, pyc transcription, and reduced-nucleotide levels is discussed.

Glutamine Synthetase II Constitutes a Novel Taxonomic Marker in Rhizobium etli and Other Rhizobium Species

Different Rhizobium species may be identified by using polymorphisms in their glutamine synthetases (GSII) but not by their GSI profiles. We analyzed the GSs of various Rhizobium tropici and Rhizobium etli strains (which are capable of nodulating and fixing nitrogen in Phaseolus vulgaris beans), as well as strains of other species included for comparison. The GS polymorphisms were determined by identifying variations in native enzyme mobility (revealed by GS activity staining) and in the isoelectric points of the monomers (revealed by immunodetection with antibodies against the GS proteins) by using gel electrophoresis. Restriction fragment length polymorphism patterns obtained by hybridizing an internal fragment of the GSII gene obtained from R. etli with total fragmented DNAs from different strains clearly distinguished the different groups. GSII is a novel and useful marker for Rhizobium groups and species, and GSII data support R. tropici and R. etli as bona fide species.

The naringenin-induced exoproteome of Rhizobium etli CE3

Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular “moonlighting” roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.

Thiamine limitation determines the transition from aerobic to fermentative‐like metabolism in Rhizobium etli CE3

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169::miniTn5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-beta-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.