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Translocon  Pulling of Nascent SecM Controls the Duration of Its Translational Pause and Secretion-Responsive secA Regulation.Martha E. Butkus, 2003.SecA is an ATPase and motor protein that drives protein translocation across the bacterial plasma membrane . In Escherichia coli SecA levels are regulated by the secretion needs of the cell utilizing secM, which encodes a secreted protein . Previous studies demonstrated that this regulation requires a translational pause within secM, whose duration regulates the accessibility of the secA Shine-Dalgarno sequence on secM secA mRNA . Here we provide evidence that translocon pulling of nascent SecM is what regulates the duration of the secM translational pause, and thus secA expression levels, thereby providing direct support for this model .
Modified Pathway To Synthesize Ribulose 1,5-Bisphosphate in Methanogenic Archaea. Michael W. Finn, 2004.Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea . RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms . Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo . To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction . Homology searches, as well as direct enzymatic assays with M . jannaschii, failed to reveal the presence of PRK . The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo . In the present study, direct enzymatic assays performed with alternative substrates and extracts of M . jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M . jannaschii and other methanogenic archaea . Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M . jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli . Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK . We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme . Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli. Carl-Alfred Alpert, 2003.The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined . Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication . The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons . A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L . sakei for plasmid replication . The L . sakei replicon alone could not support replication in E . coli . Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L . sakei . pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L . sakei, making it an interesting basis for developing vectors . Sequence relationships with other plasmids are described and discussed .
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