Rhizobium tropiciteu genes involved in specific uptake of Phaseolus vulgaris bean-exudate compounds

Rhizobium tropici nodulates and fixes nitrogen in bean. In the R. tropici strain CFN299 we identified and characterized teu genes (tropiciexudate uptake) induced by bean root exudates, localized by insertion of a promoter-less Tn5-gusA1 transposon. teu genes are present on a plasmid of around 185 kb that is conserved in all R. tropici strains. Proteins encoded by teu genes show similarity to ABC transporters, specifically to ribose transport proteins. No induction of the teu genes was obtained by treatment with root exudates from any of several other plants tested, with the exception of Macroptilium atropurpureum, which is also a host plant for R. tropici. It appears that the inducing compound is characteristic of bean and closely related legumes. It is present in root exudates, but not in seeds. This compound is removed, presumably by metabolism, from the exudates by the majority of bean-nodulating rhizobia (such as R. etli, R. leguminosarum bv. phaseoli and R.␣giardinii). The principal inducing compound has not been identified, but some induction was obtained using trigonelline. The CFN299 strain seems to have an additional uptake system, as no phenotype is observed in two different mutants. R. tropici strain CIAT899, on the other hand, must have only one uptake system, since a mutant bearing an insertion in the teu genes could not remove the compound from the exudates as efficiently as the wild type, and it showed diminished nodulation competitiveness. Received: 21 November 1997 / Accepted: 18 March 1998     

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2FGC transporter through a rigid-body mechanism to extract LPS

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB₂FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

MDR1 P-glycoprotein transports endogenous opioid peptides

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K_m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.     

Current mechanistic understanding of intermembrane lipid trafficking important for maintenance of bacterial outer membrane lipid asymmetry

The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry that makes it an effective permeability barrier against toxic molecules, including antibiotics. Central to the maintenance of OM lipid asymmetry is the OmpC-Mla (maintenance of lipid asymmetry) system, which mediates the retrograde transport of phospholipids from the outer leaflet of the OM to the inner membrane. The molecular mechanism(s) of this lipid trafficking process is not fully understood; however, recent advances in structural elucidations and biochemical reconstitutions have provided detailed new insights. Here, we present an integrated understanding of how the OmpC-Mla system transports mislocalized phospholipids across the bacterial cell envelope.     

Cloning and Nucleotide Sequence of the Gene Encoding a Serine Proteinase Inhibitor Named Marinostatin from a Marine Bacterium,Alteromonas sp. Strain B-10-31

The gene (mstI) encoding a serine proteinase inhibitor named marinostatin from marine Alteromonas sp. strain B-10-31 was cloned and its nucleotide sequence was analyzed. A short open reading frame of 192 bp encoded 63 amino acids with a molecular weight of 6,985. Furthermore, the initial product of marinostatin (marinostatin L) was purified and its amino acid sequence was analyzed. These results indicate that marinostatin is produced as a unique precursor consisting of the mature peptide and the leader peptide for an ATP-binding cassette (ABC) transporter, and furthermore the initial product of marinostatin is dehydrated and processed by proteolysis to give homologous forms of marinostatin.     

Mechanisms of iron and haem transport by Listeria monocytogenes

Abstract

Listeria monocytogenes, the causative agent of listeriosis, is a virulent foodborne Gram-positive bacterial pathogen, with 20–30% mortality. It has a broad ability to transport iron, either in the form of ferric siderophores, or by extracting it from mammalian iron binding proteins. In this review we focus on the mechanisms of ferric siderophore and haem transport into the listerial cell. Despite the fact that it does not synthesize siderophores, L. monocytogenes transports ferric siderophores in the wild environment by the actions of cytoplasmic membrane ABC-transporter systems. The bacterium acquires haem, on the other hand, by two mechanisms. At low (nanomolar) concentrations, sortase B-dependent, peptidoglycan-anchored proteins scavenge the iron porphyrin in human or animal tissues, and transfer it to the underlying ABC-transporters in the cytoplasmic membrane for uptake. At concentrations at or above 50 nM, however, haem transport becomes sortase-independent, and occurs by direct interactions of the iron porphyrin with the same ABC-transporter complexes. The architecture of the Gram-positive cell envelope plays a fundamental role in these mechanisms, and the haem acquisition abilities of L. monocytogenes are an element of its ability to cause infectious disease.     

The in ovo administration of l-trans pyrrolidine-2,4-dicarboxylic acid regulates small intestinal growth in chicks

Glutamate, which is one of the most important contributors to oxidative metabolism in the intestinal mucosa, is mainly transported by the excitatory amino acids transporters (EAATs) that are expressed in enterocytes. The objective of this study was to evaluate the effects of in ovo administration of l-trans pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a potent competitive inhibitor of glutamate uptake by EAATs, on the growth of the small intestine in chicks. Two series of experiments were conducted with hatching eggs; 100 μl of various l-trans-PDC solutions (0, 0.075 or 0.225 mg/egg for the Control group, low-dose l-trans pyrrolidine 2,4-dicarboxylic acid group (L-PDC) or high-dose l-trans pyrrolidine 2,4-dicarboxylic acid group (H-PDC), respectively) was injected into the albumen sac of these hatching eggs before incubation. Hatchlings were sacrificed by cervical dislocation to determine the embryonic development in Experiment I, whereas the birds in Experiment II were raised or sampled at hatching, days 7 and 14 (D7 and D14) for further study. Gene expression in the small intestines was determined by real-time RT-PCR; and serum concentration of free amino acids was determined by an amino acid analyzer. The results showed that the hatchability was decreased by in ovo administration of l-trans-PDC. The small intestinal weights of the H-PDC group were decreased (P<0.05) at hatching and increased (P<0.05) on D7 and D14 compared with those in the Control group. In addition, the gene expression of EAAT2 in the completed or segmental small intestines was not changed (P>0.05); EAAT3 gene expression in the duodenum (P<0.05), jejunum (P=0.084) and ileum (P=0.060) on D14 was lower in the H-PDC group than in the Control group. Furthermore, the serum concentrations of free proline, threonine and phenylalanine but not glutamate or aspartate were increased (P<0.06) in H-PDC group. In conclusion, this paper is the first to report that in ovo administration of l-trans-PDC induces small intestinal growth retardation during the embryonic period and catch-up growth after hatching.