A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria.

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

Isolation and identification of a human intestinal bacterium capable of daidzein conversion.

Equol, which produced from daidzein (one of the principal isoflavones), is recognized to be the most resultful in stimulating an estrogenic and antioxidant response. The daidzein transformation was studied during fermentation of five growth media inoculated with feces from a healthy human, and a daidzein conversion strain was isolated. To enrich the bacterial population involved in daidzein metabolism in a complex mixture, fecal samples were treated with antibiotics. The improved propidium monoazide combined with the quantitative polymerase chain reaction (PMAxx-qPCR) assay showed that the ampicillin treatment of samples did result in a reduction of the total visible bacteria counts by 52.2% compared to the treatment without antibiotics. On this basis, the newly isolated rod-shaped, Gram-positive anaerobic bacterium, named strain Y11 (MN560033), was able to metabolize daidzein to equol under anaerobic conditions, with a conversion ratio (equol ratio: the amount of equol produced/amount of supplemented daizein) of 0.56 over 120 h. The 16S rRNA partial sequence of the strain Y11 exhibited 99.8% identity to that of Slackia equolifaciens strain DZE (NR116295). This study will provide new insights into the biotransformation of equol from daidzein by intestinal microbiota from the strain-level and explore the possibility of probiotic interventions.

Clostridium saccharogumia sp. nov. and Lactonifactor longoviformis gen. nov., sp. nov., two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside.

Two anaerobic bacteria involved in the conversion of the plant lignan secoisolariciresinol diglucoside were isolated from faeces of a healthy male adult. The first isolate, strain SDG-Mt85-3Db, was a mesophilic strictly anaerobic Gram-positive helically coiled rod. Based on 16S r RNA gene sequence analysis, its nearest relatives were Clostridium cocleatum (96.7% similarity) and Clostridium ramosum (96.6%). In contrast to these species, the isolate was devoid of alpha-galactosidase and -glucosidase and did not grow on maltose, melibiose, raffinose, rhamnose and trehalose. The hypothesis that strain SDG-Mt85-3Db represents a new bacterial species of the Clostridium cluster XVIII was confirmed by DNA-DNA hybridisation experiments. The G+C content of DNA of strain SDG-Mt85-3Db (30.7+/-0.8 mol%) was comparable with that of Clostridium butyricum, the type species of the genus Clostridium. The name Clostridium saccharogumia is proposed for strain SDG-Mt85-3Db (=DSM 17460T=CCUG 51486T). The second isolate, strain ED-Mt61/PYG-s6, was a mesophilic strictly anaerobic Gram-positive regular rod. Based on 16S rRNA gene sequence analysis, its nearest relatives were Clostridium amygdalinum (93.3%), Clostridium saccharolyticum (93.1%) and Ruminococcus productus (93.0%). The isolate differed from these species in its ability to dehydrogenate enterodiol. It also possessed alpha-arabinosidase and -galactosidase and had a higher G+C content of DNA (48.0 mol%). According to these findings, it is proposed to create a novel genus, Lactonifactor, and a novel species, Lactonifactor longoviformis, to accommodate strain ED-Mt61/PYG-s6. The type strain is DSM 17459T (=CCUG 51487T).

[A new hydrogen-producing strain and its characterization of hydrogen production].

An anaerobic, thermophilic, hydrogen-producing strain T42 was obtained from a hot spring of South Mountain District, Tibet. Cells are Gram-positive, mobile rod-shaped. Spores were not observed. Temperature range for growth is 32 degrees C to 69 degrees C (optimum temperature, 60 degrees C - 62 degrees C), and pH range for growth is 5.0 to 8.8 (optimum pH, 7.0 - 7.5). The generation time is around 30 min. Organic nitrogen sourc is required for growth. Strain T42 utilizes a wide range of carbohydrates, including starch, dextrin, sucrose, cellobiose, fructose, maltose, ribose, glycogen and galactose. Acetate, ethanol, H2 and CO2 are the end products of glucose fermentation. The (G + C) content of strain T42 is 31.2 mol%. Phylogenetic analysis based on the 16S rDNA sequence similarity indicates that strain T42 is the closest relative to Thermobrachium celere and Caloramator indicus. Biological characteristics and phylogenetic analysis of the 16S rDNA gene indicate the new strain belongs to the genus Thermobrachium. Strain T42 produces H2 from glucose at maximal level when growing at 62 degrees C and initial pH 7.2, the hydrogen yields and maximal hydrogen production rate are 1.06 mol H2/mol glucose and 24.0 mmol H2/gDW/h, respectively. Strain T42 also produced H2 by fermentating from a variety of carbohydrates. 20 mmol/L Magnesium and 2 mmol/L iron increase the hydrogen production content by 20% and 23.3%, respectively, but nickel has no effect on the hydrogen production. In the co-culture of strain T42 and methane-producing strain M. thermautotrophicus Z245, hydrogen pressure is dramatically decreased, meanwhile deduced H2 production and the consumption of glucose are increased markedly by 2.8 fold and 1 fold, and the ratio of acetate/ethanol is enhanced froml to 1.7.

The apical border plaque in severe periodontitis. An ultrastructural study.

This study concerns the apical border (AB) plaque in relation to severe forms of periodontitis (SP), including juvenile, post-juvenile, and rapidly progressing periodontitis. Twenty-four (24) teeth from 16 patients with SP were examined by transmission electron microscopy (TEM). The AB was not discrete, with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB the established plaque consisted of a layer of Gram-positive cocci and ghost cells and a superficial layer mainly of Gram-negative morphotypes, including cocci, rods, filaments, fusiforms, and spirochetes. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with ghost cells in abundance. Ruthenium red, alcian blue-lanthanum nitrate, and safranin O were used to label matrix polyanionic macromolecules, and periodic acid (thiosemicarbazide) silver proteinate for intracellular polysaccharide (IPS). The matrix components were mainly fibrillar. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall, and cytoplasmic IPS granules. The latter varied in distribution and were evident even in the most apically advanced intact microorganisms. The results indicate that IPS and some matrix features of the apical border plaque in severe periodontitis in certain aspects resemble those of sub-contact area plaque on children's teeth, in health or associated with early chronic gingivitis, and with those in chronic adult periodontitis. They also suggest the establishment of acidic regions in the microniche at the bottom of the periodontal pocket in the various forms of periodontitis differing in rate of progression. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in severe periodontitis, similar to those in chronic adult periodontitis.

Degradation of arginine and other amino acids by butyrate-producing asaccharolytic anaerobic Gram-positive rods in periodontal pockets.

The use of 20 amino acids by butyrate-producing asaccharolytic anaerobic Gram-positive rods (AAGPRs) in periodontal pockets, i.e. Eubacterium minutum, Filifactor alocis, E. infirmum, E. sulci and E. saphenum, was studied. E. minutum used only arginine and lysine, and produced substantial amounts of butyrate and ammonia as the main metabolic products from arginine, and acetate, butyrate and ammonia from lysine. Fi. alocis used arginine alone and produced butyrate and ammonia. E. infirmum, E. sulci and E. saphenum used lysine alone and produced acetate, butyrate and ammonia. The growth of these bacterial species was supported and enhanced by arginine and/or lysine enriched to culture media, but not by the other amino acids. Arginine deiminase, ornithine carbamoyltransferase and carbamate kinase activity were detected in the cell-free extract of E. minutum, suggesting that arginine was metabolised to citrulline initially, and subsequently to ornithine and carbamoyl phosphate. Ornithine and carbamoyl phosphate were further converted to butyrate, and carbon dioxide and ammonia, respectively. Enzymatic activity of arginine deiminase and ornithine carbamoyltransferase was not detected in Fi. alocis, indicating that Fi. alocis converted arginine to ornithine directly, not via citrulline, and further to butyrate.

Carbon isotope effects associated with autotrophic acetogenesis.

The carbon kinetic isotope effects associated with synthesis of acetate from CO2 and H2 during autotrophic growth of Acetobacterium woodii at 30 degrees C have been measured by isotopic analyses of CO2, methyl-carbon, and total acetate. Closed systems allowing construction of complete mass balances at varying stages of growth were utilized, and the effects of the partitioning of carbon between CO2 and HCO3- were taken account. For the overall reaction, total carbonate --> total acetate, isotope effects measured in replicate experiments ranged from -59.0 +/- 0.9% to -57.2 +/- 2.3%. Taking into account all measurements, the weighted mean and standard deviation are -58.6 +/- 0.7%. There is no evidence for intramolecular ordering in the acetate. The carbon isotopic composition of sedimentary acetate, otherwise expected to be near that of sedimentary organic carbon, is likely to be depleted in environments in which autotrophic acetogenesis is occurring.

Acetogenic bacteria: what are the in situ consequences of their diverse metabolic versatilities?

The four decades of the now classic studies by Harland G. Wood and Lars G. Ljungdahl lead to the resolution of the autotrophic acetyl-CoA 'Wood/Ljungdahl' pathway of acetogenesis. This pathway is the hallmark of acetogens, but is also used by other bacteria, including methanogens and sulfate-reducing bacteria, for both catabolic and anabolic purposes. Thus, the pathway is wide spread in nature and plays an important role in the global turnover of carbon. Because most historical studies with acetogens focused on the biochemistry of the acetyl-CoA pathway, the metabolic diversity and ecology of acetogens remained largely unexplored for many years. Although acetogens were initially conceived to be a somewhat obscure bacteriological group with limited metabolic capabilities, it is now clear that acctogens are arguably the most metabolically diverse group of obligate anaerobes characterized to date. Their anaerobic metabolic arsenal includes the capacity to oxidize diverse substrates, including aromatic, C1, C2, and halogenated compounds, and engage a large number of alternative energy-conserving, terminal electron-accepting processes, including classic fermentations and the dissimilation of inorganic nitrogen. In this regard, one might consider acetogens on a collective basis as the pseudomonads of obligate anaerobes. By virtue of their diverse metabolic talents, acetogens can be found in essentially all habitats. This review evaluates the metabolic versatilities of acetogens relative to both the engagement (regulation) of the acetyl-CoA pathway and the ecological roles likely played by this bacteriogical group.

Characteristics of granular sludge in a single upflow sludge blanket reactor treating high levels of nitrate and simple organic compounds.

Simultaneous denitrification and methanogenesis were accomplished in a single upflow sludge blanket (USB) reactor. More than 99% and 95% of nitrate and chemical oxygen demand (COD) removal rates were obtained at a loading of 600 mg NO3-N/L x d and 3,300 mg COD/L x d, respectively. The specific denitrification rate (SDR) increased as COD/NO3-N ratios decreased. Maximum SDR with acetate could reach 1.05 g NO3-N/gVSS x d. Significant sludge flotation was observed at the top of the reactor due to the change of microbial composition and the formation of hollow granules. Granules became fluffy and buoyant due to the growth of denitrifiers. Microscopic examination showed that granules exhibited layered structure and they were mainly composed of Methanosarcina sp., Pseudomonas sp., and rod-shaped bacteria.

Performance and microbial community analysis of two-stage process with extreme thermophilic hydrogen and thermophilic methane production from hydrolysate in UASB reactors.

The two-stage process for extreme thermophilic hydrogen and thermophilic methane production from wheat straw hydrolysate was investigated in up-flow anaerobic sludge bed (UASB) reactors. Specific hydrogen and methane yields of 89 ml-H(2)/g-VS (190 ml-H(2)/g-sugars) and 307 ml-CH(4)/g-VS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total hydraulic retention time (HRT) of 4 days . The energy conversion efficiency was dramatically increased from only 7.5% in the hydrogen stage to 87.5% of the potential energy from hydrolysate, corresponding to total energy of 13.4 kJ/g-VS. Dominant hydrogen-producing bacteria in the H(2)-UASB reactor were Thermoanaerobacter wiegelii, Caldanaerobacter subteraneus, and Caloramator fervidus. Meanwhile, the CH(4)-UASB reactor was dominated with methanogens of Methanosarcina mazei and Methanothermobacter defluvii. The results from this study suggest the two stage anaerobic process can be effectively used for energy recovery and for stabilization of hydrolysate at anaerobic conditions.

Plasmid-mediated transfer of the bla(NDM-1) gene in Gram-negative rods.

The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase NDM-1 (New Delhi metallo-ß-lactamase) producers, mostly in Enterobacteriacae. Five bla(NDM) (-1) -positive plasmids of different incompatibility groups (IncL/M, FII, A/C and two untypeable plasmids) from clinical Enterobacteriaceae were evaluated for conjugation properties and host specificity. Successful conjugative transfers were obtained using all tested enterobacterial species as recipients (Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Proteus mirabilis) and all plasmid types. Conjugation frequencies varied from 1 × 10(-4) to 6 × 10(-8) transconjugants per donor. Higher conjugation rates were obtained for two plasmids at 30 °C compared with that observed at 25 and 37 °C. Carbapenems used as selector did not lead to higher conjugation frequencies. None of the five plasmids was transferable to Acinetobacter baumannii or Pseudomonas aeruginosa by conjugation. This work underlines how efficient the spread of the carbapenemase bla(NDM) (-1) gene could be among Enterobacteriaceae.

Salsuginibacillus halophilus sp. nov., a halophilic bacterium isolated from a soda lake.

A Gram-stain-positive, rod-shaped, endospore-forming, halophilic, alkalitolerant bacterium, designated halo-1(T), was isolated from sediment of Xiarinaoer soda lake, located in the Inner Mongolia Autonomous Region of China. Strain halo-1(T) grew in the presence of 9-30 % (w/v) NaCl (optimum 19 %) and at pH 5-10 (optimum pH 9). The cell-wall peptidoglycan contained meso-diaminopimelic acid and the major respiratory isoprenoid quinone was MK-7. The predominant cellular fatty acids of the isolate were anteiso-C(15 : 0) (58.35 %), anteiso-C(17 : 0) (12.89 %) and C(16 : 0) (6.52 %). The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, glycolipid and a phospholipid of unknown structure. The DNA G+C content of the strain was 46.4 mol%. On the basis of 16S rRNA gene sequence similarity, strain halo-1(T) showed the highest similarity (93.9 %) to Salsuginibacillus kocurii CH9d(T). Strain halo-1(T) could be clearly differentiated from its closest phylogenetic relative on the basis of several phenotypic, genotypic and chemotaxonomic features. Therefore, strain halo-1(T) represents a novel species, for which the name Salsuginibacillus halophilus sp. nov. is proposed, with the type strain halo-1(T) (=CGMCC 1.7653(T) =NBRC 104934(T)).

Antibiotic resistance in lactic acid bacteria and Micrococcaceae/Staphylococcaceae isolates from artisanal raw milk cheeses, and potential implications on cheese making.

Antibiotic susceptibility against 19 antimicrobial agents was evaluated in isolates of the genera Lactococcus (46 isolates), Leuconostoc (22), Lactobacillus (19), Staphylococcus (8), Enterococcus (7), and Microccoccus/Kocuria (5) obtained from the predominant microflora of nonrecent and recent types of artisanal raw cow's milk cheeses. Beta-lactams showed broad activity against all genera, although leuconostocs and lactobacilli were highly resistant to oxacillin (80% to 95.5%). Resistance to aminoglycosides was frequent for lactococci and enterococci (particularly for streptomycin), whereas lower rates of resistance were detected for lactobacilli and leuconostocs. Technologically interesting traits for the food industry were distributed among isolates that showed different degrees of resistance to common antibiotics. However, isolates showing resistance to less than 2 antibiotics were mainly those with properties of greatest technological interest (acidifying activity, proteolytic/lipolytic activities, or diacetyl production).

[Radioisotopic assays of rates of carbon monoxide conversion by anaerobic thermophilic prokaryotes].

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H2-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of 14CO by the bacterium was estimated at 38-56 micromol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm3 sediment per day. 85% of the utilized 14CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell bio mass production; and only just over 0.001% was converted to methane.

Carboxydocella sporoproducens sp. nov., a novel anaerobic CO-utilizing/H2-producing thermophilic bacterium from a Kamchatka hot spring.

A novel anaerobic, thermophilic, CO-utilizing bacterium, strain KarT, was isolated from a hot spring of Karymskoe Lake, Kamchatka Peninsula. The cells of the novel isolate were Gram-positive, spore-forming, short rods. The bacterium grew chemolithoautotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO + H2O --> CO2 + H2), and in the absence of CO, under N2 in the gas phase, chemoorganoheterotrophically with yeast extract, sucrose or pyruvate. Growth was observed in the temperature range 50-70 degrees C, with an optimum at 60 degrees C, and in the pH range 6.2-8.0, with an optimum at pH 6.8. The micro-organism did not grow on solid media; it was able to grow only in semi-solid medium containing 0.5 % agar. The generation time under optimal conditions for chemolithoautotrophic growth was 1 h. The G+C content of the DNA was 46.5+/-1 mol%. Growth was completely inhibited by penicillin, novobiocin, streptomycin, kanamycin and neomycin. Analysis of the 16S rRNA gene sequence showed that the isolate should be assigned to the genus Carboxydocella. On the basis of the results of DNA-DNA hybridization and morphological and physiological analyses, strain KarT represents a novel species of the genus Carboxydocella, for which the name Carboxydocella sporoproducens sp. nov. is proposed. The type strain is KarT (=DSM 16521T = VKM B-2358T).

Brochocin-C, a new bacteriocin produced by Brochothrix campestris.

Brochotrix campestris ATCC 43754 produces a bacteriocin inhibitory towards Brochothrix thermosphacta, lactobacilli, Listeria spp., and other gram-positive bacteria. This antimicrobial agent is heat stable, sensitive to proteases, catalase insensitive, and free of organic acids. No phage particles were detected by transmission electron microscopy. Muramidase activity was not detected in the preparations. On the basis of established criteria, the antimicrobial agent was classified as a bacteriocin and named brochocin-C.

An identification scheme for rapidly and aerobically growing gram-positive rods.

An identification scheme for aerobically growing Gram-positive rods (genera Actinomyces, Arcanobacterium, Aureobacterium, Bacillus, Brevibacterium, Cellulomonas, Corynebacterium, Dermabacter, Erysipelothrix, Gardnerella, Lactobacillus, Listeria, Microbacterium, Oerskovia, Propionibacterium, Rhodococcus, Rothia, Turicella, as well as unnamed CDC groups, Clostridium tertium, and Mycobacterium fortuitum/chelonae) is presented. It is derived from the Hollis-Weaver scheme and uses catalase, oxidative/fermentative carbohydrate metabolism and motility as primary reactions. Tests for lipophilism, nitrate reduction, urease, esculin hydrolysis, the CAMP reaction, acid formation from five carbohydrates, as well as for some facultative reactions should lead to a correct diagnosis based on information available at the end of 1995.

Fermentative degradation of triethanolamine by a homoacetogenic bacterium.

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9 +/- 1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B12) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.

Biochemical characteristics and fatty acid composition of Gilardi rod group 1 bacteria.

Fifteen strains of eugonic, nonoxidative, gram-negative rods isolated primarily from human wounds of the extremities and blood formed a distinct group which was designated Gilardi rod group 1. The phenotypic characteristics of Gilardi rod group 1 were most similar to those of CDC group M-5, with the major difference that nitrite reduction was observed with CDC group M-5. All 15 strains of Gilardi rod group 1 possessed a distinct fatty acid profile which was characterized by large amounts (> 15%) of cis-vaccenic (18:1 omega 7c), palmitic (16:0), myristic (14:0), and lactobacillic (19:0 cyc11,12) acids and moderate amounts (3 to 5%) of lauric (12:0), 3-hydroxylauric (3-OH-12:0), and palmitoleic (16:1 omega 7c) acids. This fatty acid profile is unique compared with the profiles of CDC group M-5 and other bacteria we have tested and is useful for the rapid identification of Gilardi rod group 1 isolates.