Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo.

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.

Direct Identification of Urinary Tract Pathogens From Urine Samples Using the Vitek MS System Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

BACKGROUND: We evaluated the coincidence rate between Vitek MS system (bioMérieux, France) and Vitek 2 in identifying uropathogens directly from urine specimens. METHODS: Urine specimens submitted to our microbiology laboratory between July and September 2013 for Gram staining and bacterial culture were analyzed. Bacterial identification was performed by using the conventional method. Urine specimens showing a single morphotype by Gram staining were processed by culturing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Of 2,370 urine specimens, 251 showed a single morphotype on Gram staining, and among them, 202 were available for MALDI-TOF MS. RESULTS: In these 202 specimens, colony growth was observed in 189 specimens, and 145 specimens had significant growth of single-colony morphotype in culture. One hundred and ten (75.9%) of them had colony counts of ≥10(5) colony-forming units (CFU)/mL and included 71 enteric gram-negative bacteria (GNB), 5 glucose-non-fermenting GNB, 9 gram-positive cocci (GPC), and 25 yeasts. Furthermore, 70 (98.6%), 3 (60.0%), 4 (44.4%), and 5 (20.0%), respectively, of these were correctly identified by Vitek MS. Thirty-one specimens (21.4%; 11 GNB, 7 GPC, 12 yeasts, and 1 gram-positive bacillus) had colony counts of 10(4)-10(5) CFU/mL. Four specimens (2.8%) yielded colony counts of 10(3)-10(4) CFU/mL. CONCLUSIONS: Vitek MS showed high rate of accuracy for the identification of GNB in urine specimens (≥10(5) CFU/mL). This could become a rapid and accurate diagnostic method for urinary tract infection caused by GNB. However, for the identification of GPC and yeasts, further studies on appropriate pre-treatment are warranted.

Rothia marina sp. nov., isolated from an intertidal sediment of the South China Sea.

A novel non-sporulating, non-motile, catalase-positive, oxidase-negative, facultatively anaerobic, Gram-positive coccus, designated strain JSM 078151(T), was isolated from an intertidal sediment sample collected from Naozhou Island in the South China Sea, China. Growth was found to occur in the presence of 0-15 % (w/v) NaCl (optimum 0.5-3 % (w/v) NaCl), at pH 6.5-10.5 (optimum pH 7.0-8.0) and at 5-35 °C (optimum 25-30 °C). The peptidoglycan type was determined to be A3a, containing lysine, glutamic acid and alanine. The major cellular fatty acid identified was anteiso-C15:0 and the predominant menaquinones are MK-7 and MK-8. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unidentified phospholipid. The genomic DNA G+C content of strain JSM 078151(T) was determined to be 55.2 mol%. A phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 078151(T) should be assigned to the genus Rothia, and was most closely related to Rothia nasimurium CCUG 35957(T) (98.3 % sequence similarity), followed by Rothia amarae J18(T) (97.5 %) and Rothia terrae L-143(T) (97.3 %). A combination of phylogenetic analysis, DNA-DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the suggestion that strain JSM 078151(T) represents a novel species of the genus Rothia, for which the name Rothia marina sp. nov. is proposed. The type strain is JSM 078151(T) (= DSM 21080(T) = KCTC 19432(T)).

Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine diagnostics.

This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Gram-positive, catalase-negative cocci not belonging to the Streptococcus or Enterococcus genus and benefits of database extension.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software was used singly or in combination with a database extension generated in this study with 51 collection strains from 16 genera. Most strains were identified by using both databases individually, and some were identified only by applying the combined database. Thus, the methodology is very useful and the generated database extension was helpful.

Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry.

We evaluated Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 305 clinical isolates of staphylococci, streptococci, and related genera by comparing direct colony testing with preparatory extraction. Isolates were previously identified by use of phenotypic testing and/or 16S rRNA gene sequencing. Manufacturer-specified score cutoffs for genus- and species-level identification were used. After excluding 7 isolates not present in the Biotyper library, the Biotyper correctly identified 284 (95%) and 207 (69%) isolates to the genus and species levels, respectively, using extraction. By using direct colony testing, the Biotyper identified 168 (56%) and 60 (20%) isolates to the genus and species levels, respectively. Overall, more isolates were identified to the genus and species levels with preparatory extraction than with direct colony testing (P < 0.0001). The analysis was repeated after dividing the isolates into two subgroups, staphylococci, streptococci, and enterococci (n = 217) and "related genera" (n = 81). For the former subgroup, the extraction method resulted in the identification of 213 (98%) and 171 (79%) isolates to the genus and species levels, respectively, whereas the direct colony method identified 136 (63%) and 56 (26%) isolates to the genus and species levels, respectively. In contrast, for the subgroup of related genera, the extraction method identified 71 (88%) and 36 (44%) isolates to the genus and species levels, respectively, while the direct colony method identified 32 (40%) and 4 (5%) isolates to the genus and species levels, respectively. For both subgroups, preparatory extraction was superior to direct colony testing for the identification of isolates to the genus and species levels (P < 0.0001). Preparatory extraction is needed for the identification of a substantial proportion of Gram-positive cocci using the Biotyper method according to manufacturer-specified score cutoffs.

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry.

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.

Bacteria-based controlled assembly of metal chalcogenide hollow nanostructures with enhanced light-harvesting and photocatalytic properties.

Herein, a general bottom-up approach is proposed for the controlled assembly of metal chalcogenide nanoparticles into biomorphic porous hollow nanostructures by a sonochemical method using bacteria as morph-biotemplates. Biomorphic PbS and ZnS hollow nanostructures have been successfully synthesized with two species of bacteria cocci and bacillus as morph-templates. The biomorphic hollow assemblies possess shape-controllable, size-tunable and shell-thickness-adjustable characteristics. Thus, the structure and morphology of the hollow assemblies may be varied in a controllable way to tailor their properties over a broad range. A preliminary study on the light-harvesting properties of PbS and ZnS hollow spheres revealed that the hollow and porous structure is clearly far more favorable for the absorption of light than solid counterparts, which accounts for both multiple scattering effects at the large voids (hollow cavities) and Rayleigh scattering by nanovoids of the exterior shells. Furthermore, photocatalytic studies of ZnS nanostructures by degradation of acid fuchsine under solar irradiation have proved that the hollow structures possess superior photocatalytic activity to the corresponding solid counterparts. This versatile approach provides an effective route for the further extensive study of the distinct properties imparted by hollow nanostructures and extends their application potentials in photocatalysis and solar energy storage/conversion.

The presence of bacteria species in semen and sperm quality.

PURPOSE: To verify the prevalence of semen bacterial contamination and whether the contamination could decrease sperm quality. METHODS: Spermiogram, semen culture, and sperm transmission electron microscopy (TEM) analysis were performed. TEM data were elaborated using a mathematical formula that calculates a fertility index (FI)--able to define patients as fertile or infertile--and the percentage of sperm apoptosis, immaturity and necrosis. We aligned the amino acid sequence of beta-tubulin with protein of the most frequent species isolated from semen. RESULTS: Patients were divided according to the contaminating species; in each group, we observed fertile individuals, in whom the semen quality was similar to that of controls and infertile men whose sperm quality was significantly decreased, in terms of motility, FI, apoptosis and necrosis. Partial homology between beta-tubulin and bacterial proteins was observed. CONCLUSION: Sperm bacterial contamination is quite frequent and could contribute to the deterioration of the sperm quality of infertile men.

A polysaccharide of Alloiococcus otitidis, a new pathogen of otitis media: chemical structure and synthesis of a neoglycoconjugate thereof.

Alloiococcus otitidis is a recently discovered Gram-positive bacterium that has been linked with otitis media (middle ear infections). In this study, we describe the structure of a novel capsular polysaccharide (PS) expressed by the type-strain of A. otitidis, ATCC 51267, and the synthesis of a glycoconjugate composed of the capsule PS and bovine serum albumin (BSA). The capsule PS of A. otitidis type-strain was determined to be a repeating trisaccharide composed of 3-substituted N-acetyl-D-glucosamine (GlcpNAc), 6-substituted N-acetyl-D-galactosamine (GalpNAc), and 4-substituted D-glucuronic acid (GlcpA), of which the majority was amidically decorated with L-glutamic acid (Glu): {-->6)-beta-GalpNAc-(1-->4)-[Glup-->6]-beta-GlcpA-(1-->3)-beta-GlcpNAc-(1}n. Monomeric analysis performed on other A. otitidis strains revealed that similar components were variably expressed, but Glu appeared to be a regular constituent in all the strains examined. Due to the suitable presence of GlcpA and Glu, our approach for glycoconjugate synthesis employed a carbodiimide-based strategy with activation of available carboxyl groups by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), which afforded direct coupling between the capsule PS and BSA. Analysis by mass spectrometry indicated that this A. otitidis capsule PS-BSA conjugate was composed of BSA units that carried up to seven capsule PSs. This work represents the first report in the literature describing an A. otitidis cell-surface carbohydrate and the synthesis of a glycoconjugate preparation thereof. Presently, we are formulating plans to immunologically evaluate this A. otitidis glycoconjugate vaccine in animals.

Howardella ureilytica gen. nov., sp. nov., a Gram-positive, coccoid-shaped bacterium from a sheep rumen.

An unidentified obligately anaerobic, fastidious, Gram-positive, non-motile, non-spore-forming, non-fermentative coccoid-shaped bacterium (designated strain GPC 589(T)) was isolated from the rumen fluid of a sheep. The major fatty acid constituents (>5 %) were C(16 : 0) (29.2 %), C(18 : 0) (40.7 %) and an unidentified compound (19.7 %) with an equivalent chain-length of 13.523. The G+C content of the DNA was 34 mol%. The organism was strongly ureolytic and generated ATP through the hydrolysis of urea. Comparative 16S rRNA gene sequence analysis demonstrated that strain GPC 589(T) was far removed, phylogenetically, from the ruminococci and related Gram-positive anaerobic cocci but exhibited a phylogenetic association with Clostridium rRNA cluster XIVa [as defined by Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). Int J Syst Bacteriol 44, 812-826]. Sequence divergence values of 12.5 % or more were observed between strain GPC 589(T) and all other recognized species within this and related rRNA clostridial clusters. Phylogenetic analysis showed that strain GPC 589(T) represents a new genus within cluster XIVa. On the basis of both phylogenetic and phenotypic evidence, it is proposed that strain GPC 589(T) should be classified as representing a new genus and novel species, Howardella ureilytica gen. nov., sp. nov. The type strain is strain GPC 589(T) (=DSM 15118(T)=JCM 13267(T)).

In silico comparison of bacterial strains using mutual information.

Fast-sequencing throughput methods have increased the number of completely sequenced bacterial genomes to about 400 by December 2006, with the number increasing rapidly. These include several strains. In silico methods of comparative genomics are of use in categorizing and phylogenetically sorting these bacteria. Various word-based tools have been used for quantifying the similarities and differences between entire genomes. The simple di-nucleotide frequency comparison, codon specificity and k-mer repeat detection are among some of the well-known methods. In this paper, we show that the Mutual Information function, which is a measure of correlations and a concept from Information Theory, is very effective in determining the similarities and differences among genome sequences of various strains of bacteria such as the plant pathogen Xylella fastidiosa, marine Cyanobacteria Prochlorococcus marinus or animal and human pathogens such as species of Ehrlichia and Legionella. The short-range three-base periodicity, small sequence repeats and long-range correlations taken together constitute a genome signature that can be used as a technique for identifying new bacterial strains with the help of strains already catalogued in the database. There have been several applications of using the Mutual Information function as a measure of correlations in genomics but this is the first whole genome analysis done to detect strain similarities and differences.

Planococcus columbae sp. nov., isolated from pigeon faeces.

An orange-pigmented, Gram-positive bacterial strain, designated PgEx11(T), was isolated from pigeon faeces. Analysis of the 16S rRNA gene sequence of the isolate indicated that it had 94.2-98.2 % sequence identity with respect to those of seven recognized species of the genus Planococcus. The strain PgEx11(T) contained anteiso-C(15 : 0) as a major cellular fatty acid and MK-7 and MK-8 as the major menaquinones. The DNA G+C content of strain PgEx11(T) was 50.5 mol%. Furthermore, analysis of the 16S rRNA gene sequence indicated high levels of similarity with Planococcus rifietoensis (98.2 %), Planococcus maitriensis (97.6 %), Planococcus citreus (97.5 %) and Planococcus maritimus (97.1 %). However, the mean value for DNA-DNA relatedness between PgEx11(T) and these four closely related species was in the range 45.4-16.8 %, respectively. Moreover, strain PgEx11(T) also differs from its close relatives with regard to biochemical and chemotaxonomic characteristics. On the basis of phenotypic, chemotaxonomic and genotypic differences, strain PgEx11(T) represents a novel species of the genus Planococcus, for which the name Planococcus columbae sp. nov. is proposed. The type strain is PgEx11(T) (=MTCC 7251(T)=DSM 17517(T)).

Humicoccus flavidus gen. nov., sp. nov., isolated from soil.

A Gram-positive, non-motile, spherical, non-spore-forming bacterial strain, DS-52(T), was isolated from soil from Dokdo, Korea, and its taxonomic position was investigated by using a polyphasic approach. It grew optimally at 25 degrees C and pH 6.0-7.0. Strain DS-52(T) had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and galactose, mannose, xylose and rhamnose as whole-cell sugars. It contained MK-8(H(4)) and MK-9(H(4)) as the predominant menaquinones and anteiso-C(15 : 0), iso-C(15 : 0) and C(17 : 0) as major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyldimethylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DS-52(T) is most closely related to the genus Nakamurella of the suborder Frankineae. Strain DS-52(T) exhibited 16S rRNA gene sequence similarity values of 96.5 % to Nakamurella multipartita JCM 9543(T) and 92.0-93.9 % to other members of the suborder Frankineae. The diagnostic diamino acid type and polar lipid profile of strain DS-52(T) were the same as those of the genus Nakamurella. However, strain DS-52(T) could be clearly distinguished from the genus Nakamurella by differences in predominant menaquinones, major fatty acids and cell-wall sugars. Accordingly, based on combined phenotypic, chemotaxonomic and phylogenetic data, strain DS-52(T) (=KCTC 19127(T)=CIP 108919(T)) is proposed as the type strain of a novel species in a new genus, Humicoccus flavidus gen. nov., sp. nov.

Evidence of local antibody response against Alloiococcus otitidis in the middle ear cavity of children with otitis media.

Alloiococcus otitidis is a recently discovered bacterium frequently associated with otitis media. However, no study is available as to whether A. otitidis has a pathogenic role and induces local immune response in the middle ear as a true pathogen. Whole bacterial sonicate of A. otitidis was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. Then, Western blot analysis was performed with supernatant of the middle ear effusions from children with A. otitidis-positive otitis media. SDS-PAGE of the bacterial sonicate showed several protein bands, designated A1-A11. Western blot analysis revealed the presence of IgG, secretory IgA, IgG2, and IgM against A. otitidis in the middle ear effusions. Absorption of the specimens with sonicates of other major middle ear pathogens did not alter the reactivity of antibodies against the alloiococcal antigens. The results suggest that specific local immune response against A. otitidis is induced during middle ear infection of the organism as a true pathogen. A5, A6 or A11 is expected to be a main antigenic determinant. This is the first report to show evidence of local antibody response against A. otitidis and to disclose antigenic components of A. otitidis.

Survey of extreme solvent tolerance in gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene.

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.

Tetragenococcus koreensis sp. nov., a novel rhamnolipid-producing bacterium.

A Gram-positive, non-motile, non-spore-forming coccus (strain JS(T)) was isolated from kimchi (a traditional Korean food) and investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between strain JS(T) and its closest relative, Tetragenococcus halophilus IAM 1676(T), was 98.1%. The level of DNA-DNA relatedness between the two strains was 9.7%. Strain JS(T) had a DNA G+C content of 38.3% and a cellular fatty acid profile containing 16:0, 18:1 and cyclo fatty acids. Phylogenetic data and genomic and phenotypic features demonstrated that strain JS(T) represents a novel species, for which the name Tetragenococcus koreensis sp. nov. is proposed. The type strain is JS(T) (=KCTC 3924(T)=DSM 16501(T)=LMG 22864(T)).

Atopococcus tabaci gen. nov., sp. nov., a novel Gram-positive, catalase-negative, coccus-shaped bacterium isolated from tobacco.

A novel Gram-positive, aerobic, catalase-negative, coccus-shaped organism originating from tobacco was characterized using phenotypic and molecular taxonomic methods. The organism contained a cell wall murein based on L-lysine (variation A4alpha, type L-lysine-L-glutamic acid), synthesized long-chain cellular fatty acids of the straight-chain saturated and monounsaturated types (with C(16:1)omega9, C(16:0) and C(18:1)omega9 predominating) and possessed a DNA G+C content of 46 mol%. Based on morphological, biochemical and chemical characteristics, the coccus-shaped organism did not conform to any presently recognized taxon. Comparative 16S rRNA gene sequencing studies confirmed the distinctiveness of the unknown coccus, with the bacterium displaying sequence divergence values of greater than 7% with other recognized Gram-positive taxa. Treeing analysis reinforced its distinctiveness, with the unidentified organism forming a relatively long subline branching at the periphery of an rRNA gene sequence cluster which encompasses the genera Alloiococcus, Allofustis, Alkalibacterium, Atopostipes, Dolosigranulum and Marinilactibacillus. Based on phenotypic and molecular phylogenetic evidence, it is proposed that the unknown organism from tobacco be classified as a new genus and species, Atopococcus tabaci gen. nov., sp. nov. The type strain of Atopococcus tabaci is CCUG 48253(T) (=CIP 108502(T)).

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni.

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO(2)) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-(13)C]glucose, [U-(13)C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive ( K(i)=180 microM) and uncompetitive ( K'(i)=350 microM) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively ( K(i)=1.3 microM) with a low contribution of uncompetitive inhibition ( K'(i)=13 microM). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 microM) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 microM). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.

Salinicoccus alkaliphilus sp. nov., a novel alkaliphile and moderate halophile from Baer Soda Lake in Inner Mongolia Autonomous Region, China.

A novel alkaliphilic and moderately halophilic gram-positive coccus, designated strain T8T, was isolated from Baer Soda Lake in Inner Mongolia Autonomous Region, China. Strain T8T grew in the presence of 0-25% (w/v) NaCl and at pH 6.5-11.5, with optimum growth at 10% (w/v) NaCl and pH 9.0. It grew at 10.0-46.0 degrees C, with an optimum growth temperature of 32.0 degrees C. The organism was strictly aerobic, non-motile, non-sporulating and catalase- and oxidase-positive. The DNA G+C content was 49.6 mol%. The cell wall contained Lys and Gly. The major isoprenoid quinone was menaquinone 6 (MK-6). Phylogenetic analyses based on 16S rDNA sequence comparisons indicate that strain T8T is a member of the genus Salinicoccus. DNA-DNA relatedness of less than 50% with the described species of Salinicoccus supported the view that this organism represents a novel species of the genus Salinicoccus. The name Salinicoccus alkaliphilus sp. nov. is proposed for this novel species. The type strain is T8T (= AS 1.2691T = JCM 11311T).