Evaluation of autoscan-4 for identification of members of the family Enterobacteriaceae.

A study was performed to compare the Autoscan-4 (MicroScan, Inc., Mahwah, N.J.) with conventional biochemical methods for identifying clinical isolates of the family Enterobacteriaceae. The Autoscan-4 yielded correct identification of 95.4% of the isolates at the species level and 98.4% at the genus level. Only one misidentification was observed. The identification of both common and less-common isolates of Enterobacteriaceae makes this system highly efficient.

Cytochemical study of the nucleolus of the slime mold Dictyostelium discoideum.

The nucleus of the slime mold Dictyostelium discoideum is characterized by the presence of several large dense masses which are all in tight contact with the nuclear membrane. These dense masses, considered as nucleoli, present a rather homogeneous texture, in which dense chromatin, fibrillar, and granular material are not easily detected. The autoradiographic study of [3H]uridine pulse-labeled cells showed that the majority of the silver grains were located inside these masses. The use of EDTA regressive-staining, acetylation and enzymatic digestion indicated that they are mostly composed of RNP and are totally devoid of dense chromatin as the rest of the nucleus is. After treatment with actinomycin D, fibrillar and granular material segregated but no chromatin could be found. All these observations confirmed that the dense masses correspond to nucleoli despite their peculiar ultrastructure. It can also be concluded that this type of nucleoli cannot be considered as a taxonomic character of the slime molds because it does not exist in all slime molds and was observed in some dinoflagellates, and ascomycetes.

Cloning and expression of a DNA fragment carrying a his nifA fusion and the nifBQ operon from a nif constitutive mutant of Klebsiella pneumoniae.

From the nifc mutant plasmid pPC868, previously shown to carry a DNA duplication responsible for the Nifc phenotype, a 10 kb HindIII fragment was cloned into the multicopy vector pBR325. Restriction analysis of the resulting plasmids and in vitro deleted derivatives confirmed that the mutation was a fusion between his and nifLA. The order was hisG-hisD'-'nifL-nifA so that nifA was transcribed under the control of the his promoter and the nifL gene was altered. In addition the cloned fragment contained the adjacent nifBQ operon, and complementation data revealed that the nifA, nifB and hisG genes were expressed. Synthesis of nifA product under the transcription control of the his (or cat [CmR]) promoter enabled complementation of nifA and nifB mutations either in the absence or the presence of ammonia, but did not restore nitrogen fixation in a glnF mutant. Therefore, the nifA gene product requires glnF for its positive control function in a manner analogous to ntrC. Protein content analysis of minicells containing various multicopy nif plasmids confirmed the genetic organization mentioned above. A new polypeptide of 51,500 daltons was found whose synthesis was observed at 30 degrees C but not at 37 degrees C. According to the physical map, this protein could be the nifB gene product. Our results are in agreement with nifB transcription being under the control of a thermolabile nifA product. Moreover we obtained results suggesting that the presence of multiple copies of a functional nifB gene inhibited nitrogen fixation.

Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense.

Homology was detected between the structural genes for the nitrogenase complex of K. pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains. Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A. brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K. pneumoniae nif probe. Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K. pneumoniae nif genes was found to be approximately 5 kb. Proteins encoded by the AbRI fragment were examined after infection of E. coli minicells.