Summary auto-generated
This study developed a quantitative manometric assay to measure terminal oxidase activity in bacteria using TMPD (a tetramethyl-p-phenylenediamine derivative) as an electron donor. The researchers tested 79 bacterial strains representing 34 genera, all grown heterotrophically under standardized conditions. Results were analyzed by plotting corrected TMPD oxidation rates (Qo2 values) against TMPD-to-endogenous respiration ratios on a double logarithmic scale. The quantitative assay allowed researchers to subdivide bacteria into four groups based on oxidase activity. Groups I and II contained all Kovacs oxidase-positive bacteria with high to moderately high TMPD oxidation rates (Qo2 values of 27-2,164). Groups III and IV contained oxidase-negative bacteria with low or unmeasurable TMPD activity. The study identified specific thresholds separating oxidase-positive from oxidase-negative bacteria: a minimum corrected Qo2 value of 33 and a minimum TMPD-to-endogenous ratio of 5. These quantitative results correlated perfectly with the traditional qualitative Kovacs oxidase test, but provided greater sensitivity for distinguishing bacterial groups, particularly among organisms previously considered intermediate in oxidase activity.
Key findings
- A quantitative TMPD oxidase assay using manometry can precisely measure terminal oxidase activity in bacterial whole cells and correlates perfectly with the standard qualitative Kovacs oxidase test
- Four distinct bacterial groups were identified based on TMPD oxidation rates, with oxidase-positive bacteria (groups I-II) showing Qo2 values of 27-2,164 and oxidase-negative bacteria (groups III-IV) showing values of 0-33
- The threshold separating oxidase-positive from oxidase-negative bacteria is a corrected TMPD Qo2 value of 33 and a TMPD-to-endogenous respiration ratio of 5
- The assay demonstrated sufficient sensitivity to subdivide traditionally classified oxidase-positive bacteria into two groups with markedly different TMPD oxidation rates
- Oxidase activity correlates with the presence of integrated terminal oxidase complexes containing both c-type cytochromes and terminal oxidase components such as cytochrome o or a+a3
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Abstract
A manometric assay system employing ascorbate and N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD) was used to quantitate terminal oxidase activity in bacterial non-proliferating whole cells. A wide variety of physiologically diverse bacteria, all of which were grown heterotrophically, was tested by this assay. For this survey study, 79 bacterial strains, which represented 34 genera, were used. Turbidimetrically standardized resting (non-proliferating) cell suspensions were prepared from cells harvested at the late logarithmic growth phase; all cells were grown under identical nutritional conditions. The TMPD oxidase activity obtained quantitatively correlated exceptionally well with results of the standard Kovacs oxidase test. In fact, the increased sensitivity of this quantitative assay allowed for further reclassification within the two major divisions of Kovacs oxidase-positive and -negative groups. Groups I and II contained all of the oxidase-positive microorganisms and the bacteria listed in group I had the highest TMPD oxidase rates, the Qo2 values (microliters of O2 consumed per hour per milligram [dry weight] at 30 C) ranging from 393 to 2, 164. The organisms listed in group II still had moderately high TMPD oxidase activity, the Qo2 values ranging from 27 to 280. All oxidase-negative bacteria fell into groups III and IV. Bacteria in group III had low but still measurable TMPD oxidase rates, the Qo2 values ranging from 3 to 33, whereas the bacteria found in group IV were inert and unable to oxidize TMPD. A grouping analysis allowed for the resolution of that point which separates oxidase-positive from oxidase-negative bacteria. This point, for non-proliferating cells, was found to be an absolute TMPD oxidation Qo2 value of 33 (after correcting for the endogenous rate by subtraction) and a Qo2 (TMPD/endogenous) ratio of 5; the latter parameter indicated that the uncorrected TMPD oxidation Qo2 value had to be five times greater than the rate for endogenous respiration. All Kovacs oxidase-positive organisms were found to have TMPD oxidase Qo2 values greater than these two metabolic parameters, whereas all Kovacs oxidase-negative organisms had lower values.