Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov.

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.     

Biochemical properties of Fusobacterium naviforme and phenotypically similar isolates

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.     

Deoxyribonucleic acid homologies among strains of Bacteroides melaninogenicus and related species

Saccharolytic, black-pigmented Bacteroides strains, which at present belong to the species Bacteroides melaninogenicus were classified on the basis of deoxyribonucleic acid (DNA) base ratios and DNA hybridization studies. These strains were divided into several DNA homology groups, which showed no or low mutual DNA homology. A DNA homology group with a percentage guanine plus cytosine (G + C) of 42–43% was formed by three strains of Bact. melaninogenicus subsp. melaninogenicus ; the type strain of this subspecies, strain ATCC 25845, had about 60% DNA homology with this group. Strain ATCC 15930, which has been assigned to this subspecies, had a percentage G + C of 47% and showed no DNA homology with the former group. All strains of Bact. melaninogenicus subsp. intermedius had a percentage G + C of 39–45%. A DNA homology group was formed by eight strains of this subspecies. The type strain of Bact. melaninogenicus subsp. intermedius , ATCC 25611, showed relatively low DNA homology with this main DNA homology group. A strain of Bact. melaninogenicus subsp. intermedius serotype C1 showed no DNA homology with the other strains tested. Furthermore two strains labelled 'Bact. melaninogenicus subsp. levii' were found to form a distinct DNA homology group. On the basis of the DNA homology results, the strains, which at present are classified in the species Bact. melaninogenicus , were clearly distinguished from strains of Bact. asaccharolyticus and Bact. gingivalis , and also from strains of related non-pigmented Bacteroides species.     

Effects of temperature on germination and mitochondrial dehydrogenases in two soybean (Glycine max) cultivars

The effects of eight germination temperatures from 10°C to 35°C on germination and dehydrogenase activities of two soybean (Glycine max [L.] Merr.) cultivars were investigated after 48 h of seedling growth. Axis fresh weights of cv. Chippewa increased as germination temperature increased from 10°C to 35°C. In contrast, axis fresh weights for the cv. Wells increased more slowly with increasing temperature and reached a maximum at c. 25°C. In general, in vitro activities of glutamate dehydrogenase (GDH), NADP-isocitrate dehydrogenase (NADP-ICDH), and malate dehydrogenase (MDH) from the axes of cv. Chippewa correlated well with increases in axis fresh weights. GDH and MDH activities from axes of the cv. Wells also reflected increases in axis fresh weights although the correlation was not as evident as for the cv. Chippewa. NADP-ICDH activity from ‘Wells’ axes was highest at 35°C even though germination was poor at this temperature. GDH and MDH activities from cotyledons of both cultivars were not correlated with axis weight increases. No GDH activity was detected in ‘Wells’ cotyledons from seeds germinated at 35°C.     

Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level

Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.     

Relationships among isolates of oral haemophili as determined by DNA-DNA hybridization

In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G+C) content of DNAs from strains of Haemophilus segnis and Haemophilus para-influenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G+C content of 39.0 to 42.9% and were 49–92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70–81% homologous with the ATCC 9796 DNA probe and had a G+C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G+C content of 42.4%, and was 65–78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.     

体外培养骨髓巨核细胞脱氢酶活性的细胞化学观察

本文采用液体培养体系结合酶细胞化学方法,对体外培养不同发育阶段的小鼠肾髓巨核细胞乳酸脱氢酶、苹果酸脱氢酶、谷氨酸脱氢酶的活性变化进行了动态观察。在9天培养期间,巨核细胞的增殖数在5—7天达到高峰,并随时间有不同程度分化。对培养3、5、7、9天的巨核细胞进行酶细胞化学研究,结果表明,巨核细胞在发育成熟前,三种酶活性均有增高。提示巨核细胞在分化过程中,糖酵解及三羧酸循环代谢均有增强。     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract 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°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ 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.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

Glutamate dehydrogenase activity profiles for type strains of ruminal Prevotella spp.

The glutamate dehydrogenase (GDH) activities for the type strains of Prevotella ruminicola (strain 23), Prevotella brevis (strain GA33), and Prevotella bryantii (strain B(1)4) were assessed by a combination of enzyme assays and analysis of migration patterns of GDH proteins following nondenaturing polyacrylamide gel electrophoresis. Unlike results with most other prokaryotes, but similar to results with other members of the family Bacteroidaceae, NADPH-utilizing specific activity was greatest in all species following ammonia-limited growth. Similar also to previous findings with P. bryantii, the NAD(P)H-utilizing GDH activity of P. ruminicola can be attributed to a single protein. However, P. brevis produces an additional GDH protein(s) in response to growth with peptides. These results conclusively demonstrate that all type strains of the ruminal Prevotella sp. grouping possess GDH activity.     

RAPD fingerprinting for the distinction of Prevotella intermedia sensu stricto from Prevotella nigrescens

A collection of 70 oral strains including reference strains and clinical isolates identified as Prevotella intermedia sensu lato was constituted to cover a large clinical and geographical diversity. Electrophoresis of the enzyme malate dehydrogenase allowed the identification of the 70 study strains as Prevotella intermedia sensu stricto (n= 36), Prevotella nigrescens (n= 31) and three unclassified strains. By using four primers, DNA fingerprints were generated from 20 strains as random amplified polymorphic DNA (RAPD). Matching co-migrating amplicon positions by pairwise comparison allowed the clustering of the fingerprints as two groups coincident with the P. intermedia/P. nigrescens assignment by enzyme electrophoresis of malate dehydrogenase. Our data suggest that isolates identified asP. intermedia sensu lato by conventional criteria can be speciated asP. intermedia sensu stricto or P. nigrescens by RAPD fingerprinting.     

Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of fusobacterium nucleatum subsp. nucleatum ATCC 25586T

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (F. nucleatum ATCC 25586T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586T, especially with regard to the determination of the authenticity of the strain.     

Relations of enzymes inAspergillus repens grown under sodium chloride stress

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2m sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2m NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na⁺ (as NaCl) when added up to 100mm in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.     

Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.     

In vivo fluxes in the ammonium-assimilatory pathways in corynebacterium glutamicum studied by 15N nuclear magnetic resonance

Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)-glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.     

Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.     

Characteristics of glutamate dehydrogenase, a new diagnostic marker for the genus Fusobacterium

Enzymes representative of carbohydrate and nitrogen metabolism were screened for their presence and activity amongst species of the genus Fusobacterium. Glutamate dehydrogenase (GDH) was reliably detected in all 25 strains studied. The pH profile of this enzyme and the DNA base composition of selected strains were also determined. DNA base composition of selected strains ranged between 28-32.9 mol% G + C. GDH was active between pH 7.5-11.5 but two pH profiles of activity, with optima at 9.5 and 10.5, were discernible among species. Apart from Fusobacterium nucleatum, which had a heterogeneous enzyme pattern, the GDH electrophoretic mobility was constant within a species but in a few cases the enzyme bands overlapped. A combination of the pH profile, the GDH electrophoretic pattern and the DNA base composition provided clear separation of the test organisms into discrete groups; however, a larger number of strains must be examined before the full potential of these tests can be evaluated.     

Clostridia from Grana Cheese

Forty strains of proteolytic and saccharolytic clostridia isolated from Grana cheese were re-identified by DNA-DNA homology. Thirty culture collection strains were also examined for comparison. Organisms of the species Clostridium tyrobutyricum which present variability in phenotypic characteristics were found to constitute a homogeneous group, genetically unrelated to all the reference or competitor strains used. The proteolytic clostridia from cheese show a high level of homology with the reference strains C.sporogenes ATCC 319 and C. sporogenes ATCC 3584 and negligible homology with C. bifermentans ATCC 19299. Three strains with phenotypic characters very similar to those of the species C. butyricum present a level of DNA homology with C. butyricum ATCC 19398 ranging from 61–71%. In the light of the results obtained some taxonomic conclusions have been drawn.