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The chapter is on development and application of matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF) to identification and and classification of bacteria.

The soluble proteome of three Clostridium difficile strains of varying pathogenic potential, designated B-1, Tra 5/5 and 027 SM, were compared using differential in-gel electrophoresis in which the proteins of each strain were labelled with CyDyes. This enabled visual inspection of the 2D profiles of strains and identification of differentially expressed proteins using image analysis software. Unlabelled protein reference maps of the predominant proteins were then generated for each strain using 2D gel electrophoresis followed by protein sequencing of each spot using a Reflectron matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. Increased coverage of the proteome was achieved using 1D gel electrophoresis in a bottom-up approach using LC-MS/MS of 1 cm gel slices. A total of 888 different proteins were detected by comparative analysis of isolates grown in parallel for 64 h on blood agar plates. Of these, only 38 % were shared between all isolates. One hundred and ten proteins were identified as showing ≥2-fold difference in expression between strains. Differential expression was shown in a number of potential virulence and colonization factors. Toxin B was detected in the more virulent strains B-1 and 027 SM, but not in the lower virulent strain Tra 5/5, despite all strains possessing an intact pathogenicity locus. The S-layer protein (Cwp2) was identified in strains 027 SM and Tra 5/5 but not strain B-1, and differences in the post-translational modification of SlpA were noted for strain B-1. The variant S-layer profile of strain B-1 was confirmed by genomic comparison, which showed a 58 kb insertion in the S-layer operon of strain B-1. Differential post-translation modification events were also noted in flagellar proteins, thought to be due to differential glycosylation. This study highlights genomic and proteomic variation of different Clostridium difficile strains and suggests a number of factors may play a role in mediating the varying virulence of these different strains.

Objective: The current increasing interest in the application of mass spectrometry, in particular MALDI-TOF MS, to identification of bacteria and fungi calls for a need to utilise this technology for identification of other infectious agents such as viruses. The aim of the present study was to develop a rapid and reliable mass spectrometry-based proteomic method for identification of Escherichia coli phages.\ud Methods: The approach was based on rapid in-solution tryptic digestion of suspensions of plaque-purified bacteriophage followed by mass spectral analysis. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography – tandem mass spectrometry (LC-MS) were used to analyse the tryptic digests. Processing of tandem mass spectrometry data and interpretation of results were achieved using Mascot software and the Swiss-Prot database.\ud Results: Five bacteriophage species (Enterobacteria phages P2, T4, T5, T7 and Lambda) isolated in E. coli cultures were identified. The viral proteins were identified from a complex mixture of host bacterial proteins. In addition, using a single ion monitoring method, a Lambda prophage derived protein was also identified.\ud Conclusion: The data obtained demonstrate that LC-MS/MS can be used for accurate identification of E.coli- specific bacteriophages in both lytic and lysogenic cycles\ud Keywords: Bacteriophage virus; Mass-spectrometry; Liquid chromatography; MALDI; LC-MS/MS; Lytic; Lysogenic; Enterobacteria; E.coli; Phage; Viruses

Strains representing the species Campylobacter coli, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, and Staphylococcus aureus were randomly selected to assess the consistency of cells preserved on lenticule discs to those archived in traditional freeze-dried ampoules. Each matched pair was cultured using identical conditions and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to profile the surface-associated molecules of the cells. In addition, the cytosolic/membrane-bound proteins of C. coli and S. aureus strains were further analysed by surface-enhanced laser desorption/ionization time-of-flight MS. The mass spectral profiles in all cases showed a high degree of concordance between cells preserved by both methods and suggest that the properties of cells preserved on lenticule disc are consistent with those archived by the traditional method of freeze-drying.

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.

The ability of Porphyromonas gingivalis to biosynthesize tetrapyrroles de novo has been investigated. Extracts of the bacterium do not possess activity for 5- aminolevulinic-acid dehydratase or porphobilinogen deaminase, two key enzymes involved in the synthesis of uroporphyrinogen III. Similarly, it was not possible to detect any genetic evidence for these early enzymes with the use of degenerate polymerase chain reaction. However, the bacterium does appear to harbor some of the enzymes for cobalamin biosynthesis since cobyric acid, a pathway intermediate, was converted into cobinamide. Furthermore, degenerate polymerase chain reaction with primers to cbiP, which encodes cobyric-acid synthase, produced a fragment with a high degree of identity to Salmonella typhimurium cbiP. Indeed, the recently released genome sequence data confirmed the presence of cbiP together with 14 other genes of the cobalamin pathway. A number of these genes were cloned and functionally characterized. Although P. gingivalis harbors all the genes necessary to convert precorrin-2 into cobalamin, it is missing the genes for the synthesis of precorrin-2. Either the organism has a novel pathway for the synthesis of precorrin-2, or more likely, it has lost this early part of the pathway. The remainder of the pathway may be being maintained to act as a salvage route for corrin synthesis.

Matrix-Assisted Laser Desorption:lonisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) as a tool for differentiating bacterial species was examined using reference strains representing gram-positive and gram-negative taxa. Initially, the effect of differences in medium composition on spectral profile was examined. The results indicated that growth on Columbia blood agar resulted in a larger spectrum of ionized residues and was therefore used for the cultivation of all strains in the rest of the study. The stability of the obtained mass spectral profiles against differences in batch and media processing suggested that no significant alterations to the profiles occurred in response to changes in media sources. The established conditions from these initial experiments were used to standardize subsequent experiments. The MALDI-TOF-MS profiles of 15 reference strains were compared and species characteristic markers were identified. The potential of using MALDI-TOF-MS as a tool for probing clonal diversity...

The mass spectral profiles of three major/emerging Gram positive pathogens belonging to the genera Clostridium, Listeria and Propionibacterium were investigated using four MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization- Time of Flight) Mass Spectrometers from Waters, Shimadzu, Bruker and Ciphergen Biosystems. These instruments have been extensively used for developing a diagnostic platform for high throughput, low cost, near sample-free preparation for microbial identification. The results demonstrate the marked effect of spectral quality obtained by altering the matrix and the added value of simple preparative extraction procedures. While microbial spectral data are generally collected in the mass range 2 to 20 kDa for diagnostic signatures, most of the significant mass ions are

There is now increasing evidence that surface-associated enzymes, previously considered to be involved in intermediary metabolism or virulence, play a role in physiological reactions such as signal transduction, transport systems, and metabolic processes. Herein we report the molecular aspects of two such enzymes, the cysteine proteinase gingivain and NAD-dependent glutamate dehydrogenase of Porphyromonas gingivalis. The gdh gene comprises an open reading frame of 1,335 base pairs that encodes a 49,000-M(r) protein of 445 amino acids. The gdh gene showed high homology (78.3%) with that of Clostridium symbiosum. Optimal codons accounted for 35.9% of the total codon usage, indicating high expression of this enzyme. These data are currently being used to carry out targeted mutagenesis, which was established here for gingivain. Conditions for targeted mutagenesis within the histidine domain of the catalytic site of gingivain using Tn 4351 was successfully achieved. Consequently, the catalytic functions, such as gingivain's capacity to hydrolyze the synthetic substrate alpha-benzoyl-arginine-4-nitroanilide, were disrupted.

Cell free extracts from a wide variety of Gram-negative, anaerobic, non-sporeforming rods were screened for dehydrogenase enzymes of carbohydrate and nitrogen metabolism. Four enzymes, malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were found to be of diagnostic importance. The ‘Bacteroides fragilis’ group possessed all four enzymes whereas the ‘B. melaninogenicus-B. oralis’ group and the asaccharolytic, pigmented species were characterised by the presence of only malate and glutamate dehydrogenases. The saccharolytic and asaccharolytic pigmented species could be differentiated by the wide difference in pH optimum for malate dehydrogenase. Fusobacterium species and Leptotrichia buccalis both possessed only glutamate dehydrogenase, but there were differences in enzyme activity between both taxa. Other genera such as Anaerorhabdus, Megamonas, Mitsuokella. Rikenella, Sebaldella and Tissierella had characteristic enzyme profiles. These enzymatic data are of diagnostic value within the Bacteroidaceae and may serve as useful markers for studying the ecological interrelationships of these bacteria.Keywords: Bacteroidaceae; Dehydrogenases; Enzyme patterns.

The growth response of Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola to peptides (supplied as trypticase) and amino acids (supplied as casamino acids) was measured over 24 hours by monitoring changes in alternating-current conductivity at 37 degrees C. All species utilized peptides preferentially over amino acids. These results are consistent with those obtained previously with conventional growth-response experiments. The duration of the linear growth response to trypticase of P. gingivalis was 9.7 hours, whereas that of both F. nucleatum and T. denticola was24 hours. By contrast, there was more uniformity in the utilization of amino acids from the casamino acid mixture, which previously has been shown to be a poor growth substrate. Furthermore, subtle differences in growth patterns, such as the ability of F. nucleatum to metabolize its storage glycopolymers before utilizing amino acids, were clearly evident. The present method provides an excellent means of studying bacterial growth kinetics and delineating bacterial/substrate specificities of both synthetic and natural substrates.

A total of 31 strains of Prevotella intermedia were subjected to DNA-DNA hybridization and were characterized by performing physiological tests and by performing a multilocus enzyme analysis, using malate dehydrogenase and glutamate dehydrogenase. All of the strains assigned to P. intermedia fermented glucose and sucrose, hydrolyzed starch but not esculin, and produced indole, acetic, isobutyric, isovaleric, and succinic acids as metabolic end products. The results of DNA reassociation experiments performed with the reference probe permitted separation of the strains into two well-defined homology groups. In addition, strains with DNAs that hybridized with DNA from strain ATCC 25611T (T = type strain) had high levels of peptidase activity and cleaved lipid substrates (4-methylumbelliferyl laurate and 4-methylumbellifelyl elaidate). Multilocus enzyme electrophoresis revealed two electromorphic profiles, one characteristic of strain ATCC 25611T and the other characteristic of strain ATCC 33563T. We propose that a new species, Prevotella nigrescens, should be created for the genetically distinct group of strains that hybridized with strain ATCC 33563T. Strain ATCC 33563 is designated the type strain of P. nigrescens.

Strains of Prevotella intermedia which have been characterized previously by a variety of biochemical and chemical techniques were subjected to multilocus enzyme electrophoresis and DNA-DNA reassociation. Two separate groups of strains were discernible. One had high homology with the type strain ATCC 25611 DNA probe and electrophoretically fast migrating malate dehydrogenase (MDH) (3.8–4.0 cm) and glutamate dehydrogenase (GDH) (3.2–3.4 cm) bands. The other group of strains hybridized with the DNA probe of reference strain ATCC 33563 and possessed slower moving enzymes (MDH, 3.0 cm; GDH, 1.4–1.6 cm). These results indicate that strains currently identified as P. intermedia comprise at least two geno-species, and that the criteria used to define this species are inadequate.

In order to survive in the host and initiate infection,Salmonella entericaneeds to undergo a transition between aerobic and anaerobic growth by modulating its central metabolic pathways. In this study, a comparative analysis of the proteome ofS. entericaserovar Typhimurium grown in the presence or absence of oxygen was performed. The most prominent changes in expression were measured in a semiquantitative manner using difference in-gel electrophoresis (DIGE) to reveal the main protein factors involved in the adaptive response to anaerobiosis. A total of 38 proteins were found to be induced anaerobically, while 42 were repressed. The proteins of interest were in-gel digested with trypsin and identified by MALDI TOF mass spectrometry using peptide mass fingerprinting. In the absence of oxygen, many fermentative enzymes catalysing reactions in the mixed-acid or arginine fermentations were overexpressed. In addition, the enzyme fumarate reductase, which is known to provide an alternative electron acceptor for the respiratory chains in the absence of oxygen, was shown to be induced. Increases in expression of several glycolytic and pentose phosphate pathway enzymes, as well as two malic enzymes, were detected, suggesting important roles for these in anaerobic metabolism. Substantial decreases in expression were observed for a large number of periplasmic transport proteins. The majority of these are involved in the uptake of amino acids and peptides, but permeases transporting iron, thiosulphate, glucose/galactose, glycerol 3-phosphate and dicarboxylic acids were also repressed. Decreases in expression were also observed for a superoxide dismutase, ATP synthase, inositol monophosphatase, and several chaperone and hypothetical proteins. The changes were monitored in two different isolates, and despite their very similar expression patterns, some variability in the adaptive response to anaerobiosis was also observed.

Human isolates of Fusobacterium nucleatum subspecies appear to colonize different niches in the oral cavity, which may be reflected in their nutritional properties. Consequently the utilization of nitrogenous substrates, their sources of energy (supplied here as amino acids) were compared between the 3 subspecies using the reference strain and fresh clinical isolates of each subspecies. All strains incorporated mainly acidic and basic amino acids but significant differences occurred between subspecies. Both reference and clinical isolates of F. nucleatum subspecies polymorphum utilized all amino acids in the medium but the levels of glutamate, arginine and cysteine were noticeably higher in the reference strain. By contrast, F. nucleatum subspecies fusiforme used a very restricted range of amino acids, of which only glutamate, arginine, histidine and cysteine were taken up at greater than 0.5 mM. F. nucleatum subspecies nucleatum utilized fewer amino acids than F. nucleatum subspecies polymorphum but higher concentrations were taken up by the former. Clinical isolates of F. nucleatum subspecies nucleatum incorporated polar and nonpolar neutral amino acids poorly but their levels increased steadily as a clinical isolate was subcultured over a period of 4 months, and was eventually similar to the reference strain. The effect of adding the key catabolic substrate, glutamate (10 mM), on the amino acid uptake profile of F. nucleatum subspecies nucleatum resulted in the complete suppression of the dibasic amino acids arginine, ornithine and histidine. Strains of this subspecies could grow on glutamate as a major source of carbon and energy but, morphologically, the cells appeared somewhat distended and had a tendency to clump.

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 × 10−5 and 0.4 × 10−5, respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2′-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45°C for 10 min, while incubation at 60°C abolished all activity.

Glutamate and aspartate are key amino acids for catabolism byFusobacterium nucleatum subspecies andPorphyromonas gingivalis respectively. However, peptides such as yeast extract are their preferred sources of energy. To determine more precisely the possible nature of these peptides, we examined the utilization of these amino acids and their corresponding peptides by cell suspension experiments with a fluorescamine labeling technique. High molecular weight (M.W.) polyglutamate (>40,000) was poorly utilized by all taxa, whereas 95% of its low-M.W. peptide (2,000–5,000) was used byF. nucleatum subspeciesnucleatum, but the remaining two subspecies utilized 90% polyaspartate within the same period. ForF. nucleatum subspeciesnucleatum as the test organism, T0.5 (the time taken to use 50% of the test substrate) was 1.7 h longer for glutamate than for the homopolymer. Furthermore, in the presence of both substrates, polyglutamate suppressed the uptake of glutamate until about 50% (ca. 1.5 mmol/L) of the peptide was used, after which the incorporation of the free amino acid started. A similar pattern of utilization was observed inP. gingivalis with its preferred peptide polyaspartate, for which the T0.5 was three times shorter than its monomer, aspartate. Both species had the capacity to utilize the heteropolymer, poly aspartate/glutamate, but at a significantly slower rate than the corresponding homopolymer.

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) has been applied in large numbers of oncological studies but the microbiological field has not been extensively explored to date. This paper describes the application of SELDI-TOF MS in concert with a multi-layer perceptron artificial neural network (ANN) with a back propagation algorithm for the identification of Neisseria gonorrhoeae. N. gonorrhoeae, the aetiological agent of gonorrhoea, is the second most common sexually transmitted disease in the UK and USA. Analysis of over 350 strains of N. gonorrhoeae and closely related species by SELDI-TOF MS facilitated the design of an ANN model and revealed 20 ion peak descriptors of positive, negative and secondary nature that were paramount for the identification of the pathogen. The model performed with over 96 % efficiency when based on these 20 ion peak descriptors and exhibited a sensitivity of 95.7 % and a specificity of 97.1 %, with an area under the curve value of 0.996. The technology has the potential to link several ANN models for a comprehensive rapid identification platform for clinically important pathogens.

Three strains which resemble the type strain of Fusobacterium naviforme (ATCC 25832) by morphological and physiological criteria were isolated from human clinical specimens. All were non-fermentative, produced indole and, in common with other members of the genus Fusobacterium, butyrate was a major end-product of metabolism. Glutamate dehydrogenase and 2-oxoglutarate reductase were present in both taxa, but the enzymes of the test strains migrated to only about half the distance of that of strain ATCC 25832. The latter contained meso-diaminopimelic acid as its peptidoglycan dibasic amino acid whereas the test strains possessed meso-lanthionine. The wide divergence in DNA base composition between strain ATCC 25832 (49 mol% G + C) and the clinical isolates (ca 30–31 mol% G + C) was reflected in their low DNA-DNA homology (ca 5–15%). The present study therefore revealed major differences between F. naviforme (ATCC 25832) and the new isolates and indicate that the latter may belong to a hitherto undescribed taxon within the genus Fusobacterium.

1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit beta-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin. The K1, value (3 x 10(-5) M) determined by kinetic analysis of the hydrolysis of N alpha-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 degrees C, I = 0.1, catalysed by beta-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low Mr inhibitors of this enzyme. Computer modelling of the beta-trypsin-E64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson-Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (beta-trypsin numbering from Ile-1) in the S1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group. The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and alpha-gingivain (one of the extracellular enzymes from phyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.

Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10-11) for up to 6 h and 3 h at 25 degrees C, respectively, while at pH 6.5, 50% of their activities were lost within 2-3 h. The Km for malate oxidation catalysed by MDH was 5.8 x 10(-4) M while that for glucose-6-phosphate oxidation was 2.0 x 10(-4) M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.

Over the last decade, biochemical and chemical analyses have been used widely to study the intrageneric structure of Bacteroidaceae. New chromogenic substrates (e.g., naphthylamide-linked compounds) and fluorogenic substrates (e.g., 4-methylumbelliferyl or 7-amido-4-methyl-coumarin compounds) can be used to identify certain species within a few hours under aerobic conditions. Clarification of the taxonomy of many oral anaerobic species that are now considered important putative periodontal pathogens has permitted the development of panels of both polyclonal and monoclonal antibodies for their detection. Similarly, both DNA and RNA gene probes, derived through nucleic acid sequence analysis, have been constructed for several species; many such probes are now commercially available. In the long term, the application of these techniques will lead to a better understanding of the distribution, transmission, and ecological and clinical importance of many species that have hitherto remained poorly characterized.

The extracellular proteinase produced by Porphyromonas gingivalis, which has often been described as ‘trypsin-like’, was isolated by thiol-disulphide interchange covalent chromatography. Evidence from the method of isolation, thiolspecific inhibition reactivation cycle and thiol-specific reactivity probe kinetics, all involving 2-pyridyl disulphides, compels the view that this enzyme, for which the name gingivain is here proposed, is a cysteine proteinase. All of the hydrolytic activity towards α-N-benzoyl-L-arginine-4-nitroanilide(L-BAPNA) and azocasein in the P. gingivalis vesicle/supernatant mixture was shown to be thiol dependent by its complete inhibition by 2,2’-dipyridyl disulphide and complete reactivation by 2-mercaptoethanol. The vesicles and all of the hydrolytic activity were isolated by precipitation in 70 per cent saturated ammonium sulphate, centrifugation for 22 h at 150,000 g and 4°C. The cysteine proteinase was prepared in fully active form by sequential elution covalent chromatography on Sepharose-glutathione 2-pyridyl disulphide gel, which separated the enzyme from the vesicles and from other thiol-containing protein devoid of catalytic activity towards L-BAPNA. The fully active isolated enzyme was: (a) completely inhibited by reaction with 2,2’-dipyridyl disulphide with complete reactivation by 2-mercaptoethanol, and (b) shown to possess a key catalytic site characteristic, typical of many cysteine proteinases. Thus, stopped-flow kinetic analysis of the reaction of its catalytically essential thiol group with 2,2’-dipyridyl disulphide showed the reactivity to be minimum at ca. pH 6, behaviour characteristic of the existence of a catalytic site cysteine-histidine interactive system.Keywords: Gingivain; a cysteine proteinase; P. gingivalis proteinase.

It was recently proposed that the genus Bacteroides should be restricted to Bacteroides fragilis (the type species) and closely related organisms (viz., B. caccae, B. distasonis, B. eggerthii, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis, and B. vulgatus). By contrast, the moderately saccharolytic, predominantly oral Bacteroides species, which include B. melaninogenicus, B. oralis, and related species, form a phenotypically and phylogenetically coherent group of species which differ so significantly from the emended description of the genus Bacteroides that they should not be classified in the same genus. Therefore, we formally propose that these species be reclassified in a new genus, Prevotella. The type species is Prevotella melaninogenica.

Natural mixtures of bacterial menaquinones were separated according to the length and degree of saturation of the polyisoprenyl side-chain using ready made Merck RP-18F254 reverse phase thin-layer chromatography plates. The system described affords a simple and rapid means of menaquinone characterization.

Aspartate as asparagine catabolism was studied in representative strains ofBacteroides intermedius strain T588 andB. gingivalis strain W83. Cell suspensions of both species deamidated asparagine. The enzyme asparaginase was constitutive and was unaffected by the addition of ammonium ions to the culture medium. The enzyme aspartase was not detected, but since malate dehydrogenase was known to occur and succinate was present as a major end product of metabolism, aspartate catabolism was postulated to occur via oxaloacetate, malate, and fumarate to succinate. All enzymes of this pathway were present in cell-free extracts, and some of the major properties of these enzymes were examined. The electron carriers cytochrome b and menaquinone-9 were present inB. gingivalis, whereasB. intermedius possessed cytochrome c and menaquinone-11. The membrane-bound enzyme fumarate reductase utilized NADH as an electron donor, but the reaction was inhibited by short wave ultraviolet radiation and 2-n-heptyl-4-hydroxyquinoline-N-oxide.

Growth ofBacteroides intermedius was promoted only moderately by glucose, and the incorporation of14C-glucose into cells was limited. WithBacteroides gingivalis growth promotion was negligible and glucose incorporation even more restricted. Both species grew prolifically on protein hydrolysates containing peptides, but grew poorly on acid-hydrolyzed casein even when supplemented with amino acids. These results are discussed in relation to the ecological distribution of these species compared to saccharolytic bacteroides.

We describe the comparative ultrastructures of bile-sensitive Bacteroides species which were isolated from oral cavities and ferment xylose and arabinose. Reference strains Bacteroides buccae ATCC 33574T(T = type strain), Bacteroides capillus ATCC 33690Tand ATCC 33691, and Bacteroides pentosaceus NP333Tand WPH61 and Bacteroides sp. strains ES42 and ES57 all had an extra surface layer (S-layer) outside the outer membrane. No S-layer was detected in Bacteroides oris ATCC 33573Tand ATCC 27518, Bacteroides oralis, Bacteroides veroralis, or Bacteroides sp. strains ES2759 and ES2834. The deoxyribonucleic acid guanine-plus-cytosine contents and both the malate dehydrogenase and glutamate dehydrogenase mobilities of the strains with an S-layer were identical. We suggest that the oral pentose-fermenting Bacteroides isolates with an S-layer may belong to the same species.

Bacteroides oulorum, a new nonpigmented, saccharolytic species from oral cavities, is described. This species differs both biochemically and chemically from related taxa, such as Bacteroides oralis, Bacteroides buccalis, Bacteroides denticola, Bacteroides veroralis, and Bacteroides pentosaceus. The major fermentation products in glucose broth are acetate and succinate. In common with other members of the genus Bacteroides, all strains contain malate dehydrogenase and glutamate dehydrogenase, but they differ from some species, such as Bacteroides fragilis, in lacking glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The long-chain fatty acids are predominantly of the straight-chain saturated, iso- and anteiso-methyl branched types, with 12-methyltetradecanoic acid constituting the major acid. Strains of B. oulorum can be clearly distinguished from other oral Bacteroides species by the presence of major amounts of unsaturated menaquinones with 10 isoprene units. The deoxyribonucleic acid base composition of B. oulorum is 45 to 46 mol% guanine plus cytosine. The type strain is WPH 179 (= NCTC 11871).

The growth yields of 10 strains ofBacteroides fragilis isolated from a variety of clinical sites were determined in (a) basal medium, (b) basal medium plus heme, and (c) basal medium plus heme and menadione. The molar growth yield values, expressed as a function of glucose (YG) and ATP produced (YATP) for 24 h and 48 h were used for a comparison of different strains. Considerable variation occurred among strains, but in general only the results from 24-h grown cells were reproducible. After this period, the microscopic appearance of cells changed dramatically from well-formed, intact cells to large collections of extracellular vesicles and lysed cells. All strains were stimulated by heme, but marked differences occurred among strains. The addition of heme and menadione to the basal medium increased the YG values of some strains, whereas others were unaffected. Heme-cultured cells produced acetate, propionate, and succinate as major metabolic end products and possessed cytochrome b, menaquinone-10, and fumarate reductase activity. Strain NCTC 9343 grown without added heme by continuous culture or batch culture produced cells that were morphologically and biochemically similar. Under both conditions these cells lacked cytochromes, menaquinones, and fumarate reductase activity, but produced high levels of lactate and fumarate together with lower levels of acetate, propionate, and succinate.

The electrophoretic mobilities of glutamate dehydrogenase and 2-oxoglutarate reductase were compared for three reference strains and 30 human, oral isolates of Fusobacterium nucleatum. Both enzymes allowed the same strains to be grouped into three electrophoretic clusters, designated groups Fn-1, Fn-2, and Fn-3. Group Fn-1 contained the type strain of F. nucleatum, strain ATCC 25586, and nine clinical isolates. Group Fn-2 comprised 20 strains and appeared to contain the strains of F. nucleatum that are isolated most commonly from oral cavities. Strain NCTC 10953 (formerly “Fusobacterium polymorphum”) was a member of this cluster. Strains of group Fn-3 were rarely isolated; this group contained three isolates and reference strain NCTC 11362, which was listed previously as “Fusobacterium fusiforme.” The deoxyribonucleic acid (DNA) base compositions of all strains were between 25 and 27 mol% guanine plus cytosine. Under optimal conditions of DNA-DNA hybridization, all of the strains exhibited high levels of DNA homology (73 to 99%) to the three reference DNA probes belonging to groups Fn-1, Fn-2, and Fn-3. However, under stringent DNA hybridization conditions there was evidence of more genetic homogeneity within each group.

The cysteine proteinase produced in the culture supernatant of Porphyromonas gingivalis was extensively purified. Haemagglutination type assays in which the enzyme was titrated against a fixed concentration of erythrocytes, showed that low levels of enzyme directly caused lysis of the red blood cells. However, using the same assay, the presence of stoichiometric amounts of the thiol blocking agent, 2,2′-dipyridyl disulphide (2-PDS) specifically inhibited the action of the enzyme or its haemagglutination with W83 cells or vesicles. In all cases, electron micrographs revealed that in the presence of 2-PDS the erythrocytes remained intact. Thiol activator free enzyme or aerated, inactivated enzyme had no effect on the red blood cells. These results show conclusively that the secreted cysteine proteinase of P. gingivalis causes lysis of erythrocytes and must now be regarded as a potent virulence determinant of P. gingivalis.

DNA from representative strains of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 was digested with restriction enzymes Eco RI and Taq I and the electrophoretically separated fragmetns hybridized with a 32 P-16S rRNA gene probe from E. coli . The rRNA gene restriction patterns from DNA digested with either enzyme allowed the clustering of strains into the three subgroups. However, Taq I digested DNA yielded a wider distribution of taxonomically useful bands (ca 0.65 ± 14.3 kbp) and the pattern produced was characteristic of each subgroup. The present method is a simple and reliable means of identifying the three subgroups of F. nucleatum and provides a useful method for further studies of the heterogeneity of F. nucleatum .

The lipid compositions of Rothia dentocariosa was investigated. All of the strains tested possessed closely related lipid profiles consisting of predominantly straight-chain saturated and methyl branched long-chain fatty acids, unsaturated menaquinones with seven isoprene units and a polar lipid composition comprising diphosphatidylglycerol, phosphatidylglycerol and a diglycosyldiacylglycerol. The results of the present study indicate Rothia dentocariosa is a good and distinct taxon. The lipid data however does not support the classification of Rothia dentocariosa in the family Actinomycetaceae.

A chemically defined medium (BGDM) has been developed specifically forBacteroides gingivalis. The medium contains 4 amino acids, 5 mineral salts, cysteine hydrochloride as a reducing agent, and the growth factors hemin and menadione. Eight strains ofB. gingivalis have been subcultured repeatedly in this medium with no apparent changes in colonial or cellular morphology. The metabolic end products of strains grown in this medium were reproducible and yielded patterns similar to those produced by cells cultured in complex media. The growth rates were about 50% slower than those of cells grown in a complex medium, and the growth rate constants ranged between 0.013 and 0.067 H−1. When the defined medium was supplemented with protein hydrolysates such as trypticase, proteose peptone, bactocasitone, or yeast extract, at concentrations up to 1.0%, growth increased. No such growth increase was observed in the medium supplemented with casamino acids. Thus a minimal medium can be formulated by adding one of the growth-enhancing protein hydrolysates to the defined medium at varying concentrations depending upon the growth yield required.

Various subspecies of Bacteroides melaninogenicus differ in their pigmentation. Subsp. asaccharolyticus produces protohaem almost exclusively, subsp. intermedicus both protohaem and a smaller proportion of protoporphyrin, and subsp. melaninogenicus mainly protoporphyrin with a trace of protohaem. As a consequence young colonies can be differentiated by their red fluorescence in u.v. light (365nm): subsp. asaccharolyticus does not fluoresce, subsp. intermedicus shows a limited fluorescence, and subsp. melaninogenicus shows a bright fluorescence. The pigments were isolated as the dimethyl esters of protohaemin and of protoporphyrin and identified by electronic spectroscopy, mass spectrometry and comparisons by t.l.c. Incorporation of delta-aminolaevulinate into these pigments was not detected, nor was porphobilinogen formation observed. Subsp. melaninogenicus grown in the presence of [14C]protohaemin formed [14C]protoporphyrin. This appears to represent a novel biological demetallation.

Cell surface ultrastructure and other surface properties of Porphyromonas gingivalis strain W50 and pleiotropic mutants W50/BP1 (brown), and W50/BE1 (beige) were studied. The percentage hydrophobicity of strains W50, W50/BP1, W50/BR1, and W50/BE1 gradually decreased from 24 to 9. Ruthenium red stained cells studied by transmission electron microscopy revealed a layer of extracellular polymeric material of varying thickness depending on the strain. The layer was thickest in W50/BP1 (15-20 nm), strains W50 and W50/BR1 both had a layer of 12-15 nm, while strain W50/BE1 completely lacked this layer. The results clearly showed that the hydrophobicity of P. gingivalis was related not only to the thickness of the layer but also to other factors like the composition of the capsular material, such that only strain W50/BE1, for example, showed no haemagglutinating activity. The surface properties of the pleiotropic mutants appeared to be stable characteristics as cells grown on either solid or in liquid media gave comparable results. The loss of virulence of the beige strain (W50/BE1) is probably partly due to the alteration of these surface properties. Both virulent and avirulent strains, however, possessed extracellular vesicles.