Robert F. Ramaley

Thermomicrobium, a New Genus of Extremely Thermophilic Bacteria

A new, extremely thermophilic bacterium from an alkaline hot spring in Yellowstone National Park is described. The organism is gram negative and occurs as pleomorphic rods, often appearing in pairs. It is an obligate aerobe and grows optimally at 70 to 75 C at a pH of 8.2 to 8.5 with a generation time of 5.5 h. When grown in culture, the organism has a very pronounced pink color due to cellular carotenoid pigments. The deoxyribonucleic acid base ratio as determined by cesium chloride density gradient ultracentrifugation was found to be 64.3 mol% guanine plus cytosine. Nutritional studies with the new organism demonstrated a more restricted growth in comparison to organisms belonging to the genus Thermus. In studies involving defined carbon sources, it was shown that the new organism grew well only on sucrose or glycerol when glutamate served as an additional nutritional source. The organism is regarded as belonging to a new genus, for which the name Thermomicrobium is proposed. This genus is placed in the family A chromobacteraceae. The type species of the new genus is Thermomicrobium roseum sp. nov., of which ATCC 27502 is the type strain.

Molecular biology of extracellular enzymes.

Publisher Summary This chapter discusses the process of secretion of enzymatic and nonenzymatic proteins (for example, binding proteins) in both gram-positive and gram-negative bacteria. The chapter examines the increasing evidence that indicates that these secretory proteins are synthesized from membrane-bound polyribosomes in precursor form and are secreted directly through the membrane as they are being synthesized. The identification of cytoplasmic enzymes released by lysis versus true extracellular enzymes is performed by determination of the cytoplasmic versus medium level of the particular enzyme during growth of the bacteria, and demonstration of the lack of release of other cytoplasmic enzymes. It has been discovered that the membrane-bound form of penicillinase has phosphatidic acid covalently bound to the serine of its amino terminal end. Because the other amino acids of the peptide are hydrophilic, it is the acyl fatty acids of the phosphatidylserine-penicillinase, which enable the hydrophobic binding of the precursor form of penicillinase to the cytoplasmic membrane, and presumably assists its secretion through the cytoplasmic membrane.

Isolation of a New, Pink, Obligately Thermophilic, Gram- Negative Bacterium (K-2 Isolate)

A pink, obligately themophilic (60 C), gram-negative, nonmotile, nonsporeforming, rod-shaped bacterium with a deoxyribonucleic acid base ratio of 64 to 65 mol% guanine plus cytosine has been repeatedly isolated from slightly alkaline, man-made and natural thermal aquatic environments. This bacterium (K-2 isolate) does not appear to have been described previously and it has been deposited with the American Type Culture Collection as an unidentified bacterium with the accession no. 27599.

Presence of Thermophilic Naegleria Isolates in the Yellowstone and Grand Teton National Parks

During a study of the extremely thermophilic bacteria of Yellowstone, we became aware of the published report (Scaglia et al., 1983) of the isolation of a thermophilic amoeba (Naegleria australiensis) from the bathing pools of a commercial Italian hot spring spa and their observation that some of these newly isolated amoebae strains were as virulent for mice as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis in humans (John, 1982; Marciano-Cabral, 1988). Because of the possibility that similar types of thermophilic pathogenic amoebae might be present in the Yellowstone and Grand Teton National Park thermal areas and possible exposure to park visitors and employees, we conducted a survey for Naegleria isolates, concentrated on those pools or outflows where it was known that visitors and employees used these pools for unauthorized bathing and soaking (Whittley, 1995).

Draft Genome Sequence of the Moderately Thermophilic Bacterium Schleiferia thermophila Strain Yellowstone (Bacteroidetes)

ABSTRACT The draft genome sequence of the moderately thermophilic bacterium Schleiferia thermophila strain Yellowstone (Bacteroidetes), isolated from Octopus Spring (Yellowstone National Park, WY, USA) was sequenced and comprises 2,617,694 bp in 35 contigs. The draft genome is predicted to encode 2,457 protein coding genes and 37 tRNA encoding genes and two rRNA operons.

Histidine decarboxylase from rat and rabbit brain: Partial purification and characterization

Histidine decarboxylase, the synthetic enzyme for histamine, was partially purified from regions of rat or rabbit brain rich in the enzyme. The enzyme was purified using ion exchange and hydrophobic column chromatography and chromatofocusing. Approximately 70-fold and 110-fold enrichments were attained from rat and rabbit brain, respectively. Rat and rabbit brain histidine decarboxylase had isoelectric points of pH 5.4 and 5.6, Km values of 80 μM and 120 μM histidine and Vmax values of 210 and 625 pmol histamine formed/hr-mg protein, respectively. The partially purified histidine decarboxylase from both sources was dependent on pyridoxal phosphate for maximal activity and was inhibited by α-fluoromethylhistidine, nickel chloride and cobaltous chloride but was not inhibited by impromidine, α-methyldopa, DTNB, zinc chloride or mercuric chloride. The enzyme had a broad pH optimum between pH 7.2 and 8.0. These studies provide further information on the characteristics of mammalian histidine decarboxylase from brain.

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli.

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.