Pleiomorphism in Mycobacterium.

Morphological variants in mycobacterial cultures under different growth conditions, including aging of the culture, have been shown to include fibrous aggregates, biofilms, coccoids, and spores. Here we discuss the diversity in shape and size changes demonstrated by bacterial cells with special reference to pleiomorphism observed in Mycobacterium spp. in response to nutritional and other environmental stresses. Inherent asymmetry in cell division and compartmentalization of cell interior under different growth conditions might contribute toward the observed pleiomorphism in mycobacteria. The regulatory genes comprising the bacterial signaling pathway responsible for initiating morphogenesis are speculated upon from bioinformatic identifications of genes for known sensors, kinases, and phosphatases existing in mycobacterial genomes as well as on the basis of what is known in other bacteria.

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions.

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.

Manganese ions induce miscleavage in the Escherichia coli RNase P RNA-catalyzed reaction.

Cleavage by the endoribonuclease RNase P requires the presence of divalent metal ions, of which Mg2+ promotes most efficient cleavage. Here we have studied the importance of there being Mg2+ in RNase P RNA catalysis. It is demonstrated that addition of Mn2+ resulted in a shift of the cleavage site and that this shift was associated with a change in the kinetic constants, in particular kcat. Our data further suggest that the influence of Mn2+ on cleavage site recognition depends on the -1/+73 base-pair in the substrate and the +73/294 base-pair in the RNase P RNA-substrate (RS)-complex. Based on our data we suggest that cleavage in the presence of Mg2+ as the only divalent metal ion proceeds through an intermediate which involves the establishment of the +73/294 base-pair in the RS-complex. By contrast, addition of Mn2+ favours an alternative pathway which results in a shift of the cleavage site. We also studied the influence of Mn2+ on cleavage site recognition and the kinetics of cleavage using various RNase P RNA derivatives carrying substitutions in the region of RNase P RNA that base-pair with the 3' terminal end of the substrate. From these results we conclude that a change in the structure of this RNase P RNA domain influences the involvement of a divalent metal ion(s) in the chemistry of cleavage.

RNase P RNA structure and cleavage reflect the primary structure of tRNA genes.

The function of RNase P RNA depends on its folding in space. A majority of RNase P RNAs from various bacteria show a similar secondary structure to that of Escherichia coli (M1 RNA). However, there are exceptions as exemplified by the RNase P RNA derived from the low GC-content Gram-positive bacteria Bacillus subtilis and Mycoplasma hyopneumoniae (Hyo P RNA). Previous studies using M1 RNA and Hyo P RNA suggest differences both with respect to the kinetics of cleavage as well as to cleavage site recognition. Here we have studied cleavage by these two structurally different RNase P RNAs as a function of changes in the 5' leader and the 3'-terminal CCA motif in the substrate. Our data suggest that the nucleotide at the -2 position in the 5' leader plays a role both for cleavage site recognition and for the rate of cleavage. However, depending on the identity of the -2 residue differences in the cleavage pattern comparing these two types of RNase P RNAs were observed. The results also suggest that the identity of the -1/+73 base-pair in the substrate influences the cleavage site recognition process. These findings will be related to differences in structure comparing these types of RNase P RNAs and the "RCCA-RNase P RNA" interaction. In addition, our findings will be discussed with respect to the primary structure of the tRNA genes in different bacteria.

Stable lead isotopes and lake sediments--a useful combination for the study of atmospheric lead pollution history.

Analysis of stable lead isotopes and lead concentrations in lake-sediment deposits, not least in varved (annually-laminated) sediments, is a useful method to study lead pollution history. This paper presents details from a study of 31 lakes in Sweden. Using a strong acid digestion of sediment samples and ICP-MS analyses, we have found that Swedish lake sediments have a high natural (pre-pollution) 206[Pb]207[Pb] ratio (mean 1.52+/-0.18, range 1.28-2.01, n=31 lakes). In contrast, atmospheric lead pollution derived from metal smelting processes, coal burning and from alkyl-lead added to petrol has a lower ratio (< 1.2). Consequently, when pollution lead deposition began approximately 3500 years ago, the lead isotope ratio of the sediments started to decline, and in modern sediments it is typically < 1.2. Using the isotope and concentration values and a mixing model, the relative contribution of pollution and natural lead in sediment samples can be calculated. The pollution lead records of the Swedish lake sediments show a consistent picture of the atmospheric lead pollution history. Some noticeable features are the Roman peak (approx. 0 AD), the large and permanent Medieval increase (approx. 1000 AD), peaks at approximately 1200 and 1530 AD, the rapid increase after World War II, the peak in the 1970s, and the large modern decline.

Metal ion cooperativity in ribozyme cleavage of RNA.

Combinations of chemical and genetic approaches were used to study the function of divalent metal ions in cleavage of RNA by the ribozyme RNase P RNA. We show that different divalent metal ions have differential effects on cleavage site recognition and rescue of cleavage activity by mixing divalent metal ions that do not promote cleavage by themselves. We conclude that efficient and correct cleavage is the result of cooperativity between divalent metal ions bound at different sites in the RNase P RNA-substrate complex. Complementation of a mutant RNase P RNA phenotype as a result of divalent metal ion replacement is demonstrated also. This finding together with other data indicate that one of the metal ions involved in this cooperativity is positioned near the cleavage site. The possibility that the Mg(2+)/Ca(2+) ratio might regulate the activity of biocatalysts that depend on RNA for activity is discussed.

Inhibition of RNase P RNA cleavage by aminoglycosides.

A number of aminoglycosides have been reported to interact and interfere with the function of various RNA molecules. Among these are 16S rRNA, the group I intron, and the hammerhead ribozymes. In this report we show that cleavage by RNase P RNA in the absence as well as in the presence of the RNase P protein is inhibited by several aminoglycosides. Among the ones we tested, neomycin B was found to be the strongest inhibitor with a Ki value in the micromolar range (35 microM). Studies of lead(II)-induced cleavage of RNase P RNA suggested that binding of neomycin B interfered with the binding of divalent metal ions to the RNA. Taken together, our findings suggest that aminoglycosides compete with Mg2+ ions for functionally important divalent metal ion binding sites. Thus, RNase P, which is an essential enzyme, is indeed a potential drug target that can be used to develop new drugs by using various aminoglycosides as lead compounds.