SUMMARY: Iodination of intact Pasteurella haemolytica serotype A2 cells labelled a sub-set of total cellular proteins. Comparison of the autoradiographic patterns obtained from iodinated cells grown on complete medium and on iron-depleted medium showed that expression of three proteins, of 100,70 and 35 kDa, respectively, was increased by growth under iron-depleted conditions. Of these proteins, that of 35 kDa had not been reported previously. Like the 100 and 70 kDa proteins, the 35 kDa protein was expressed in natural infections, since it was recognized by antiserum from sheep that had recovered from an experimental infection with P. haemolytica A2. The 35 kDa protein was partially purified by reverse-phase HPLC and was found to be antigenic in both sheep and mice. A monoclonal antibody that was specific for the 35 kDa protein was used to identify the cellular location of the protein by immunoblotting of cell fractions enriched for particular cellular components. This demonstrated that the 35 kDa protein was located mainly in the periplasm.
SUMMARY: The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum β-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum β-lactam antibiotics and with constitutively overproduced chromosomal β-lactamase in the periplasm. The binding of structurally different β-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1·5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested β-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced β-lactamase did not prevent PBP-β-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific β-lactams.
SUMMARY: The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.
SUMMARY: Growth of Fusarium oxysporum on heat-killed Bacillus subtilis cells was accompanied by the loss of bacterial cytoplasmic contents, and this ‘cytolysis’ could be catalysed in heat-treated bacteria by the fungal culture fluids. In electron micrographs the bacterial walls appeared undamaged, and the absence of wall-lytic enzymes was confirmed by use of isolated bacterial walls as substrate. Appearance of cytolytic activity in cultures was paralleled by the production of proteolytic activity in the cultures. Proteolysis and cytolysis had similar pH optima at 8·8-9·0. Cultures grown on casein, but not glucose, produced high cytolytic activity. Rapid cytolysis occurred when heat-treated B. subtilis cells were incubated with trypsin, subtilisin or pronase E. Viable bacteria, however, were not attacked, either by concentrated culture fluids or by the commercial protease preparations.
SUMMARY: A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers’ yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4·5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (K i about 5 mM), but increased TPP activity about six-fold (K a about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.
SUMMARY: This paper describes periplasmic c-type cytochromes from two strains of Paracoccus denitrificans NCIB 8944 grown in heterotrophic or methylotrophic conditions. It is shown that the functions of two monomeric, monohaem cytochromes induced during growth on methanol have been wrongly designated in previous work. The CO-reactive cytochrome c 553 (30 kDa) is not the electron acceptor for methanol dehydrogenase; this is shown to be the role of the cytochrome c 552 (22 kDa). The monomeric 45 kDa cytochrome induced in conditions of oxygen insufficiency is a dihaem c-type cytochrome and does not contain haem b as previously assumed. In addition to these cytochromes, the Oxford strain of NCIB 8944 contains two cytochrome c complexes. One of these (150 kDa), produced in relatively small amounts, consists of a non-haem protein plus four haemoproteins (28, 33, 41 and 47 kDa). The second complex is a novel dimeric multi-haem cytochrome c (46 kDa) which constitutes about 25% of the periplasmic c-type cytochrome. It reacts with CO and has no methionine ligands. One subunit (16 kDa) has two low-spin haems; the larger subunit (30 kDa) has three haems which have low-spin characteristics in the oxidized state and are high-spin in the reduced state. The subunits were readily separated at pH 12 and could be subsequently reconstituted into a complex indistinguishable from the original. The 30 kDa subunit was denatured on prolonged exposure to high pH, which also converted it to a low-spin cytochrome. No function could be designated for these novel c-type cytochrome complexes.