Rhizobium etli accumulates poly-␤-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an ⍀-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, ␤-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD ؉ /NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.Poly-␤-hydroxybutyrate (PHB) and other polyhydroxyalkanoates (PHA) are accumulated by a wide range of bacteria as carbon and reductive-power storage compounds. Several species belonging to the genera Rhizobium, Bradyrhizobium, and Azorhizobium accumulate PHB in free life (40, 43) and in symbiosis (16,23,29,48). In contrast, in other species, such as Rhizobium meliloti, the accumulation of PHB is observed only in the free-living state or in the first steps of nodule development but never in nitrogen-fixing bacteroids (20). The physiological role of these compounds in symbiosis is not completely understood. It is known that bacteroids of Bradyrhizobium japonicum may accumulate PHB and fix nitrogen simultaneously, although both functions require large amounts of reductive power (48). Bergersen et al. (1) proposed that PHB reserves in bacteroids can support some nitrogen fixation during darkness and prolong the period of nitrogen fixation. Bacteroids can also use PHB as a source of energy and reductive power for nitrogen fixation when incubated, ex planta, at a low oxygen concentration (2). In Rhizobium etli, PHB is accumulated not only in the stationary phase, like in other bacteria, but also during exponential growth. Moreover, PHB is being synthesized and degraded continuously even under conditions in which none of the polymer accumulates (10). This suggests the presence of a very sensitive regulatory mechanism that controls the accumulation or degradation of PHB, thus allowing rapid modulation of the levels of reductive power and of oxidizable substrates. This situation is especially favorable in organism...

Strains of Rhizobium etli, Rhizobium meliloti, and Rhizobium tropici decreased their capacity to grow after successive subcultures in minimal medium, with a pattern characteristic for each species. During the growth of R. etli CE 3 in minimal medium (MM), a fermentation-like response was apparent: the O 2 content was reduced and, simultaneously, organic acids and amino acids were excreted and poly-␤-hydroxybutyrate (PHB) was accumulated. Some of the organic acids excreted into the medium were tricarboxylic acid (TCA) cycle intermediates, and, concomitantly, the activities of several TCA cycle and auxiliary enzymes decreased substantially or became undetectable. Optimal and sustained growth and a low PHB content were found in R. etli CE 3 when it was grown in MM inoculated at a low cell density with O 2 maintained at 20% or with the addition of supplements that have an effect on the supply of substrates for the TCA cycle. In the presence of supplements such as biotin or thiamine, no amino acids were excreted and the organic acids already excreted into the medium were later reutilized. Levels of enzyme activities in cells from supplemented cultures indicated that carbon flux through the TCA cycle was maintained, which did not happen in MM. It is proposed that the fermentative state in Rhizobium species is triggered by a cell density signal that results in the regulation of some of the enzymes responsible for the flux of carbon through the TCA cycle and that this in turn determines how much carbon is available for the synthesis and accumulation of PHB. The fermentative state of free-living Rhizobium species may be closely related to the metabolism that these bacteria express during symbiosis.In aerobic bacteria, the tricarboxylic acid (TCA) cycle functions to generate reduced nucleotides by the complete oxidation of pyruvate, which enters the cycle in the form of acetyl coenzyme A (acetyl-CoA). The reduced nucleotides are then used to generate ATP via the electron transport system. Another major function is to produce intermediates for anabolism, and several anaplerotic reactions serve to replenish TCA cycle intermediates which are consumed in these processes (32).The accumulation of the microbial reserve polyester poly-␤-hydroxybutyrate (PHB) in bacteria is well documented (1,6,45), as is the presence of PHB in several species of Rhizobium, both in the free state (44, 46) and in symbiosis (14,19,48). While several different pathways for the production of PHB in various groups of bacteria have been characterized (1), the most common pathway begins with the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. Sequential reduction and polymerization reactions produce PHB. This product can be depolymerized and ultimately converted back to acetyl-CoA (Fig. 1). Like the TCA cycle, carbon flux through this pathway is greatly influenced by growth conditions, and the two cycles must compete for a common starting metabolite, acetyl-CoA. However, the function of PHB in cell metabolism in Rhizobium species has...

Transcription is an essential step in gene expression and its understanding has been one of the major interests in molecular and cellular biology. By precisely tuning gene expression, transcriptional regulation determines the molecular machinery for developmental plasticity, homeostasis and adaptation. In this review, we transmit the main ideas or concepts behind regulation by transcription factors and give just enough examples to sustain these main ideas, thus avoiding a classical ennumeration of facts. We review recent concepts and developments: cis elements and trans regulatory factors, chromosome organization and structure, transcriptional regulatory networks (TRNs) and transcriptomics. We also summarize new important discoveries that will probably affect the direction of research in gene regulation: epigenetics and stochasticity in transcriptional regulation, synthetic circuits and plasticity and evolution of TRNs. Many of the new discoveries in gene regulation are not extensively tested with wetlab approaches. Consequently, we review this broad area in Inference of TRNs and Dynamical Models of TRNs. Finally, we have stepped backwards to trace the origins of these modern concepts, synthesizing their history in a timeline schema.

BackgroundGlycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.ResultsTranscriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.ConclusionsThe overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

LeuO is a LysR-type transcriptional regulator that has been implicated in the bacterial stringent response and in the virulence of Salmonella. A genomic analysis with Salmonella enterica serovar Typhi revealed that LeuO is a positive regulator of OmpS1, OmpS2, AssT, and STY3070. In contrast, LeuO down-regulated the expression of OmpX, Tpx, and STY1978. Transcriptional fusions supported the positive and negative LeuO regulation. Expression of ompS1, assT, and STY3070 was induced in an hns mutant, consistent with the notion that H-NS represses these genes; transcriptional activity was lower for tpx and STY1978 in an hns background, suggesting that this global regulatory protein has a positive effect. In contrast, ompS2 and ompX expression appeared to be H-NS independent. LeuO specifically bound to the 5 intergenic regions of ompS2, assT, STY3070, ompX, and tpx, while it was not observed to bind to the promoter region of STY1978, suggesting that LeuO regulates in direct and indirect ways. In this work, a novel set of genes belonging to the LeuO regulon are described; interestingly, these genes are involved in a variety of biological processes, suggesting that LeuO is a global regulator in Salmonella.