Identification and Quantification of Ascorbic Acid in Kiwi Fruit by High-Performance Liquid Chromatography

The content of ascorbic acid in kiwi fruit (Actinidia chinensisPlanch) of various cultivars was determined by high-performance liquid chromatography (HPLC). A minimal content of ascorbic acid was found in fruits of Gaivard cultivar: in juice – 5.44, skin – 1.14, and pulp – 4.20 mg/g.     

Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD⁺) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD⁺ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C_Ts) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD⁺ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD⁺ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r² = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Succession of Selected Strains of Acetobacter pasteurianus and Other Acetic Acid Bacteria in Traditional Balsamic Vinegar

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic vinegar acetification.The use of selected starter cultures (SSC) in fermented food production is widely applied throughout the food industry, in particular for wine, dairy products, sausages, and a variety of vegetables (3, 11). The advantages of their use are related to the improvement of the process control, hygiene, and quality with respect to fermented foods obtained through indigenous fermentation. Vinegar is one of the fermented beverages produced without SSC inoculation, in both small- and large-scale production, mainly for the following reasons: (i) the majority of vinegars have low commercial value, and often technological innovation is not considered profitable, and (ii) there is limited knowledge of the ecophysiology of acetic acid bacteria (AAB) due to the difficulty in accessing, sampling, isolating, and preserving strains (2, 12, 15, 16, 17). Among vinegars, traditional balsamic vinegar (TBV) is an Italian aged condiment produced by “seed vinegar,” the so-called “mother of vinegar” that is an indigenous starter culture withdrawn from acetifying vinegar through back-slopping procedures. The raw material is a fermented and cooked grape must (here indicated as must) at a soluble solids content ranging from 20 to 60°Bx (10). TBV production is regulated by denomination of protected origin guidelines that specify procedures and final product features. In particular, the raw material characteristics, the production process (e.g., must cooking, alcoholic fermentation, acetic oxidation, and ageing), features of the production area (no environmental condition management is permitted), and analytical and sensorial parameters are stated as follows: acidity (not less than 4.5% [wt/wt], expressed as grams of acetic acid per 100 g of product), density at 20°C (not less than 1.240 g per liter), color, aroma, and taste. The production is performed in wood barrels, and the process is carried out by sequential refilling to acetify the must and replace the volume lost by evaporation. AAB grow on the surface of liquid by biofilm formation. No addition of any substance can be made except for the acetifying must as a starter (7). Microbial studies of TBV reported culture-dependent and -independent approaches to evaluating AAB occurrence in TBV musts (5, 10). These studies highlighted the occurrence of Gluconacetobacter europaeus as a widespread indigenous species, as well as Acetobacter pasteurianus, Acetobacter aceti, and Acetobacter malorum. However, no comprehensive studies of AAB diversity and the correlation between species occurrence and technological steps of TBV production have been published, due mainly to the difficulty of easy access to AAB microflora in vinegar matrix by both culture-dependent and -independent approaches.Regarding production technology, at least one drawback of current production procedures has been acknowledged. It concerns the difficulty of start-up acetification, which affects the minimum acidity value required for the final product. In fact, some studies showed that many variables regulate AAB growth and activity. Above all is the sugar concentration among substrates and the temperature among physical parameters. To efficiently control the acetification start-up, it is necessary to understand the function of AAB responsible for the initial colonization of musts and to investigate the microbial succession suitable to complete the acetification. Our previous researches on TBV showed that AAB strains exhibit different growing abilities. In particular, strains of Acetobacter pasteurianus grow quickly on laboratory synthetic media, wine, and cooked must. In contrast, strains belonging to G. europaeus do not grow or grow very slowly on cooked and fermented must (9, 10).The goal of this study was to implement a laboratory SSC to test it on a factory scale for TBV production purposes. In particular, we focused our attention on the effect of A. pasteurianus strain AB0220 on the acetification and dynamics of species at the end of the process. The SSC effectiveness was assessed by monitoring analytical parameters (acetic acid, ethanol, and pH), species succession, and strain persistence during three stages by the following molecular analyses: PCR/denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus (ERIC)/PCR sequencing using genomic DNA extracted from biofilms recovered on GYC (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) plates.     

Metabolic Status of Bacteria and Fungi in the Rhizosphere of Ponderosa Pine Seedlings

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.     

Regulation of Root-Associated Methanotrophy by Oxygen Availability in the Rhizosphere of Two Aquatic Macrophytes

The relative importance of oxygen for root-associated methanotrophy was examined by using sediment-free, intact freshwater marsh plants (Pontederia cordata and Sparganium eurycarpum) incubated in split chambers. The root medium contained approximately 100 (mu)M methane. Methane oxidation was calculated from the difference between methane loss from chambers in the presence and absence of 1 mM 1-allyl-2-thiourea, a methanotrophic inhibitor. When the root medium was oxic, methane oxidation accounted for 88 and 63% of the total methane depletion for S. eurycarpum and P. cordata, respectively; the remainder represented diffusional loss to the atmosphere via roots, stems, and leaves. Under suboxic conditions, methane oxidation was not detectable for S. eurycarpum but accounted for 68% of total methane depletion for P. cordata. The introduction of a biological oxygen sink, Pseudomonas aeruginosa, resulted in complete loss of methane oxidation in S. eurycarpum chambers under oxic conditions, while methane consumption continued (51.6% of total methane depletion) in P. cordata chambers. The differences between plant species were consistent with their relative ability to oxygenate their rhizospheres: during a suboxic incubation, dissolved oxygen decreased by 19% in S. eurycarpum chambers but increased by 232% for P. cordata. An in situ comparison also revealed greater methanotrophic activity for P. cordata than S. eurycarpum.     

Radioisotope Assay for the Quantification of Sulfate-Reducing Bacteria in Sediment and Water

A radioisotope enrichment culture method was developed to estimate the physiologically active component of a population of sulfate-reducing bacteria in environmental water and sediment samples. Aliquots of water or sediment were added to 50-ml serum bottles filled with ³⁵S-sulfate broth incubated for approximately 30 h. After incubation, the disintegration rate per milliliter of spent medium was measured, and the percentage of loss of activity resulting from bacterial sulfate reduction was determined. This loss of sulfate from the medium was then translated to a specific number of Desulfovibrio desulfuricans cells that would reduce an equivalent amount of sulfate in the same incubation time. This comparison was done using a series of growth curves of D. desulfuricans covering a range of inoculum densities between 10² and 10⁷ cells. The radioassay was used to follow the effects of a pulp mill on a small anoxic river in Florida. The activity of the sulfate-reducing bacteria in the river was greatly suppressed when the mill was closed for annual maintenance. The initiation of waste treatment resulted in improved water quality in 1 week, but the river sediments required a month to show a 10-fold reduction in the population of sulfate-reducing bacteria.     

Growth Response and Modifications of Organic Nitrogen Compounds in Pure and Mixed Cultures of Lactic Acid Bacteria from Wine

The interactions between the proteolytic X₂L strain of Oenococcus oeni and the non-proteolytic 12p strain of Pediococcus pentosaceus were assayed. The characteristics of cell growth, protein degradation, and amino acid production of both strains were determined in pure and mixed cultures. O. oeni showed poor cell growth and greater ability in the release of amino acids to the extracellular medium, whereas P. pentosaceus showed a higher yield in cell production with a decrease in the amino acid concentration in the medium. P. pentosaceus especially consumed essential amino acids for growth, and O. oeni released several of the essential amino acids important for growth of P. pentosaceus. In the mixed culture, mutualism was observed. The higher activity of the proteolytic system of O. oeni in mixed culture produced an increase in cell growth and in the amount of essential amino acids released. These findings provide new knowledge about the metabolic interactions between lactic acid bacteria isolated from wine when proteins are degraded in mixed bacterial populations.     

Degradation and Utilization of Hemicellulose from Intact Forages by Pure Cultures of Rumen Bacteria

Several pure strains of rumen bacteria have previously been shown to degrade isolated hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources. This study was undertaken to determine whether similar hemicellulose degradation or utilization, or both, occurs from intact forages. Fermentations by pure cultures were run to completion by using three maturity stages of alfalfa and two maturity stages of bromegrass as individual substrates. Organisms capable of utilizing xylan or isolated hemicelluloses could degrade and utilize intact forage hemicellulose, with the exception of two strains of Bacteroides ruminicola which were unable to degrade or utilize hemicellulose from grass hays. Intact forage hemicelluloses were extensively degraded by three cellulolytic strains that were unable to use the end products; in general, these strains degraded a considerably greater amount of hemicelluloses than the hemicellulolytic organisms. Hemicellulose degradation or utilization, or both, varied markedly with the different species and strains of bacteria, as well as with the type and maturity stage of the forage. Definite synergism was observed when a degrading nonutilizer was combined with either one of two hemicellulolytic strains on the bromegrass substrates. One hemicellulolytic strain, which could not degrade or utilize any of the intact bromegrass hemicellulose alone, almost completely utilized the end products solubilized by the nonutilizer. Similar synergism, although of lesser magnitude, was observed when alfalfa was used as a substrate.     

Fermentation of Isolated Pectin and Pectin from Intact Forages by Pure Cultures of Rumen Bacteria

Studies on the rate and extent of galacturonic acid and isolated pectin digestion were carried out with nine strains of rumen bacteria (Butyrivibrio fibrisolvens H10b and D16f, Bacteroides ruminicola 23 and D31d, Lachnospira multiparus D15d, Peptostreptococcus sp. D43e, B. succinogenes A3c, Ruminococcus flavefaciens B34b, and R. albus 7). Only three strains, 23, D16f, and D31d, utilized galacturonic acid as a sole energy source, whereas all strains except A3c and H10b degraded (solubilized) and utilized purified pectin. Nutrient composition of the basal medium and separate sterilization of the substrate affected the rate and extent of fermentation for both substrates. Pectin degradation and utilization were measured with two maturity stages each of intact bromegrass and alfalfa. For bromegrass I, all strains tested (B34b, 23, D16f, D31d, D15d, and D43e) degraded a considerable amount of pectin and, with the exception of B34b, utilized most of what was degraded. Similar, but lower, results were obtained with bromegrass II, except for the two strains of B. ruminicola, 23 and D31d, which were unable to degrade and utilize pectin from this forage. All strains were able to degrade and utilize pectin from both maturity stages of alfalfa; however, values were considerably lower for strains 23 and D31d. Synergism studies, in which a limited utilizing strain, B34b, was combined with the limited degrading strain, D31d, resulted in a slight increase in degradation and a very marked increase in utilization of the pectin in all four forages. Similar results were obtained on both alfalfa substrates with a combination of strains B34b and D16f; however, no increases were observed with this combination on bromegrass.     

Assay and Characteristics of Circadian Rhythmicity in Liquid Cultures of Neurospora crassa

Previous work on circadian rhythms of Neurospora crassa has been done almost exclusively with cultures expressing rhythmic conidiation and growing on solid agar medium. Such conditions severely restrict the kinds of biochemical experiments that can be carried out. We have now developed systems which allow indirect assay of circadian rhythmicity in liquid culture. Neurospora was grown in glucose and acetate liquid media under conditions which result in a range of growth rates and morphologies. Liquid media were inoculated with conidia and the cultures were grown in constant light for 33 or 48 hours, by which time floating mycelial pads had formed. Experimental pieces of mycelium then were cut and placed in fresh new liquid medium. As controls, other pieces of mycelium were cut and put directly on solid agar medium in race tubes. All cultures were transferred to constant darkness at this time. This light-to-dark transition set the phase of the circadian clock of both the liquid and solid cultures. At various times after the light-to-dark transition, the mycelial pieces in the liquid were transferred in the dark to solid medium in race tubes, where they grew normally and conidiated rhythmically. Comparison of the phase of the rhythm in these race tubes to the controls demonstrated that, under appropriate conditions, the circadian clock of the liquid cultures functions normally for at least two cycles in constant conditions. Using these culture systems, a significantly greater variety of biochemical studies of circadian rhythmicity in Neurospora is now possible.     

酸奶发酵剂和乳酸菌生物技术育种

由新鲜牛奶制成的酸奶由于其丰富的营养、特殊的风味、爽滑的质构和良好的生理功能 ,倍受人们青睐。对酸奶乳酸菌的微生物种类、功能及其组成的酸奶发酵剂进行了综述 ,并简要介绍了乳酸菌生物工程育种方面的研究进展及酸奶乳酸菌研究领域一些令人感兴趣的课题。     

Separation and Quantification of Abscisic Acid and its Metabolites by High-performance Liquid Chromatography

A technique for the separation and quantitative estimation ofabscisic acid and its metabolic products, phaseic acid and dihydrophaseicacid by high performance liquid chromatography (HPLC) is described.The efficiency of two HPLC columns, LiChrosorb RP-18 and LiChrosorbNH₂ is compared. The method has been tested using extracts fromtomato plants and some of the results are presented. Abscisic acid, dihydrophaseic acid, phaseic acid, chromatography, tomato, Lycopersicon esculentum, Mill     

Inhibition of Methanogenesis by Methyl Fluoride: Studies of Pure and Defined Mixed Cultures of Anaerobic Bacteria and Archaea

Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.     

Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10² CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.     

Characterization of Growth and Metabolism of Commercial Strains of Propionic Acid Bacteria by Pressure Measurement

Dairy propionibacteria are essential starters for Emmental cheese manufacture. The behavior of three commercial strains of Propionibacterium freudenreichii subsp. shermanii (P.f. 1, P.f. 2 and P.f 3) were studied in a liquid medium under air and N₂ atmosphere using an on‐line pressure measurement technique. Growth kinetics and metabolite production were characterized under conditions usually reported as “optimal conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and also evaluated under “stressful conditions” (pH 5.2, NaCl 2 %, temperature 20 °C) simulating the cheese ripening conditions. In both cases, the effects of oxygen on growth were strain‐dependent. Under “stressful conditions”, two of the three strains were inhibited by oxygen under conditions of air atmosphere, while all three strains grew under conditions of N₂ atmosphere. In the latter case, the duration of the lag phase and the maximum rate of pressure variation were significantly different, however, no significant differences were found between the strains with regard to the total fermentation time. Under “optimal conditions” metabolite production was strain‐dependent. In an air atmosphere, all strains produced more acetate and CO₂ and less propionate than in a nitrogen atmosphere.     

Synergism in Degradation and Utilization of Intact Forage Cellulose, Hemicellulose, and Pectin by Three Pure Cultures of Ruminal Bacteria

Pure cultures of ruminal bacteria characterized as using only a single forage polysaccharide (Fibrobacter succinogenes A3c, cellulolytic; Bacteroides ruminicola H2b, hemicellulolytic; Lachnospira multiparus D15d, pectinolytic) were inoculated separately and in all possible combinations into fermentation tubes containing orchard grass as the sole substrate. Fermentations were run to completion, and then cultures were analyzed for digestion of cellulose plus degradation and utilization of hemicellulose and pectin. Addition of the noncellulolytic organisms, in any combination, to the cellulolytic organism F. succinogenes had little effect on overall cellulose utilization. F. succinogenes degraded but could not utilize hemicellulose; however, when it was combined with B. ruminicola, total utilization of hemicellulose increased markedly over that by B. ruminicola alone. L. multiparus was inactive in hemicellulose digestion, alone or in any combination. Although unable to degrade and utilize purified pectin, B. ruminicola degraded and utilized considerable quantities of the forage pectin. In contrast, L. multiparus was very active against purified pectin, but had extremely limited ability to degrade and utilize pectin from the intact forage. Both degradation and utilization of forage pectin increased when F. succinogenes was combined with B. ruminicola. Sequential addition of two cultures, allowing one to complete its fermentation before adding the second, was used to study synergism between cultures on forage pectin digestion. In general, synergistic effects did not appear to be related to a particular sequence of utilization. The ability of F. succinogenes to degrade and B. ruminicola to degrade and utilize forage pectin contradicts both previous and present data obtained with purified pectin. Thus, isolation and characterization of ruminal bacteria on purified substrates may be misleading with regard to their role in the overall ruminal fermentation.     

Axenic Culture of a Candidate Division TM7 Bacterium from the Human Oral Cavity and Biofilm Interactions with Other Oral Bacteria

The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 μm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.     

Comparison of the Morphology and Deoxyribonucleic Acid Composition of 27 Strains of Nitrifying Bacteria

The gross morphology, fine structure, and per cent guanine plus cytosine (GC) composition of deoxyribonucleic acid of 27 strains of nitrifying bacteria were compared. Based on morphological differences, the ammonia-oxidizing bacteria were separated into four genera. Nitrosomonas species and Nitrosocystis species formed one homogenous group, and Nitrosolobus species and Nitrosospira species formed a second homogenous group in respect to their deoxyribonucleic acid GC compositions. Similarly, the nitrite-oxidizing bacteria were separated into three genera based on their morphology. The members of two of these nitrite-oxidizing genera, Nitrobacter and Nitrococcus, had similar GC compositions, but Nitrospina gracilis had a significantly lower GC composition than the members of the other two genera.