Complete genome sequences of Methylophaga sp. strain JAM1 and Methylophaga sp. strain JAM7

Methylophaga sp. strains JAM1 and JAM7 have been isolated from a denitrification system. Strain JAM1 was the first Methylophaga strain reported to be able to grow under denitrifying conditions. Here, we report the complete genome sequences of the two strains, which allowed prediction of gene clusters involved in denitrification in strain JAM1.     

Penicillin-binding proteins of Escherichia coli. Comparison of a strain carrying an R-factor and the parent strain.

Both from Escherichia coli K12 W3630 carrying an R-factor, R+75, and from the parent strain at least six penicillin- and cephalosporin-binding proteins were obtained as soluble forms. The molecular weights of the binding proteins of the strain carrying an R-factor were similar to those of the parent strain and not affected by the presence of an R-factor which specified the production of a beta-lactamase. Gel filtration with [14C]benzylpenicillin suggested the equimolar binding of benzylpenicillin to each binding protein. Three binding proteins of E. coli carrying R+75 and two binding proteins of the parent strain were purified by affinity chromatography followed by gel filtration. In fluorescence titration, various penicillins and cephalosporins were shown to bind to the purified binding proteins and their association constants were in the range of 0.4 to 21-10(3) M-1. The binding proteins of both strains did not react with the antibody against the beta-lactamase specified by R+75.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Calcium carbonate formation by Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807

Precipitation of CaCO3 catalyzed by the growth and physiology of cyanobacteria in the genus Synechococcus represents a potential mechanism for sequestration of atmospheric CO2 produced during the burning of coal for power generation. Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807 were tested in microcosm experiments for their ability to calcify when exposed to a fixed calcium concentration of 3.4 mM and dissolved inorganic carbon concentrations of 0.5, 1.25 and 2.5 mM. Synechococcus sp. strain PCC 8806 removed calcium continuously over the duration of the experiment producing approximately 18.6 mg of solid phase calcium. Calcium removal occurred over a two-day time period when Synechococcus sp. strain PCC 8807 was tested and only 8.9 mg of solid phase calcium was produced. Creation of an alkaline growth environment catalyzed by the physiology of the cyanobacteria appeared to be the primary factor responsible for CaCO3 precipitation in these experiments.     

Immunologic activity of myelin basic protein in strain 2 and strain 13 guinea pigs.

The resistance of Strain 2 guinea pigs to experimental allergic encephalomyelitis (EAE) induced by inoculation with whole CNS tissue in complete Freund's adjuvant (CFA) has been confirmed. The resistance is even more pronounced when myelin basic protein (BP) is used in attempts to induce EAE. Strain 2 guinea pigs are also resistant to an immunization schedule (multiple injections with BP in IFA followed by a single injection of BP in CFA) known to induce significant levels of antibody in susceptible strains. The poor response of Strain 2 guinea pigs to BP is not the result of lack of specific B cells--antibody equivalent to that produced by Strain 13 animals is obtained when the inoculum contains 0.5 mg BP and 2.5 mycobacteria.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Glycosyltransferases of Streptococcus mutans strain Ingbritt.

The glycosyltransferases of S. mutans strain Ingbritt have been resolved by SDS-polyacrylamide gel electrophoresis, followed by incubation in the presence of non-ionic detergent to restore enzyme activity. A group of high molecular weight proteins synthesizing glucans has been identified, as well as three distinct fructan-synthesizing activities. The glucan-forming enzymes have been purified by affinity chromatography on insoluble glucan, followed by gel chromatography in SDS, and antiserum to the purified enzymes has shown that they are antigenically identical within serotypes c, e and f, and cross-react strongly with serotype b.     

Isolation and characterization of Novosphingobium sp. strain MT1, a dominant polychlorophenol-degrading strain in a groundwater bioremediation system.

A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8 degrees C for over 6 years. We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis. The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity. The dominant organism, Novosphingobium sp. strain MT1, was isolated and characterized. Novosphingobium sp. strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8 degrees C. The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum. Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability. Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h(-1)), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.     

5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum.

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14C-labeled uracil or FU by the two strains was compared, the wild type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype.     

Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719.

The effect of the nitrogen source on the cellular activity of ferredoxin-nitrate reductase in different cyanobacteria was examined. In the unicellular species Anacystis nidulans, nitrate reductase was repressed in the presence of ammonium but de novo enzyme synthesis took place in media containing either nitrate or not nitrogen source, indicating that nitrate was not required as an obligate inducer. Nitrate reductase in A. nidulans was freed from ammonium repression by L-methionine-D,L-sulfoximine, an irreversible inhibitor of glutamine synthetase. Ammonium-promoted repression appears therefore to be indirect; ammonium has to be metabolized through glutamine synthetase to be effective in the repression of nitrate reductase. Unlike the situation in A. nidulans, nitrate appeared to play an active role in nitrate reductase synthesis in the filamentous nitrogen-fixing strains Anabaena sp. strain 7119 and Nostoc sp. strain 6719, with ammonium acting as an antagonist with regard to nitrate.