Studies on the Growth Physiology of Beneckea Natriegens.

Beneckea natriegens provides an interesting system for the study of respiratory control in bacteria as it apparently has four different recognisable cytochrome oxidases. From inhibitor studies on cell free extracts a branched electron transport chain has been proposed for this organism. The individual branches of the electron transport chain are distinguished by their differing affinity for oxygen and sensitivity to inhibition by cyanide. It has been suggested that the multiple oxidases present in this organism function at different medium dissolved oxygen tensions and may be associated with differing efficiencies of phosphorylation.

The respiratory control and growth physiology of Beneckea natriegens was studied using continuous culture techniques. As a preliminary to this investigation the inorganic ion requirement of this organism was examined in batch culture and a basal salts medium formulated to facilitate the operation of a carbon limited chemostat culture at a medium glucose feed concentration of 4.0 gl-1. The results obtained in batch culture were confirmed in continuous culture (D = 0.34 h-1) where a linear relationship between bacterial biomass and substrate concentration (0.5 to 4,0 gl-1 glucose) was observed, indicative of carbon limitation.

The effect of growth rate on growth physiology was investigated and the molar growth yields from glucose and oxygen were found to be dependent on, and to increase with, increasing growth rate. The in situ respiration rate of B. natriegens was directly proportional to the dilution rate. The 'potential' respiration rate, however, was independent of the growth rate and remained at a value higher than the in situ respiration rate, the latter increased with increase in the dilution rate. The cytochrome content of bacteria decreased with increasing growth rate, and no positive correlation between bacterial cytochrome content and respiration rate was observed.

To assess whether branching of the electron transport system occurred under physiological conditions, B. natriegens was grown at various medium dissolved oxygen tensions in glucose limited chemostat culture and the effect of medium dissolved oxygen tension on growth yield, respiration and cytochrome composition examined. The results indicated that the metabolic rate and growth efficiency of this organism, as indicated by the q02' qC02 and Yglc' was little affected by changes in the dissolved oxygen tension over the range <2 mm Hg to 134 mm Hg. The 'critical' dissolved oxygen tension in chemostat studies was <1.3 pM O2 and is in good agreement with values of between 0.15 and 0.25 pM O2 obtained for the Km for oxygen using the respirograph technique.

These Km values are approximately an order of magnitude lower than those previously reported for this organism. These studies failed to show any physiological evidence of a switch in pathway of electrons from a coupled to an uncoupled branch. Moreover the different branches of the respiratory chain of B. natriegens are reported to be recognised by a difference of at least an order of magnitude in cyanide sensitivity of the respective terminal oxidases. Therefore, if there is a change in the relative importance of these branches in response to dissolved oxygen tension this would be reflected in the cyanide sensitivity of the bacteria. No such change in cyanide sensitivity over the dissolved oxygen range 8 to 140 mm Hg was observed.

If the branched respiratory chain proposed for B. natriegens operates under physiological conditions then growth of this organism in the presence of cyanide would be expected to cause a switch to the cyanide resistant respiratory pathway and change the growth efficiency. However, growth of B. natriegens in the presence of cyanide and carbon monoxide had no measurable effect on respiration rate or growth efficiency. But, at high growth rates, low concentrations of cyanide (24 pM) caused the culture to wash out although the cyanide sensitivity of harvested bacteria was independent of growth rate.

A detailed study was made of the effect of respiration rate on cyanide sensitivity of bacteria harvested from chemostat culture. The respiration rate was varied either by manipulating the initial substrate concentration or the incubation temperature. The sensitivity of respiration to KCN was found to be dependent on the initial respiration rate of the bacteria. At sub-maximal respiration rates the bacteria were insensitive to KCN concentrations below a threshold value which increased as the respiration rate decreased. The same effect was obtained when respiration rate was decreased by lowering the initial substrate concentration or incubation temperature. The results are consistent with the potential respiration rate being limited by a cyanide-sensitive component. At sub-maximal respiration rates, in the absence of KCN, another factor becomes rate limiting but addition of KCN at a concentration above a threshold value caused the cyanide sensitive component to become rate-limiting for respiration once more.

Growth in the presence of cyanide caused an increased production of the CO-binding c type cytochrome. As the increased synthesis of this cytochrome did not cause an increased resistance to cyanide or an increase in respiration rate it was thought to be unrelated to cyanide sensitivity. Formate was oxidised constitutively by B. natriegens. In the methane utilising bacteria, a CO binding c type cytochrome has been implicated in the reaction that brings about the oxygenation of methane. It is postulated that formate is oxidised by B. natriegens via two separate systems which are distinguished by their differing 'apparent' Km for oxygen. One system is thought to involve a NAD-linked formate dehydrogenase, the other a CO binding c type cytochrome linked formate oxidase.