The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter flavus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 bp and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. flavus H4-14 showed that transcription of cfxL and cfxS initiated 22 bp upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.

It has been established in numerous cases that proteins which are exported from Escherichia coli are synthesized on membrane-bound polysomes in precursor forms which are proteolytically cleaved to generate the mature species. Here we present evidence that at least one step in the export of proteins requires energy. Energy requirements for processing of the precursors of both the M13 coat protein [Date, T

Aerobically grown Escherichiu coli GM48 harboring plasmid pKScitS that codes for the sodiumdependent citrate carrier from Klebsiella pneumoniae (CitS) allows initial-rate measurements of citrate uptake in whole cells. The cation stoichiometry and selectivity of CitS was studied using this experimental system. The relationship between the initial rate of uptake of citrate and the Na' concentration was sigmoidal at pH values between 5 and 7 suggesting a Na' stoichiometry higher than 1. Rates of uptake increased quadratically in a range of non-saturating Na' concentrations showing that two Na' are translocatedcatalytic cycle. Symport of Na' is absolutely required in the range pH 5-7 because no uptake could be detected in the absence of Na'. Protons cannot replace Na' in the translocation step but the decrease in apparent affinity for Na' towards lower pH suggests that protons can compete with Na' for the cation-binding sites.Li' can replace Na' in the symport reaction but it takes about a 200-fold higher concentration of Lit over Na' to achieve the same rate of uptake, showing that the affinity of CitS for Li' is much lower than for Na'. Though high Li' concentrations have an inhibitory effect on citrate uptake, the data suggest that the Li' stoichiometry is also 2.Chemiosmotic energy transduction in bacteria can depend on both proton and sodium ion cycling across the cytoplasmic membrane. Cells maintain an inward directed proton motive force (pmf) and sodium motive force (smf) across the cell membrane. The pmf is generated by primary proton pumps, the smf either, directly, by primary Na' pumps or, indirectly, through Na'/H' antiport activity. Secondary solute transporters use the free energy stored in the ion gradients to accumulate a solute inside the cell. Mechanistically, this implies that the solute is transported in symport with H + or Na'. A large group of secondary transporters has been described that exclusively couple the translocation of solute with H', e.g. most of the sugar permeases in Escherichia coli [I, 21. In extremophiles many solute transporters are exclusively driven by the Na' gradient, e.g. all amino acids in the halophile Micrococcus halobius [ 3 ] . The difference in coupling ion must reflect differences in the cation-binding site on the carrier protein. In addition to the two classes of permeases that differ in the cation usage, a number of transporters have been described that use either H' or Na' as the coupling ion. For instance, the melibiose carrier of E. coli encoded by the melB gene transports melibiose in symport with a single cation that can be either H', Na' or Li' [4, 51. The different cations compete for the cation-binding site on the permease indicating a lower selectivity of the binding Correspondence to J. S. Lolkema,

A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system Dijkstra, Durk; Broos, Jaap; Lolkema, Julius; Enequist, H.; Minke, W.; Robillard, G.T. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date : 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Dijkstra, D., Broos, J., Lolkema, J. S., Enequist, H., Minke, W., & Robillard, G. T. (1996). A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvatedependent mannitol transport system. Biochemistry, 35(21), 6628-6634. DOI: 10.1021/bi952222t Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. ReceiVed September 18, 1995; ReVised Manuscript ReceiVed March 14, 1996 X ABSTRACT: The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant . The steady-state fluorescence spectra of EII mtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I -quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I -. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 385 or ...