Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide.

The mitochondrial gene encoding yeast cytochrome oxidase subunit II (Cox2p) specifies a precursor protein with a 15-amino-acid leader peptide. Deletion of the entire leader peptide coding region is known to block Cox2p accumulation posttranscriptionally. Here, we examined in vivo the role of the pre-Cox2p leader peptide and the mRNA sequence that encodes it in the expression of a mitochondrial reporter gene, ARG8m, fused to the 91st codon of COX2. We found within the coding sequence antagonistic elements that control translation: the positive element includes sequences in the first 14 codons specifying the leader peptide, while the negative element appears to be within codons 15 to 91. Partial deletions, point mutations, and local frameshifts within the leader peptide coding region were placed in both the cox2::ARG8m reporter and in COX2 itself. Surprisingly, the mRNA sequence of the first six codons specifying the leader peptide plays an important role in positively controlling translation, while the amino acid sequence of the leader peptide itself is relatively unconstrained. Two mutations that partially block translation can be suppressed by nearby sequence substitutions that weaken a predicted stem structure and by overproduction of either the COX2 mRNA-specific translational activator Pet111p or the large-subunit mitochondrial ribosomal protein MrpL36p. We propose that regulatory elements embedded in the translated COX2 mRNA sequence could play a role, together with trans-acting factors, in coupling regulated synthesis of nascent pre-Cox2p to its insertion in the mitochondrial inner membrane.

Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo.

Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by sigmaA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the fur gene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 microM but not at 5 microM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors.

Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria. Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.

Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes.

In Bacillus subtilis, hydrogen peroxide induces the synthesis of catalase (KatA), alkyl hydroperoxide reductase (AhpCF), and a DNA-binding protein of the Dps family (MrgA). KatA, AhpCF, heme biosynthesis enzymes, and MrgA are also induced upon entry into stationary phase under conditions of iron and manganese limitation. In an effort to define the peroxide regulon repressor, PerR, we used mini-Tn10 mutagenesis to identify loci affecting the regulation of mrgA. From this screen, we isolated two mini-Tn10 insertions in ahpC, the gene encoding the small subunit of AhpCF, that increase the transcription of mrgA-lacZ even in iron-supplemented minimal medium. Indeed, these ahpC::Tn10 insertions lead to elevated expression from all peroxide regulon promoters, including those for mrgA, katA, hemAXCDBL, and ahpCF. As a result, the ahpC::Tn10 mutants display an increased resistance to H2O2. The ahpCF promoter region contains three sequences similar to the peroxide regulon consensus operator (per box). We demonstrate that the ability of ahpC::Tn10 mutations to derepress mrgA requires aerobic growth. In contrast, a second distinct trans-acting regulatory mutation bypasses this requirement for aerobic growth. Since the peroxide regulon is activated in the absence of AhpCF, which degrades alkyl hydroperoxides, we propose that organic hydroperoxides may be physiologically relevant inducers in vivo.

Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants.

Two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats, were investigated through pathology, microbiology, and DNA amplification-based techniques. Efforts were made to survey the diversity of Listeria monocytogenes strains in the silage consumed by affected animals and to verify the causal relationship between silage and disease outbreak. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples. Random amplified polymorphic DNA patterns revealed two distinct L. monocytogenes strains, one of which was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. Three brain isolates and one silage isolate, all of which had different random amplified polymorphic DNA patterns, were found in the outbreak involving goats. All isolates from both outbreaks were indistinguishable in an in vitro assay for cell-to-cell spread and growth in macrophages. All brain isolates from the goat outbreak had identical intracellular ActA patterns, which were different from the pattern for the silage isolate. While the sheep brain isolate had an ActA pattern different from that of the corresponding silage isolate, the patterns for the brain isolates from the two outbreaks were not identical. This survey demonstrates the diversity of L. monocytogenes in silage and suggests the existence of one or more selective processes by which certain strains are more prone to give rise to disease.

A combined modified reverse dot-blot and nested PCR assay for the specific non-radioactive detection of Listeria monocytogenes.

A modified reverse dot-blot assay was developed and used in combination with a nested PCR amplification of hly A for the specific detection of Listeria monocytogenes. The deoxynucleotides and digoxygenin-11-dUTP concentrations in the PCR were optimized for maximal sensitivity and economy. For the dot-blot hybridization, digoxygenin-labelled PCR products were directly spotted on nylon membrane-bound poly-dT tailed capture probes and the signal detection was either colorimetric or chemiluminescent. With crude cell lysates, the total assay could be completed in about 6 h with a detection limit between 2 and 25 colony forming units (cfu) per PCR. The assay was then tested for its applicability to environmental sampling of artificially contaminated aluminum surfaces. For environmental sampling, the assay requires an additional overnight enrichment step and has a sensitivity corresponding to 5 cfu per 25 cm2 of inoculated surface.

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.