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Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96-102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropneumoniae during growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.

Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.

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Wc have studied the effect of artifically imposed transmembrane pH gradients on the uptake of amino acids by Staphylococcus aureus.I n high potassium medium, cells treated with valinomycin and tetrachlorosalicylanilide and at Donnan equilibrium, were found to accumulate glutamate.I n low potassium medium, addition of acid to the extracellular environment was found to stimulate the uptake of glutamate and aspartate, to induce partial lysine efflux, and to cause a small and transient isoleucine uptake.I n conjunction with earlier data from this laboratory (1973), it is concluded that the basic, neutral and acidic amino acids behave, respectively, as cationic, uncharged and anionic substrates, accumulating in Staphylococcus aureus in response to (a) the membrane potential, inside negative, (b) the total protonmotive force, and (c) the transmembrane pH gradient, inside alkaline.I n previous papers [1,2] we presented evidence in favour of the hypothesis that lysine is transported in Staphylococcus aureus by means of a uniport mechanism, the monovalent cation being translocated in response to the membrane potential, inside negative. By inducing potassium efflux on the addition of valinomycin [2], we further obtained data which allowed the conclusion to be drawn that the accumulation of glycine and isoleucine is dependent on the total protonmotive force, these amino acids being transported most likely by means of protoncoupled symporters which recognise the uncharged forms of these amino acids, Such valinomycin treatment was found to have no effect on the characteristics of glutamate uptake. This negative result was, howevcr, in keeping with the previous suggestion [l] that acidic amino acids may also be translocated by means of proton-coupled symporters displaying a stoichiometry of 1 : I with respect to proton and the monovalent amino-acid anions. According to this hypothesis, the translocation of acidic amino acids would be electroneutral and dependent only on the transmembrane pH gradient, inside alkaline.I n this present investigation we set out to test this hypothesis directly. Through altering the transmembrane pH gradient by experimental manipulation of' the extracellular ionic environment, we have examined the uptake of acidic, and additionally of neutral and basic amino acids, in response to this artificially imposed driving force. We have been able to confirm the proposed role of the transmembrane pH gradient in the uptake and accumulation of amino acids by Staph. aureus. MATERIALS AND METHODS Preparation of Cell XuspensionsSuspensions of endogenously reduced Staphylococcus aureus (Colworth bacterial culture collection 273) were routinely prepared as described previPotassium-depleted cells were prepared similarly, except in the final resuspension; cells from a 100 ml culture were resuspended in 75 ml 0.25 mM N-tris-(hydroxymethyl) -methyl -2 -aminoethane -sulphonic acid, 0.1 mM Tris, p H 7 .O. Tetrachlorosalicylanilide and valinomycin (in 36 p1 volumes of acetone) were added to 1.4 pM and 1.8 pM, respectivel...