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T.C. Charles

Dept of Biology, University of Waterloo, 200 University Ave. West, Waterloo, ON, N2L 3G1 Canada

1. Introduction

Bulk soils are characteristically oligotrophic, carbon-limited environments (van Elsas, van Overbeek 1993; van Veen, van Overbeek et al. 1997), due to recalcitrance of carbon substrate to degradation, and sequestration in soil sites beyond the reach of microorganisms. Microbial cell growth rates in soil environments are therefore extremely low, on the order of a few cell divisions per year (van Elsas, van Overbeek 1993), and most cells exist in a state of carbon starvation-induced stationary phase. In contrast, rhizosphere environments are rich in carbon nutrients, due to exudation of carbon compounds from plant roots. Of particular importance to local microbial ecology is the organisms' ability to traverse between bulk soil and rhizosphere, and to take advantage of nutrients as they become available. In the absence of cell growth, the organisms are unable to compete for a particular nutrient unless they are able to take up and store that compound and then use the stored material for cell growth when conditions are more favorable. Intracellular storage compounds thus have crucial roles to play in the proliferation and persistence of bacterial cells in the environment, and in their ability to colonize plant roots. One of the best-known carbon storage compounds is poly-3-hydroxybutyrate (PHB), commonly found as deposits in bacterial cells (Anderson, Dawes

1990). These PHB deposits typically accumulate intracellularly under conditions where growth is limited by a factor other than carbon nutrient availability. If later exposed to carbon starvation conditions, these cells may catabolize these deposits as a source of carbon and energy.

Root nodule bacteria such as Sinorhizobium meliloti have complex and specific nutrition requirements before and during their interactions with the plant host (Finan, McWhirmie et al.

1991). S. meliloti is also very good at persisting in the soil as a saprophyte in the absence of the plant host. Interest in PHB metabolism in root nodule bacteria is in part due to the prominence of PHB as the predominant carbon storage compound in some bacteroids (Forsyth, Hayward et al. 1958; McDermott, Griffith et al. 1989). Possible roles for PHB are (i) fueling the extensive cell division (Gage, Bobo et al. 1996) that takes place within the infection thread, (ii) protection of nitrogenase enzyme from oxygen during periods of darkness by providing reducing power for the maintenance of the 02 diffusion barrier in the absence of photosynthesis (Bergersen, Peoples et al. 1991), (iii) as an aid in the recovery of bacteroids on their release into the rhizosphere following nodule senescence (Klucas 1975), and (iv) to increase the survival of bacteria in the soil and rhizosphere. Although S. meliloti cells within the infection thread are often observed to have PHB deposits, these deposits are not present in mature bacteroids (Paau, Bloch et al. 1980; Hirsch, Long et al. 1982; Hirsch, Bang et al. 1983). If PHB is important in the interaction of S. meliloti with the host plant, it is more likely to be at the early stages leading up to infection.

Most of the early studies of PHB metabolism were targeted at the possible direct role of PHB in the energetics of N2 fixation, and did not address the contribution of PHB to nodulation or saprophytic growth. Recent years have brought the application of genetic and molecular biological techniques to the study of PHB metabolism (Povolo, Tombolini et al. 1994; Sikora, Kuykendall et al. 1994; Tombolini, Povolo et al. 1995; Cevallos, Encarnación et al. 1996; Mandón, Michel-Reydellet et al. 1998; Willis, Walker 1998), but until recently (Charles, Cai et al. 1997; Aneja, Charles 1999; Cai, Driscoll et al. 2000), few studies have considered the degradation part of the PHB cycle. The physiological effects of PHB metabolism mutations are various. Rhizobium leguminosarum phbC mutants exhibit reduced amino acid uptake activity (Walshaw, Wilkinson et al. 1997), and S. meliloti phbC mutants have reduced growth rates on PHB cycle intermediates (Cai, Driscoll et al. 2000). While Rhizobium etli phbC mutants exhibit enhanced ^-fixation activities (Cevallos, Encarnación et al. 1996), Azorhizobium caulinodans phbC mutants are deficient in N2-fixation due to inhibition of nifA transcription (Mandón, Michel-Reydellet et al. 1998). S. meliloti phbC mutants exhibit slight deficiencies in N2-fixation, and are defective in competition for nodulation (Willis,Walker 1998).

2. Isolation of PHB Degradation Pathway Mutants

The genetic approach that was used in the initial stages of the study of the PHB degradation pathway was to screen Tn5-generated mutants for those that were unable to utilize the PHB cycle intermediates 3-hydroxybutyrate and acetoacetate as sole carbon source (Charles, Cai et al. 1997). This screen resulted in a number of mutant classes, and subsequent characterization contributed to the current understanding of the PHB cycle (see Figure 1). Members of the first class were unable to utilize 3-hydroxybutyrate but retained the ability to utilize acetoacetate and acetate as sole carbon source. They were deficient in 3-hydroxybutyrate dehydrogenase activity and the mutation mapped to megaplasmid pRmeSU47b. Members of another major class, which were not able to use acetoacetate or 3-hydroxybutyrate, were mutated in a chromosomal gene that exhibited predicted amino acid similarity to the acetyl-CoA synthetase enzymes. Another major class, with a similar growth phenotype as the second class, was determined by complementation analysis to be in a locus comprised of at least four transcriptional units. This locus mapped to megaplasmid pRmeSU47b, and corresponds to the locus first identified by deletion analysis as bhb (Charles, Finan 1991). One of these transcripts, bhbA, was characterized, and was shown to encode the enzyme methylma-lonyl-CoA mutase (Charles, Aneja 1999). Whether methylma-lonyl-CoA mutase and the other gene products in this locus are really involved in the PHB degradation pathway remains to be determined. An unexpected outcome of the screen for altered utilization of PHB cycle intermediates was the isolation of a mutation in phbC, encoding PHB synthase (Cai, Driscoll et al. 2000).

3. Acetoacetyl-CoA Synthetase

The gene encoding a protein with predicted amino acid similarity to the acetyl-CoA synthetase enzymes in fact encodes the enzyme acetoacetyl-CoA synthetase (Cai, Driscoll et al. 2000). This enzyme activates acetoacetate to acetoacetyl-CoA, and is not able to use acetate as substrate. A true synthasejff /

PHB synthase iphbCj

PHB depolymerase (phbD)


Acetoacetyl-CoA reductase

Acetoacetyl-CoA reductase


Ketothiolase (phbA)


3-Hydroxybutyrate dehydrogenase






Acetyl-Co A synthetase


Figure 1. PHB Cycle i n Sinorhizobium meliloti acetyl-CoA synthetase, with greater similarity to other bacterial acetyl-CoA synthetase enzymes, is encoded elsewhere on the chromosome, and this enzyme is not able to use acetoacetate as substrate (Thaha 1999). The genes have been designated acsA2 (for the acetoacetate-specific) and acsAl (for the acetate-specific) respectively. The acsAl mutants are able to grow on acetoacetate or 3-hydroxybutyrate, but not acetate. This is consistent with the generation of acetyl-CoA via ketothiolase activity on acetoacetyl-CoA during growth on 3-hydroxybutyrate or acetoacetate. A key outcome of this work was the realization that acetoacetate is activated by acetoacetyl-CoA synthetase, rather than by a CoA transferase, as in E. coli (Jenkins, Nunn 1987).

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