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::⍀Sm r /Sp r mutant CAR1, which is unable to synthesize poly-␤-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178: [1646][1647][1648][1649][1650][1651][1652][1653][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 ␤-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.Polyhydroxyalkanoic acids (PHAs) are a class of biodegradable plastics synthesized by many bacteria and thought to function as intracellular reserves of carbon and energy (2). Poly-␤-hydroxybutyric acid (PHB) is a type of PHA composed of polymerized 3-hydroxybutyrate units. The pathways for the biosynthesis of PHA have been studied in various bacteria, and the genes encoding the biosynthetic enzymes have also been investigated (40). The most common PHB-biosynthetic pathway consists of three enzymes, the first of which is a ␤-ketothiolase which condenses two molecules of acetyl coenzyme A (acetyl-CoA) to form acetoacetyl-CoA. The acetoacetyl-CoA is then reduced to D-(Ϫ)-3-hydroxybutyryl-CoA by an acetoacetyl-CoA reductase. Lastly, the D-(Ϫ)-3-hydroxybutyryl-CoA monomers are linked by PHB synthase. The genes encoding ␤-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase are designated phaA, phaB, and phaC, respectively.Rhizobium etli accumulates PHB both in symbiosis and in free life (7,14). PHB in rhizobia and other bacteria is thought to serve as a reserve of carbon and/or electrons to be utilized under suboptimal growth conditions (13,23). An R. etli PHBnegative mutant (CAR1) with an insertionally inactivated PHB synthase structural gene (phaC) was described previously. Physiological studies showed that CAR1 was unable to synthesize PHB and excreted more organic acids...

Rhizobium etli undergoes a transition from an aerobic to a fermentative metabolism during successive subcultures in minimal medium. This metabolic transition does not occur in cells subcultured in rich medium, or in minimal medium containing either biotin or thiamine. In this report, we characterize the aerobic and fermentative metabolism of R. etli using proteome analysis. According to their synthesis patterns in response to aerobic (rich medium, minimal medium with biotin or minimal medium with thiamine) or fermentative (minimal medium without supplements) growth conditions, proteins were assigned to five different classes: (i) proteins produced only in aerobic conditions (e.g., catalase-peroxidase KatG and the E2 component of pyruvate dehydrogenase); (ii) protein produced under both conditions but strongly induced in aerobic metabolism (e.g., malate dehydrogenase and the succinyl-CoA synthetase beta subunit); (iii) proteins that were induced equally under all conditions tested (e.g., AniA, DnaK, and GroEL); (iv) proteins downregulated during aerobic metabolism, and (v) proteins specific to only one of the conditions analyzed. Northern blotting studies of katG expression confirmed the proteome data for this protein. The negative regulation of carbon metabolism proteins observed in fermentative metabolism is consistent with the drastic physiological changes which occur during this process.

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.

The Rhizobium etli poly-␤-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.