No abstract

For the first time a single experimental approach, 16 S ribosomal RNA sequence characterization, has been used to develop an overview of phylogenetic relationships in the bacterial world. The technique permits the tracing of relationships back to the common ancestor of all extant life. This first glimpse of bacterial phylogeny reveals a world whose roots appear to span more than 3 billion years. A deep phylogenetic split exists among the bacteria, which necessitates their division into two major lines of descent, the archaebacteria and the true bacteria (or eubacteria). It is a general finding that the most ancient bacterial phenotypes are anaerobic, and that aerobic phenotypes have arisen a number of times. Photosynthetic phenotypes are also extremely ancient. Many nonphotosynthetic groups appear to have arisen from photosynthetic ancestry, which is reason to question the generally held belief that the first bacteria were anaerobic heterotrophs. The two ultimate lines of bacterial descent are no more closely related to one another than either is to the cytoplasmic aspect of the eukaryotic cell. However, in that the eukaryotic cell is a phylogenetic chimera, it itself cannot be seen as a line of descent comparable to the two bacterial lines—although some of its individual parts can be so viewed. In this way, the chloroplast and perhaps the mitochondrion are each eubacterial, and at least one ribosomal protein is archaebacterial. A third line of descent that is neither eubacterial nor archaebacterial is represented in the 18 S ribosomal RNA.

A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoylCoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.

Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.