The rne gene and ribonuclease E.

The cloned rne+ gene complements temperature sensitive RNase E mutations and directs the synthesis of a polypeptide. In vitro the RNA transcribed from the rne gene directs the synthesis of a number of polypeptides, one of which is identical in size to the in vivo product of the rne gene. A rabbit reticulocyte cell free extract programmed with this RNA produced RNase E activity. Thus, it is evident that the rne gene is the structural gene for RNase E. However, the in vivo product of the cloned RNase E gene is more thermolabile than the chromosomal gene product. When cells containing the rne plasmid were treated with chloramphenicol, the pre-existing RNase E became less heat labile with time. This leads to the suggestion that in the cell RNase E undergoes post-translational modification(s).

Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+.

A precursor to 10Sa RNA accumulates in an rne mutant. However, the present studies indicate that RNase III is the enzyme that processes this RNA. Cell extracts prepared from an rne mutant failed to cleave p10Sa RNA, whereas E coli wild type, rne and rnp cell extracts processed p10Sa RNA under specific assay conditions that require the presence of Mn2+ but not under the customary conditions used for assaying RNase III. That the p10Sa cleaving activity is solely RNase III was confirmed by comparing the increase in p10Sa and poly(A).poly(U) cleaving activities in a strain harboring a plasmid carrying an RNase III gene as compared to a normal E coli strain. It is of interest that these 2 substrates are cleaved by RNase III efficiently, but under 2 different assay conditions. In all strains tested, with normal or elevated levels of RNase III, RNase III fractionates predominantly with the membrane. Further characterization of the maturation of 10Sa RNA revealed that the processing of 10Sa RNA is a 2 step reaction involving 2 separate activities, both sensitive to heat and proteinase K treatment. The first step is catalyzed by RNase III, and results in the formation of a molecule, p10Sa', which is larger than the mature 10Sa RNA. The second activity catalyzes the conversion of p10S' to 10Sa RNA, and this step does not require a divalent cation. The second activity is not any of the known processing endoribonucleases, RNase III, E or P, but could be a new enzyme having no obligate requirement for a divalent cation.

Granulocyte-specific monoclonal antibody inhibiting cytotoxicity reactions in the chicken.

Monoclonal antibody (MAb) 1C3, specific for chicken granulocytes, is described for the first time. Treatment of peripheral blood leukocytes with this MAb markedly decreased natural cytotoxicity reaction against the target cell line. The antibody-dependent cellular cytotoxicity (ADCC) activity of purified granulocytes was also severely affected by treatment with MAb 1C3. These results suggest that 1C3 detects a functional surface antigen on chicken granulocytes and support the hypothesis that granulocytes are the main effector cells in natural cytotoxicity in the chicken.

Recombinant Mycobacterium smegmatis vaccine candidates.

Mycobacterium smegmatis is a species of rapidly growing saprophytes with a number of properties that make it an effective vaccine vector. Recombinant M. smegmatis expressing protective antigens of different pathogens and molecules modulating the immune responses offers some potential for reduction of the burden of tuberculosis, HIV and hepatitis B infections. This paper discusses the molecular methods used to generate recombinant M. smegmatis and the results obtained with some of these recombinants.

Production and purification of low calcium response protein H of Chlamydophila pneumoniae.

Chlamydophila pneumoniae possesses a type III secretion system (TTSS), which allows the bacteria to secrete effector molecules into the inclusion membrane and into the cytosol of the host cell. Low calcium response protein H (LcrH), as a part of the TTSS, is a chaperone protein expressed from the middle to late stages of the chlamydial developmental cycle. Gene of LcrH (CPn0811) in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin. The purity was checked with mass spectrometry. The samples were used for immunization of BALB/c mice. The inducible E. coli clone, which over-expresses the chlamydial LcrH, permits the study of the biological properties of this protein.

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv.

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.

The entire rne gene: the remaining questions.

A 30-fold overexpression of the entire rne gene results in a 3-fold increase in RNase E activity. The overexpression of cloned rne fragments suggests that synthesis of the Rne protein is autoregulated and this regulation is related to the RNase E activity of the gene product. The enzyme produced by the cloned gene has an enhanced thermolability. The thermal stability of the temperature-sensitive enzyme increases when the cells are cultivated in the presence of chloramphenicol, which suggests posttranslational alterations.

Porphyrin and corrinoid mutants of Bacillus subtilis.

Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemin-containing medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B12 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.

Porphobilinogen-accumulation by a porphyrin auxotrophic strain of Bacillus subtilis.

The amount of porphobilinogen accumulated by a Bacillus subtilis hemC mutant strain is dependent on time and exogenic aminolevulinic acid addition. hemC mutant seems to be suitable for porphobilinogen production on a large scale.

RNA processing in prokaryotic cells.

RNA processing in Escherichia coli and some of its phages is reviewed here, with primary emphasis on rRNA and tRNA processing. Three enzymes, RNase III, RNase E and RNase P are responsible for most of the primary endonucleolytic RNA processing events. The first two are proteins, while RNase P is a ribozyme. These three enzymes have unique functions and in their absence, the cleavage events they catalyze are not performed. On the other hand a relatively large number of exonucleases participate in the trimming of the 3' ends of tRNA precursor molecules and they can substitute for each other. Primary processing is the first event that happens to the nascent RNA molecule, while in secondary RNA processing, the substrate is a product of a primary processing event. Although most RNA processing occurs in RNP particles, it seems that only in secondary RNA processing is the RNP particle required for the reaction. Bacteria and especially bacteriophages contain self-splicing introns which in cases were probably acquired from other species.

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene.

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.

Mapping the uroporphyrinogen III cosynthase locus in Bacillus subtilis.

The gene hemD taking part in the formation of uroporphyrinogen III from porphobilinogen was mapped by two- and three-factor transduction crosses in Bacillus subtilis. This gene codes uroporphyrinogen III cosynthase. The gene hemD is linked to the hemA locus and is located between the hemA and pheA loci.

Membrane characteristics of the hemA mutant of Bacillus subtilis.

The membrane of the delta-aminolaevulinic acid synthase (EC. 2.3.1.37) deficient mutant of Bacillus subtilis growing in the presence of delta-aminolaevulinic acid differs only to a limited extent from the wild type. In haemin-containing medium, however, significant differences are observed as regards the osmotic stability of the protoplasts and the membrane protein composition.

Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis.

Three genes hemE, hemF, hemG taking part in the porphyrin biosynthesis of Baccillus subtilis were mapped by two- and three-factor transduction crosses. The gene hemE determines uroporphyrinogen decarboxylase (EC 4.1.1.37) the gene hemF coproporphyrinogen oxidase (EC 1.3.3.3) and the gene hemG ferrochelatase (EC 4.99.1.1) enzymes. The loci hemE, hemF, hemG, are not linked to hemA locus and located near the argC and metD loci.

Sequencing and expression of the rne gene of Escherichia coli.

RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli. We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame. Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site. We have translated this gene using a T7 RNA polymerase/promoter system. We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence. The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein. The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.

Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell.

Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm. The RNA-processing enzymes were associated with the membrane fraction. The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction. By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane. The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity. The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed.

RNA processing: new mutants that affect endonucleolytic processing of RNA.

A strain of Escherichia coli carrying the rne-3071 mutation that affects the RNA processing enzyme ribonuclease E, was mutagenized, and double mutants deficient in RNA processing were isolated. The isolation was based on the appearance of a particular RNA precursor molecule upon infection of an rne mutant with a specific bacteriophage T4 deletion strain. From one of the double mutants the rne mutation was removed, and the new single mutant, designated rng, was examined. In this mutant the maturation of host RNA as well as of bacteriophage T4 RNA is affected. The effect of the rng mutation on RNA synthesis is unique and can be distinguished from the effects of the other established mutations in RNA processing. The effects of the rng mutation can be recognized in vivo and in vitro.

Genetic and biochemical analysis of haemin dependent mutants of Bacillus subtilis.

Numerous porphyrin auxotrophic mutants have been isolated from the 168 trpC2 strain of Bacillus subtilis by selection with streptomycin. Some of them could be supplemented with ALA while the majority grew only in the presence of haemin. Among the latter strains, the syntropism test allowed to distinguish two groups different in phenotype, viz., feeders accumulating ALA and non-feeders accumulating instead of ALA other porphyrin intermediates. On the basis of transductional studies, feeders and non-feeders could be divided into two and four groups, respectively. Biochemical investigation revealed that, with one exception, one enzyme of the porphyrin biosynthesis was coordinated to each hem locus. The following genes were identified:hemB yields ALA-dehydrase;hemC yields PBG-deaminase; hemE yields uroporphyrinogen decarboxylases; hemF yields coproporphyrinogen oxidase; hemG yields protoporphyrin-iron-chelatase.

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis.

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

Cloning, expression and purification of SmpB from Mycobacterium tuberculosis.

SmpB, a small tmRNA binding protein, is essential for trans-translation. 6His and FLAG tagged SmpB was cloned from Mycobacterium tuberculosis H37Rv. It was expressed in Escherichia coli using the T7 promoter-polymerase system. Anti-FLAG M2 agarose was used for its purification. Mycobacterial SmpB copurifies with other proteins. We identified elongation factor EF-Tu in the purified SmpB preparations.