Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes.

We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.

Selective effects of histidine-rich polypeptides on the aggregation and viability of Streptococcus mutans and Streptococcus sanguis.

Enriched preparations of histidine-rich polypeptides (HRPs) and isolated HRP pairs (1-2, 3-4 and 5-6) degrade in the presence of fresh autologous whole saliva to a series of low-molecular-weight cationic peptides (HRPs 6a-c and 7). Analysis of the HRPs during degradation indicates that: HRP 1 is not the parent molecule of the HRPs; the HRP pairs do not convert to each other in a cascade-like sequence in saliva; and the HRPs can be separated into 2 groups consisting of HRPs 1-2 and 3-7. Preparations containing HRPs 1-7, 1-2, and 3-7 were obtained by fractionation and separation on Bio-Rex 70, and tested for aggregating and antibacterial effects against Streptococcus mutans BHT, S. mutans GS-5 and Streptococcus sanguis G9B. HRPs 1-2 had significant aggregating effects on all 3 strains but the other HRPs had little to no agglutinating ability. The HRPs did not inhibit the growth of S. sanguis, and HRPs 1-2 enhanced its growth. No growth enhancement by the HRPs was observed for the 2 S. mutans strains. However, significant bacterial inhibition of the S. mutans strains was noted after incubation with HRPs 3-7. The data suggest that the dissimilar effects of HRPs 1-2 and 3-7 may be of importance in the colonization and growth of S. mutans and S. sanguis in vivo.

Facile autoplast generation and transformation in Bacillus thuringiensis subsp. kurstaki.

We describe a method for maximizing the rate of conversion of Bacillus thuringiensis subsp. kurstaki vegetative cells to osmotically fragile forms in the absence of exogenously added enzymes. Optimal generation of autoplasts occurred in 50 mM sodium acetate buffer (pH 7.0) at 37 degrees C with 10% (wt/vol) polyethylene glycol as an osmotic stabilizer. The maximum autolytic rate resulted in a conversion of greater than 90% of bacilli to spherical autoplasts in 6 min. Autoplasts regained bacillary morphology upon plating on DM3-G regeneration medium, with reversion frequencies ranging from 1.2 x 10(-1) to 5.3 x 10(-3). The autoplasts could efficiently take up exogenously added plasmid DNA. The presence of plasmids was verified by Southern hybridization analysis.

Lysozyme-mediated de-chaining of Streptococcus mutans and its antibacterial significance in an acidic environment.

The ability of physiological amounts of lysozyme to de-chain two serotype c strains of Streptococcus mutans was determined. Both human and hen lysozymes were equally effective in chain breakage of S. mutans DPR and S. mutans DJR. De-chaining did not affect growth of cultures, but resulted in finely dispersed suspensions, at stationary phase, which were visibly different from untreated cultures. Less than 50 micrograms lysozyme per ml culture medium reduced chain length to virtually all diplococci and single cells, and this chain disruption increased total viable cell count. De-chaining required an active enzyme indicating that a degree of hydrolysis of the peptidoglycan occurred at the septae of the streptococci. De-chained S. mutans did not survive as well as streptococci of normal chain length when incubated under acidic conditions (pH 5.5), but gross cellular lysis was not apparent. The reduced aciduric property of the disrupted chains may have been due to a participation of autolysins or to a lethal triggered by the lysozyme-damaged peptidoglycan. De-chaining may be a mechanism by which lysozyme could regulate the levels of S. mutans in acidogenic plaque samples.