SummaryMany recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp-lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the 'Uniform Expression Model (UEM)' where expression is evenly distributed in all cells or (ii) the 'Two Population Model (TPM)' where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter ( sulAp ) to the green fluorescent protein ( gfp ) reporter gene and inserting it at att l l l l on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp-gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA , dnaT , recG , uvrD , dam , ftsK , rnhA , polA and xerC mutants were explained best by the TPM while only lexA (def) , lexA3 (ind -) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ⌬pyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum ⌬pta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.

In Escherichia coli, repair and restart of collapsed replication forks is thought to be essential for cell growth. The replication restart proteins, PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT and Rep, form redundant pathways that recognize repaired replication forks and restart them. Recognition, modulation of specific DNA structures and loading of the replicative helicase by the replication restart proteins, is likely to be important for replication restart. It has been hypothesized that PriB and PriC function with PriA in genetically separate and redundant PriA–PriB and PriA–PriC pathways. In this study, the del(priB)302 or priC303::kan mutations were used to isolate the PriA–PriB and PriA–PriC pathways genetically so that the effects of three priA missense mutations, priA300 (K230R), priA301 (C479Y) and priA306 (L557P), on these pathways could be assessed. In a wild‐type background, the three priA mutations had little, if any, effect on the phenotypes of UV resistance, basal levels of SOS expression and cell viability. In the priB mutant, priA300 and priA301 caused dramatic negative changes in the three phenotypes listed above (and others), whereas the third priA mutant allele, priA306, showed very little negative effect. In the priC mutant, all three priA mutations behaved similarly, producing little, if any, changes in phenotypes. We conclude that priA300 and priA301 mostly affect the PriA–PriC pathway and do so more than priA306. We suggest that PriA's helicase activity is important for the PriA–PriC pathway of replication restart.

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active ␤-galactosidase was obtained in a single step using the above described method.We have previously reported the development of a novel high cell density protein expression platform based on the gram-negative bacterium Ralstonia eutropha (22,23). This system has been developed to overcome some of the shortcomings associated with recombinant protein expression in Escherichia coli (e.g., poor fermentation performance at high cell density, and inclusion body formation). Expression of organophosphohydrolase, an enzyme originally isolated from Pseudomonas diminuta (20) and prone to inclusion body formation in Escherichia coli (4,28,29), was demonstrated at high levels. Titers of active, soluble organophosphohydrolyase, in excess of 10 g/liter were obtained in high cell density fermentation (3), representing at least a 100-fold increase over those previously reported in E. coli.While the successful expression of a recombinant protein is a necessary requirement, recovery and purification still remain a significant cost in recombinant protein production. We thus sought to integrate the existing R. eutropha protein expression platform with a protein purification strategy to simplify the expression and purification of recombinant proteins. This specific approach uses the natural ability of R. eutropha to produce a polymer known as polyhydroxybutyrate (PHB), which accumulates as insoluble granules within the cell. PHB is a member of the polyhydroxyalkanoate class of polymers, synthesized by many bacteria, as carbon storage compounds (2,15,16,26,30,31,32). Polyhydroxyalkanoates have received attention as biodegradable polymers and can be obtained by fermentation processes utilizing cheap, abundant renewable carbon sources (2, 24). Polyhydroxyalkanoates have been produced industrially by ZENECA Bioproducts (26) and Monsanto (10).PHB synthesis in R. eutropha has been the model system for studying polyhydroxyalkanoate biosynthesis in bacteria (10,15,16,18,26). The biogenesis of polyhydroxyalkanoate granules involves two distinct proteins, the polyhydroxyalkanoate synthase...