Dissecting the diversity of the genus Peptostreptococcus using genomic and proteomic analyses
The genus Peptostreptococcus comprises a heterogeneous collection of species that are gram-positive cocci, non-spore forming and obligately anaerobic. They represent a significant component of the normal human flora but remain poorly studied. They are asaccharolytic or weakly saccharolytic and therefore not easily delineated by conventional biochemical tests. No virulence mechanisms have been reported to date, yet peptostreptococci are nearly always present in human anaerobic infections, and growing evidence indicate that they are likely to play a key role in disease development. In order to study the role of these organisms in disease, it is necessary to reliably identify isolates and gain insight into their metabolic activities. Consequently, the aim of this study was two fold: undertake a 'polyphasic' approach to the taxonomic structure of the genus, and to study aspects of their metabolism with particular reference to the genes that regulate haem biosynthesis. It was shown early in this study that glutamate may have a central role in metabolism and the enzymic properties of glutamate dehydrogenase was used as a preliminary method to study the intra-generic structure of the genus. Several species possessed the enzyme and the electrophoretic mobilities correlated with some. The glutamate dehydrogenase gene was sequenced in 21 strains of peptostreptococci and phylogenetically closely related Clostridium species (sp.) and carried out in parallel with 16S rDNA sequencing. Comparison of the phylogenetic trees constructed using gdh sequences and 16S rDNA sequence of Peptostreptococcus showed similarities. P. anaerobius, the type species of the genus clustered with some species of Clostridium in both trees indicating that P. anaerobius is not only distantly related to other peptostreptococci but is more closely related to clostridia. Consequently, during the course of this work, it was proposed that the genus Peptostreptococcus be restricted to only P. anaerobius and other species were designated incertae sedis. Further extensive analysis on 44 strains of peptostreptococci and several related Clostridium sp. were undertaken using gas chromatography of their long chain fatty acid methyl esters (FAME), while MALDI-TOF (cell surface-associated molecules) and SELDI-TOF (intracellular proteins) mass spectrometry were also explored in an attempt to find new characters to delineate species. MALDI-TOF-MS was successfully used to discriminate between rough and smooth morphotypes of Peptostreptococcus micros strains, but these analyses remain inconclusive at the species level. However, there was sufficient data generated to begin the reclassification of the remaining taxa. Consequently, the following new genera were created to accommodate these; Finegoldia magna comb. nov. (Prevot 1933; Holdeman and Moore 1972) for Peptostreptococcus magnus\ Micromonas micros comb. nov. (Prevot 1933; Smith 1957) for Peptostreptococcus micros', Schleiferella asaccharolytica comb. nov. (Distaso 1912; Ezaki, Yamamoto, Ninomiya, Suzuki and Yabuuchi 1983) for Peptostreptococcus asaccharolyticus; Schleiferella indolica comb. nov. (Christiansen 1934; Ezaki, Yamamoto, Ninomiya, Suzuki and Yabuuchi 1983) for Peptostreptococcus indolicus; Schleiferella lacrimalis comb. nov. (Li, Hashimoto, Adnan, Miura, Yamamoto and Ezaki 1992) for Peptostreptococcus lacrimalis and Schleiferella harei comb. nov. (Murdoch, Collins, Willems, Hardie, Young and Magee 1997) for Peptostreptococcus harei. The second phase of this study focussed on investigating the diversity of the tetrapyrrole biosynthetic pathway among Peptostreptococcus. This pathway leads to the synthesis of haem, cobalamin (vitamin 612) and other metal ion chelated tetrapyrrole molecules. Haem is an iron chelated tetrapyrrole derivative that is essential for several physiological functions and is one of the prerequisites for the pathogenicity of many bacteria. Intracellular levels of haem are known to regulate bacterial biological properties and their ability to initiate infection. Molecular characterisation of important haem regulatory genes including hemA, hemB, hemH, cysG, cbiP and btuR were investigated. Gene fragments of hemB, cbiF and cbiP were amplified, cloned and sequenced. The presence of the hemB, cbiF and cbiP which encodes for aminolevulinic acid dehydratase (ALAD), precorrin-4 C-ll methyltransferase and cobyric-acid synthase respectively indicated the presence of a branched pathway leading to cobalamin biosynthesis. In-vitro transcriptional expression of hemB and cbiF were investigated by reverse-transcriptase (RT)-PCR in parallel with enzymatic assays for ALAD activity. The results of this study provided unambiguous evidence for the presence of a functional tetrapyrrole biosynthetic pathway. This is in contrast to many anaerobic taxa such as Porphyrinmonas gingivalis where only vestiges of a tetrapyrrole biosynthetic pathway occur so that the organism has an absolute dependence on in vitro haem. Peptostreptococci are therefore able to biosynthesise haem from basic precursors and this may confer significant advantages for this species in deep-seated infections where it thrives, and where the basic tetrapyrrole precursors are available.