The formation of cyclopropane fatty acids (CFAs) in Escherichia coli is a post-synthetic modification of the phospholipid bilayer that occurs predominantly as cultures enter the stationary phase of growth. The mechanism of this growth phase-dependent regulation of CFA synthesis was unclear, since log-phase and stationary-phase cultures had been reported to contain similar levels of the enzyme catalysing the reaction (CFA synthase). We report that the timing of CFA synthesis can be explained by two unusual features. Fist, the gene encoding CFA synthase (cfa) was found to be transcribed from two promoters and the 5' ends of both transcripts were mapped by primer extension. One of the promoters was active only during the log-to-stationary phase transition and depended on the putative sigma factor encoded by the rpoS(katF) gene whereas the other promoter had a standard sigma 70 promoter consensus sequence and was expressed throughout the growth curve. Second, CFA synthase activity was shown to be unstable in vivo and a Cfa fusion protein was found to have a half life of < 5 min. The combination of these factors meant that, although CFA synthase was synthesized throughout the growth curve, a large increase in activity occurred during the log-to-stationary phase transition. As stationary phase progressed, the increased CFA synthase activity rapidly declined to the basal level. This transient increase in CFA synthase activity coupled with the cessation of net phospholipid synthesis in stationary phase provides an explanation for the unusual time course of CFA synthesis.

The activity of Escherichia coli pyruvate oxidase (PoxB) was shown to be growth-phase dependent; the enzyme activity reaches a maximum at early stationary phase. We report that PoxB activity is dependent on a functional rpoS(katF) gene which encodes a sigma factor required to transcribe a number of stationary-phase-induced genes. PoxB activity as well as the beta-galactosidase encoded by a poxB::lacZ protein fusion was completely abolished in a strain containing a defective rpoS gene. Northern and primer extension analyses showed that poxB expression was regulated at the transcriptional level and was transcribed from a single promoter; the 5' end of the mRNA being located 27 bp upstream of the translational initiation codon of poxB. The poxB gene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genes arcA, arcB or fnr were not involved in anaerobic poxB gene expression. Expression of the rpoS(katF) gene has been reported to be affected by acetate, the product of PoxB reaction. However, we found that poxB null mutations had no effect on rpoS(katF) expression. Inactivation of two genes involved in acetate metabolism, ackA and pta, had no effect on either poxB or rpoS(katF) expression.

The spatial and temporal distribution of sucrose synthase (RSuS) in rice (Oryza sativa L.) was studied by Western and immunohistochemical analyses using the monospecific antibodies for three RSuS isoforms. In leaf tissues, RSuS1 was localized in the mesophyll while RSuS2 was in the phloem in addition to the mesophyll. In the roots, only RSuS1 was found in the phloem. No RSuS3 could be detected in any parts of etiolated seedlings. The expression of each RSus gene is closely linked to the seed development. RSuS1 was present in the aleurone layers of developing seeds, and at a low level in endosperm cells. RSuS2 was evenly distributed in seed tissues other than the endosperm. RSuS3 was localized predominantly in the endosperm cells. The tissue specific localizations of the three gene products suggest that RSuS1 plays a role in sugar transport into endosperm cells where the reaction catalyzed by RSuS3 provides the precursor of starch synthesis. RSus2, which is ubiquitously expressed, may play a housekeeping role.

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Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.