A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 ␤-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both ␤-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-␤-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ ␤-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 ␤-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.Glycosyl hydrolases (EC 3.2.1 to 3.2.3) cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and another moiety. Traditionally, glycosyl hydrolases were classified based on functional similarity. More recently, however, Henrissat and his coworkers have organized these enzymes into 90 glycosyl hydrolase families characterized by hydrophobicity plots, amino acid sequence similarities, and reaction mechanisms (10-12). This system also identifies possible common structural domains, thereby defining evolutionary connections and suggesting reaction mechanisms for the glycosyl hydrolases. Based on these criteria, the once-unified group of enzymes that exhibit ␤-galactosidase activity (EC 3.2.1.23) are now subdivided into four different families: 1, 2, 35, and 42.Of these, the best studied is family 2, which includes the well-characterized ␤-galactosidase from Escherichia coli that is encoded by the lacZ gene. Although there is considerable information about the regulation (1), biochemistry (18,23,35,47), reaction mechanism (17, 45), and structure (16) of this LacZ ␤-galactosidase, few other ␤-galactosidases within this family have been characterized biochemically (4, 7, 13-15, 26, 27, 43), while most examples exist only as a published sequence. Because of the emphasis on the E. coli LacZ enzyme, opportunities to lea...

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.

A novel, psychrophilic, Gram-positive bacterium (designated strain LV3 T ) from a lake near the McMurdo Ice Shelf, Antarctica, has been isolated and characterized. This organism formed red-pigmented colonies, had an optimal growth temperature of 18˚C and grew on a variety of media between "2 and 21˚C. Scanning electron micrographs of strain LV3T that showed small rods with unusual bulbous protuberances during all phases of growth were of particular interest. The G+C content of the genomic DNA was approximately 62 mol%. The cell walls contained ornithine as the diamino acid. The major fatty acids were anteiso-C 15 : 0 , iso-C 16 : 0 and anteiso-C 17 : 0 . Cells grown at "2˚C contained significant amounts of anteiso-C 15 : 1 . The major menaquinones found in strain LV3 T were MK-11 and MK-12. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain LV3 T was a member of the family Microbacteriaceae and related to, but distinct from, organisms belonging to the genera Agreia, Leifsonia and Subtercola. In addition, alignments of 16S rRNA sequences showed that the sequence of strain LV3 T contained a 13 bp insertion that was found in only a few related sequences. Based on the low growth temperature, unusual cell shape, distinct 16S rRNA gene sequence and structure and cell-wall amino acid and menaquinone compositions, Rhodoglobus vestalii gen. nov., sp. nov. is proposed, with the type strain LV3 T (=ATCC BAA-534 T =CIP 107482 T ).

A Gram-negative ultramicrobacterium (designated strain UMB49 T ) was isolated from a 120 000-year-old, 3042 m deep Greenland glacier ice core using a 0.2 mm filtration enrichment procedure.

Standardized procedures must be followed when characterizing, officially describing, and validly naming novel bacteria. For species descriptions, DNA-DNA hybridization still is needed for whole-genome comparisons between close relatives, but many established hybridization methods have drawbacks, such as requiring labeled or large amounts of DNA. We evaluated a new technique based on the spectrophotometric method in which renaturation rates are used for calculating the degree of binding, which estimates relatedness. In this new approach, DNA is denatured and reassociated in a real-time PCR thermal cycler and the process monitored fluorimetrically using SYBR Green I dye that selectively binds to double-stranded DNA. We investigated the effects of different parameters on the renaturation rates, such as the quantities of DNA and SYBR Green I used. Then using this technique, we calculated the percent binding for pairs of selected bacterial species representing different taxonomic groups and compared our results with published values. We demonstrated that the SYBR Green I method is useful for describing new species and as a screening tool to quickly identify the relatedness of uncharacterized isolates with similar 16S rRNA gene sequences.

Three novel orange, ultramicrobacterial isolates, UMB10, UMB14, and UMB34(T) were isolated from enrichment cultures inoculated with a melted 3,043 m deep Greenland ice core sample. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the isolates belonged to a single species within the genus Chryseobacterium. They were most closely related to Chryseobacterium aquaticum (99.3%), Chryseobacterium soli (97.1%), and Chryseobacterium soldanellicola (96.9%). Genomic hybridization showed low levels of relatedness between UMB34(T) and C. aquaticum and C. soldanellicola (19-30%) and C. soli and Chryseobacterium jejuense (45-56%). Comparative genomic fingerprinting analysis using the enterobacterial repetitive intergenic consensus (ERIC) sequence showed nearly identical banding patterns for the three isolates and these patterns were distinct from those of C. aquaticum, C. soldanellicola, C. soli, and C. jejuense. The cells were short rods, lacked flagella, had cell volumes of <0.1 mum(3), formed buds and smaller protrusions (blebs), produced copious extracellular material and a flexirubin type pigment. UMB34(T) produced acids from carbohydrates and utilized glucose and maltose although it did not assimilate mannose. The DNA G + C was 39.6-41.6 mol%. Based on the differences from validly named Chryseobacterium species, it was concluded that these isolates represent a new species for which the name, Chryseobacterium greenlandense is proposed. The type strain is UMB34(T) (=CIP 110007T = NRRL B-59357).

We surveyed our collection of psychrophilic bacteria to determine the types of phosphatases they produce and whether any had heat-labile activities with potential applications. Assays at different temperatures showed that the activity from one isolate was optimal at 45؇C and decreased dramatically above 55؇C. This isolate, D10, had the rod-coccus morphological cycle and cell wall amino acids associated with members of the Arthrobacter genus. Interestingly, we found that this strain made two extracellular phosphatases that could be separated by ammonium sulfate fractionation and migration during polyacrylamide gel electrophoresis. One enzyme, designated D10A, hydrolyzed both X-phos (5-bromo-4-chloro-3-indolyl phosphate) and para-nitrophenyl phosphate as substrates and had activity over a broad pH range of 7 to 11. The second enzyme, D10B, lacked activity against X-phos and had a narrow pH range of about 8 to 9. In addition, the D10B enzyme required calcium for activity. The levels of activity of both enzymes decreased for cells grown in media containing more than 100 M P i. These results not only demonstrate the existence of different enzymes from one Arthrobacter strain but also suggest ways in which other studies may have missed phosphatases with unknown requirements.