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SummaryRecent research has accumulating evidence to support that gut microbiota can be a newly discovered organ/tissue for humans. Although the functions of gut microbiota have been linked to almost all aspects of human physiological, most findings so far are still either belonged to correlational research or at preliminary stage of causal study due to the complexity of gut microbiota as well as lack of proper tools and methods. Thus, development of new approaches is essential and necessary. Lipid droplet is a cellular organelle governing cell lipid homeostasis that is directly related to human metabolic syndromes. Previously we and other groups found lipid droplets in several bacterial strains. We wonder if gut bacteria have lipid droplets. If so, these bacteria may play key roles to regulate gut lipid content, remove unnecessary lipids and produce useful lipid molecules for host. Here we identified lipid droplet-like structures in freshly isolated bacteria from mouse small and large intestines, as well as mouse and human feces. We also purified and analyzed lipid droplets from a cloned human gut bacterium Streptomyces thermovulgaris. Together, we demonstrate the existence of lipid droplets in gut microbiota and established an approach to study them.

A thermostable xylanase gene ( stx I) obtained from Streptomyces thermonitrificans NTU-88 on domain analysis revealed an N-terminal catalytic domain featuring homology to a known xylanase within the glycoside hydrolase family 11. Recombinant STXI retained more than 60% of its activity following its incubation for at 60 °C for 24 h. These characteristics were close to thermophile and mesophile Streptomyces strains. The main hydrolysis products of xylan degraded by STXI included large xylooligosaccharide fragments. These results indicated that STXI was a typical endoxylanase. As regards the phylogenetic relationships of GH11, STXI and the other xylanase deriving from Streptomyces were included in a subgroup of the aerobic bacterial group. This result implied that the evolutionary relationships between the various xylanases deriving from Streptomyces strains were convergent.

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.

To the Editor: Invasive infections with Streptomyces spp. are rare; in reference to two cases reported in Emerging Infectious Diseases (1,2), we describe here the first documented case of bacteremia with Streptomyces thermovulgaris. An 81-year-old woman was admitted to the emergency room of Diakonessenhuis, Utrecht, the Netherlands, with severe abdominal pain in the right lower quadrant and feculent vomitus. The patient had a history of Crohn's disease, for which she had undergone resection of the ileum and cecum, and was receiving high-dose corticosteroid therapy (prednisone 25 mg daily). The patient also had steroid-induced osteoporosis and an internal pacemaker. On admission, the patient had a temperature of 35.6°C, a leukocyte count of 8.4 x 109/L with 30% bandforms, and a C-reactive protein level of 18 mg/L. Moreover, the patient had severe metabolic acidosis and was hemodynamically unstable, suggesting septic shock subsequent to a presumed bowel perforation. Blood samples were drawn and cultured, and empiric treatment was initiated with ceftriaxone and metronidazole. Exploratory laparotomy showed colonic inflammation. The patient was transferred to the intensive care unit postoperatively. On day 9 of hospitalization, the blood culture taken on day 1 before antimicrobial drug was started, turned positive in the automatic blood culture system. Gram staining showed gram-positive nocardioform rods, and subculture on tryptic soy agar with 7% sheep blood yielded polymorphous colonies that sunk into the agar and showed filamentous growth. Despite intensive antimicrobial and supportive therapy, sepsis progressed into multiple organ failure with severe neuropathy. On day 25 of hospitalization, the patient died. Autopsy demonstrated no possible infective focus except severe inflammation of the colon and distal ileum. Permission for cerebral section was not granted. The blood isolate showed strictly aerobic growth at 37°C and 50°C with β-hemolysis and was positive for catalase, caseinase, gelatinase, and nitrate reduction; the isolate was negative for oxidase, urease, and esculin hydrolysis and was nonmotile at 37°C. The isolate was sent to the National Institute of Public Health, Bilthoven, the Netherlands, for identification. Biochemical analysis, fatty acid analysis, and 16S rRNA typing identified the strain as S. thermovulgaris. The strain was susceptible to ceftriaxone (MIC 0.32 µg/mL) by Etest (BA Biodisk, Solna, Sweden). Further susceptibility testing performed by agar diffusion showed that the organism was also susceptible to amoxicillin, vancomycin, a combination of trimethoprim and sulfamethoxazole, and erythromycin. Bacteremia produced by a strain of S. thermovulgaris, as we report here, is the first documented case of isolation of this microorganism from human material. The streptomycetes are classified as a separate genus within the aerobic actinomycetes and are most well known for the many antimicrobial substances isolated from the approximately 600 different species (3). Streptomycetes, aerobic, spore-forming, gram-positive bacteria with filamentous growth, are ubiquitous in soil and can cause mycetomas (4). Streptomycetes are widely distributed in terrestrial and aquatic habitats with soil, fodder, and compost as their primary reservoirs. The amount of actinomycetes (the taxonomic group to which the streptomycetes belong) in soil is estimated to be 107–108 microorganisms per gram (5), and in total biomass equal to that of all other bacteria together and slightly less than that of fungi. S. thermovulgaris belongs to the thermophilic streptomycetes, which do not grow at temperatures

Bed performance of an immobilized biocatalyst composed of Streptomyces cells immobilized on a protein carrier susceptible to compaction, is studied in a fixed bed bioreactor with agitator at low or zero rotation. Adherence to the plug flow mixing regime and no significant external mass transfer were registered in the range of liquid velocities observed.

Cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 was cultivated in a basal medium with rice straw as sole source of carbon and as an inducible substrate. Variable medium components were selected in accordance with the Plackett-Burman experimental design. The optimization conditions of physical factors (pH and temperature levels) were then combined in further studies through the response surface methodology approach. Only two significant components, rice straw and yeast extract, were chosen for the optimization studies. A second-order quadratic model was constructed by central composite design (CCD). The model revealed that both pH and temperature levels were significant, and were dependent on xylanase production. Under these experimental designs, the xylanase yield increased from 51.11 to 274.49 U/mL (3,400 to 10,000 U/g of rice straw) or about 537% higher than an unoptimized basal medium. The optimum conditions to achieve maximum yield of xylanase were 27.45 g/L of rice straw and 5.42 g/L of yeast extract under relatively neutral conditions of pH 7.11, 50.03 °C, and a incubation period.

Industrially, the use of high temperatures (40-60 degrees C) in the L: -malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50 degrees C and highly stable even after 24 h of incubation at 40 degrees C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30 degrees C and was rapidly denatured at 50 degrees C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10 degrees C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.

SUMMARY: Fifty thermophilic streptomycetes from diverse habitats were compared through 135 unit characters with 201 representative mesophilic streptomycetes from an earlier numerical phenetic study. Data were examined using the simple matching (S SM), Jaccard (S J) and pattern (D P) coefficients, and clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) technique. In all three analyses, the thermophilic streptomycetes formed an aggregate taxon composed of three major (seven to nineteen strains), five minor (two to three strains) and two single-member clusters. Cluster composition was not affected by the statistics used. The numerical phenetic data showed that thermophilic streptomycetes form several distinct centres of variation, four of which correspond to previously described species; a further taxon was also considered to merit species status. It is proposed that Streptomyces thermolineatus sp. nov. be recognized and the name Streptomyces macrosporus Krassilnikov et al. 1968 be revived. Emended descriptions are given for Streptomyces megasporus (Krassilnikov et al., 1968) Agre 1983, Streptomyces thermoviolaceus Henssen 1957 and Streptomyces thermovulgaris Henssen 1957.