Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

Effects of novobiocin on heat shock protection against chromatid aberration induction by triethylenemelamine (TEM) and maleic hydrazide (MH) in Vicia faba.

Heat shock (10 min 40 degrees C) prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) significantly reduced the frequency of induced chromatid aberrations in Vicia faba main root meristems. Novobiocin treatment before heat shock did not prevent heat shock protection against both clastogens; novobiocin application after heat shock prevented protective effects. These results and those obtained earlier for heat shock protection against X-ray challenge are used to discuss possible causes underlying the protective effects triggered by heat shock.

Effects of ethidium bromide and nalidixic acid pretreatment on the induction of chromatid aberrations by TEM and maleic hydrazide in Vicia faba main root meristems.

Pretreatment of Vicia faba main root meristems with ethidium bromide (EB) or nalidixic acid (NA) significantly reduced the yield of metaphases with chromatid aberrations induced by maleic hydrazide (MH), i.e., triggered clastogenic adaptation to MH. No such protection occurred when the alkylating agent triethylenemelamine (TEM) was used for challenge treatment. The differential response of pretreated cells to MH on the one hand (protection) and to TEM (no protection) on the other supports the conclusion that clastogenic adaptation is due to different inducible (repair?) functions, which eventually exert protection against clastogenic impacts.

Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes.

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.

Mutations in rDNA. 1. Dependence of chromosome mutation induction on positions and activity of nucleolus organizer regions.

Studies on chromatid aberration induction in NORs of standard and reconstructed karyotypes, as well as in the single translocation lines of barley, indicate a correlation between synthetic activity (transcription) of rDNA and frequency of chromosome mutations induced by HU. Experimental evidence in favour of this inference arises from analyzing karyotypes with NORs located at their original sites and karyotypes with NORs translocated from their original sites. The close correlation between the different rate of synthetic activity (transcription) in nucleolus formation and the comparable range of variation in aberration involvement of NORs observed in translocation lines are discussed.

Mutations in rDNA : 3. Modulatory action of α-amanitin on aberration induction in nucleolus organizer regions.

The action of α-amanitin, an inhibitor of RNA synthesis, on the induction by hydroxyurea (HU) of chromosomal aberrations in nucleolus organizer regions of barley was studied. The data obtained show that α-amanitin can effectively modify aberration induction in rDNA. Administered before mutagen treatment or in combination with the mutagen, the toxin significantly decreased the HU-induced aberration frequencies in NORs. The data obtained provide further evidence that α-amanitin is an effective modulator of aberration induction in NORs either by interfering with RNA synthesis or by modifying chromatin structure.

Effects of chromosome reconstruction on deletion clustering in Hordeum vulgare.

The barley standard karyotype, two reconstructed karyotypes with all chromosomes interdistinguishable, and four translocation lines were treated with maleic hydrazide. A specific chromosomal site in satellite chromosome 7 (segment 44 adjacent to the nucleolus organizer region) of the standard karyotype was found to represent a deletion hot spot. A sample of specifically reconstructed karyotypes were used to check whether or not transposition of the hot spot region, or changes of its neighborhood, would affect its involvement in deletions. One of the seven karyotypes (translocation line T 505 with a pair of chromosomes having both nucleolus organizer regions and satellites in opposite arms) was without deletion clustering in segment 44. At the same time, a prominent Giemsa band close to the secondary constriction was absent from segment 44. These data show that the involvement in deletions of a certain chromosome segment is modifiable in certain cases by chromosome reconstruction. Similar observations have been made in Vicia faba.

Mutations in rDNA. 2. Effects of Actinomycin D on chromatid aberration induction in nucleolus organizer regions.

A reconstructed karyotype of barley with all chromosomes interdistinguishable was treated with hydroxyurea (HU) and Actinomycin D (Act D). The distribution pattern of chromatid aberrations after treatment with HU alone is characterized by a marked preferential involvement in chromatid translocations of segments 36 (NOR of satellite chromosome 6) and 43 (NOR of satellite chromosome 7). Act D applied at the low concentration of 0.05 µg/ml (4.10(-8) M) before HU treatment, or combined with HU, was found to cause an apparent decrease of HU-induced aberration frequencies in NORs. The exchanges in both segments proved to be approximately a half lower after Act D application when compared to the respective controls (treatment with HU alone). A recovery period of 5 h between the prolonged pretreatment with Act D (15 h) and the HU treatment eliminated the effect of the drug. The possible dependence of mutation induction upon the transcriptional activity of rDNA in NORs after Act D application is discussed.

In Lactobacillus plantarum, carbamoyl phosphate is synthesized by two carbamoyl-phosphate synthetases (CPS): carbon dioxide differentiates the arginine-repressed from the pyrimidine-regulated CPS.

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO(2). Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37-43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN). Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon. CP biosynthesis in L. plantarum was studied with three mutants (DeltaCPS-P, DeltaCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO(2)-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type L. plantarum without CO(2) enrichment may be due to the low affinity of CPS-A for its substrate CO(2) or to regulation of the CP pool by the cellular CO(2)/bicarbonate level.

'Nucleolar dominance' as observed in barley translocation lines with specifically reconstructed SAT chromosomes.

Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.

Revision of SAT chromosome number in Triticum monococcum with respect to nucleolar organizers activity.

Six varieties of Triticum monococcum were analysed by means of the nucleolar test; i.e., estimation of the maximum number of primary nucleoli per nucleus. All of the varieties exhibited 4 primary nucleoli in telophase and early interphase. Following detailed karyological analysis four SAT chromosomes in all six karyotypes were found in accordance with the maximum nucleolar number. Secondary constrictions and microsatellites were localised on the short arms of chromosome pairs 3 and 5. A new order of the chromosomes in the idiogram of Tr. monococcum is proposed.

Sister chromatid exchanges in barley.

A mean frequency of 20.6 sister chromatid exchanges (SCEs) per cell has been observed in a reconstructed karyotype of Hordeum vulgare by application of the FPG technique after unifilar incorporation of BrdU into chromosomes. The involvement in SCEs of the 48 segments into which the chromosome set had been subdivided was, with a single deviation, length proportional and independent of the segment's heterochromatin content. Asymmetric bands, indicative of an uneven distribution of adenine and thymidine between the DNA strands in adenine (A)-thymidine (T) rich chromosome regions, could not be detected after incubation of the cells in BrdU for one cycle of DNA replication.