Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH.

A gel-stabilized gradient method that employed opposing gradients of Fe2+ and O2 was used to isolate and characterize two new Fe-oxidizing bacteria from a neutral pH, Fe(2+)-containing groundwater in Michigan. Two separate enrichment cultures were obtained, and in each the cells grew in a distinct, rust-colored band in the gel at the oxic-anoxic interface. The cells were tightly associated with the ferric hydroxides. Repeated serial dilutions of both enrichments resulted in the isolation of two axenic strains, ES-1 and ES-2. The cultures were judged pure based on (i) growth from single colonies in tubes at dilutions of 10(-7) (ES-2) (ES-2) and 10(-8) (ES-1); (ii) uniform cell morphologies, i.e., ES-1 was a motile long thin, bent, or S-shaped rod and ES-2 was a shorter curved rod; and (iii) no growth on a heterotrophic medium. Strain ES-1 grew to a density of 10(8) cells/ml on FeS with a doubling time of 8 h. Strain ES-2 grew to a density of 5 x 10(7) cells/ml with a doubling time of 12.5 h. Both strains also grew on FeCO3. Neither strain grew without Fe2+, nor did they grow with glucose, pyruvate, acetate, Mn, or H2S as an electron donor. Studies with an oxygen microelectrode revealed that both strains grew at the oxic-anoxic interface of the gradients and tracked the O2 minima when subjected to higher O2 concentrations, suggesting they are microaerobes. Phylogenetically the two strains formed a novel lineage within the gamma Proteobacteria. They were very closely related to each other and were equally closely related to PVB OTU 1, a phylotype obtained from an iron-rich hydrothermal vent system at the Loihi Seamount in the Pacific Ocean, and SPB OTU 1, a phylotype obtained from permafrost soil in Siberia. Their closest cultivated relative was Stenotrophomonas maltophilia. In total, this evidence suggests ES-1 and ES-2 are members of a previously untapped group of putatively lithotrophic, unicellular iron-oxidizing bacteria.     

Isolation and characterization of a rhodococcus species strain able to grow on ortho- and para-xylene

Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1.     

Isolation and characterization of two vibrio-shaped methane-oxidizing bacteria

An enrichment method for, and the isolation of two related vibrio-shaped methane-oxidizing bacteria are described. Their morphological and physiological characteristics are given. As a name for the genus of the organisms Methy lovibrio is proposed.We wish to thank Miss W. E. de Boer and J. van der Toorn of this laboratory for making the photographs. One of the authors (P. J. Steennis) is indebted to the Royal Netherlands Fermentation Industries Ltd., Delft for a grant.     

Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.     

Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.     

A probability matrix for the identification of gram-negative, aerobic, non-fermentative bacteria that grow on nutrient agar

Results of the identification of 621 strains of Gram-negative, aerobic, non-fermentative bacteria by a computer-based probabilistic method are given. Although many of the strains were atypical and have caused difficulty in identification in the medical diagnostic laboratory, the identification rate on this matrix was 91.5%.     

Diversity and characterization of airborne bacteria at two health institutions

The aim of this study was to identify the types and abundance of airborne bacteria of two health institutions (1 and 2) and to determine the genetic association between environmental and clinical isolates of Staphylococcus spp. Environmental sampling in institutions 1 and 2 was conducted for 1 year (dry and rainy seasons) using M Air T sampler. The bacteria and their susceptibility to antibiotics were identified. The colony-forming units per cubic meter (CFU/m³) of air were quantified for all the isolates, and the diversity and abundance of species were calculated. The genetic relationship between the clinical and environmental isolates of S. aureus obtained from institution 1 was established by the UPGMA based on RAPD markers. At both of the institutions, the genera most frequently isolated were Staphylococcus and Bacillus, and the greater concentration of airborne bacteria was detected during the dry season than the rainy season. The lowest diversity and highest dominance was found in the institution 2. On the other hand, at institution 1, the genus that was resistant to antibiotics was Staphylococcus, whereas at institution 2, no isolate was found to be resistant to antibiotics. Furthermore, no association between the clinical and environmental isolates of S. aureus was found. However, one clone was found in different areas of the institution 1. The presence of airborne pathogenic bacteria in institutions 1 and 2 is important to establish the measures for the prevention and control of nosocomial outbreaks.     

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.     

Isolation of haloarchaea that grow at low salinities

Summary Archaea, the third domain of life, were long thought to be limited to environmental extremes. However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the notion of Archaea as obligate extremophiles. Until now, none of these novel Archaea has been brought into culture, a critical step for discovering their ecological roles. We have cultivated three novel halophilic Archaea (haloarchaea) genotypes from sediments in which the pore-water salinity was close to that of sea water. All previously reported haloarchaeal isolates are obligate extreme halophiles requiring at least 9% (w/v) NaCl for growth and are typically the dominant heterotrophic organisms in salt and soda lakes, salt deposits and salterns. Two of these three newly isolated genotypes have lower requirements for salt than previously cultured haloarchaea and are capable of slow growth at sea-water salinity (2.5% w/v NaCl). Our data reveal the existence of Archaea that can grow in non-extreme conditions and of a diverse community of haloarchaea existing in coastal salt marsh sediments. Our findings suggest that the ecological range of these physiologically versatile prokaryotes is much wider than previously supposed.     

Construction and Characterization of Two Recombinant Bacteria That Grow on ortho- and para-Substituted Chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISP_OHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

Background  

In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria.     

Isolation and characterization of two new homoacetogenic hydrogen-utilizing bacteria from the human intestinal tract that are closely related to Clostridium coccoides.

Two gram-positive, strictly anoxic, coccoid- to rod-shaped strains of bacteria, Clostridium coccoides 1410 and C. coccoides 3110, were isolated from human feces on the typical homoacetogenic substrates formate plus H2 plus CO2 (strain 1410) and vanillate plus H2 plus CO2 (strain 3110) in the presence of 2-bromoethanesulfonate to inhibit methanogenesis. On the basis of 16S rRNA sequencing, DNA-DNA hybridization, and physiological and morphological parameters, both isolates are closely related to C. coccoides DSM 935T. The G+C contents of the DNA were 46.1 and 46.2 mol% for C. coccoides 1410 and C. coccoides 3110, respectively. Cytochromes could not be detected. Formate was degraded exclusively to acetate, whereas vanillate was O-demethylated, resulting in acetate and 3,4-dihydroxybenzoate, the latter being further decarboxylated to catechol. In the presence of organic substrates, H2 was cometabolized to acetate, but both strains failed to grow autotrophically. Lactose, lactulose, sorbitol, glucose, and various other carbohydrates supported growth as well. Untypical of homoacetogens, glucose and sorbitol were fermented not exclusively to acetate; instead, considerable amounts of succinate and D-lactate were produced. H2 was evolved from carbohydrates only in negligible traces. Acetogenesis from formate plus H2 plus CO2 or vanillate plus H2 plus CO2 was constitutive, whereas utilization of carbohydrates was inducible. Hydrogenase, CO dehydrogenase, formate dehydrogenase, and all of the tetrahydrofolic acid-dependent, C1 compound-converting enzymes of the acetyl-coenzyme A pathway of homoacetogenesis were present in cell extracts.     

Construction and characterization of UAA-controlled recombinant Zika virus by genetic code expansion

正Dear Editor,Genetic code expansion based on orthogonal tRNA-aminoacyl tRNA synthetase is an emerging technology to sitespecifically incorporate unnatural amino acids (UAAs) into viral proteins to produce UAA-controlled replication-defective virus. Briefly, aminoacyl tRNA synthase is used to transfer specific UAAs to corresponding tRNA to form aminoacyl tRNA,     

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.     

Structural characterization of recombinant human interferon-gammas derived from two different mammalian cells

Recombinant human interferon-gammas (rHuIFN-gamma s) were obtained from two different mammalian cells (mouse C127 cells and Chinese hamster ovary, CHO, cells) cultured in a microcarrier culture system. Both rHuIFN-gamma s were purified using sequential chromatographies for their comparison of structural properties. The peptide maps of HuIFN-gamma s digested with V8 protease and Western blot analysis demonstrated that C127 cells yielded mainly about 25kDa component and CHO cells produced about 25kDa and about 20kDa components. By the identification of glycosylated peptides, it was suggested that 20kDa and 25kDa components are glycosylated at one and at two sites, respectively. C-terminal amino acid sequence analysis indicated that both rHuIFN-gamma s consisted of at least six different species lacking 2 to 16 amino acid residues from C-terminus, so that C-termini of both rHuIFN-gamma s were slightly different from each other. Amino acid sequence and composition analyses of N-terminal peptides demonstrated that N-termini of both rHuIFN-gamma s were blocked and were supposed to be identical with that of natural HuIFN-gamma. These results suggested that different molecular heterogeneities of rHuIFN-gamma s resulted from the difference of post-translational modifications of host cells.