Mapping of Asporogenous Mutations of Bacillus subtilis: a Minimum Estimate of the Number of Sporulation Operons

Sporulation-specific mutations in Bacillus subtilis have been mapped by transduction and transformation. The mutations caused blocks at stages 0, II, III, and IV of sporulation; more than one phenotype was found for each of these stages. On the basis of the criteria used to define a sporulation operon, a minimum estimate could be made of the number of operons activated during sporulation. Nine operons were identified for stage 0, eight for stage II, five for stage III, and six for stage IV. It is probable that several of these 28 operons are activated in groups so that the number of steps in the dependent sequence of sporulation events should turn out to be less than the number of operons.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Factors influencing the cultivability of lake water bacteria

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.     

Transcription of the Bacillus subtilis gerK operon, which encodes a spore germinant receptor, and comparison with that of operons encoding other germinant receptors

The gerA, gerB, and gerK operons, which encode germinant receptors in spores of Bacillus subtilis, were transcribed only in sporulation, and their mRNA levels peaked initially approximately 3 h before the initiation of accumulation of the spore's dipicolinic acid. After a rapid fall, levels of these mRNAs peaked again approximately 5 h later. In one wild-type strain (PS832), gerA mRNA was the most abundant, with levels of gerB and gerK mRNAs approximately 50% of that of gerA mRNA, whereas gerB mRNA was the most abundant in another wild-type strain (PY79). The synthesis of gerK mRNA in sporulation was abolished by loss of the forespore-specific RNA polymerase sigma factor, sigma(G), and induction of sigma(G) synthesis in vegetative cells led to synthesis of gerK mRNA. SpoVT, a regulator of sigma(G)-dependent gene expression, repressed gerK expression. The gerK promoter showed sequence similarities to sigma(G)-dependent promoters, and deletion of elements of this putative promoter abolished gerK expression in sporulation.     

Selective gene expression during sporulation of Physarum polycephalum.

The two-dimensional gel electrophoresis of polypeptides synthesized in vitro from poly(A)+ RNA showed that mRNA populations change during sporulation of Physarum polycephalum. The differential hybridization of a cDNA library prepared from poly(A)+ RNA isolated from sporulating cells revealed that of 846 clones, 64 corresponded to sporulation-specific mRNAs. Further analysis demonstrated that these clones contained seven different sequences: three abundant sequences composing 3.2, 1.8, and 1.2% of the library and four other less abundant sequences. It is probable that all the major mRNAs specifically expressed in early stages of sporulation were identified. The most abundant mRNA from this group coded for a hydrophobic protein that contained a signal peptide. This protein is 47% similar to another Physarum protein, which was encoded by the most abundant plasmodium-specific mRNA. The plasmodial mRNA was degraded during sporulation and was replaced by the sporulation mRNA. These two proteins are thus encoded by members of a gene family whose expression is developmentally regulated.     

Accuracy of a Plant-Infection Technique for Counting Populations of Rhizobium trifolii

A "plant-infection" technique for identification and estimation of populations of Rhizobium is described and compared with petri dish colony counts of the same bacterial populations. Provided that bacterial suspensions are agitated thoroughly, dilutions made at 4 C, and test plants grown on agar, the plant-infection technique is an accurate method of estimating R. trifolii in pure culture or when added to soil. The results are discussed in relation to previous investigations of the subject. Tables are presented which, when applied to the distribution of positive (nodulated) test plants in either fivefold or tenfold dilution series, permit calculation of most probable numbers, and confidence limits are stated.     

Genome holography: deciphering function-form motifs from gene expression data

Background

DNA chips allow simultaneous measurements of genome-wide response of thousands of genes, i.e. system level monitoring of the gene-network activity. Advanced analysis methods have been developed to extract meaningful information from the vast amount of raw gene-expression data obtained from the microarray measurements. These methods usually aimed to distinguish between groups of subjects (e.g., cancer patients vs. healthy subjects) or identifying marker genes that help to distinguish between those groups. We assumed that motifs related to the internal structure of operons and gene-networks regulation are also embedded in microarray and can be deciphered by using proper analysis.

Methodology/Principal Findings

The analysis presented here is based on investigating the gene-gene correlations. We analyze a database of gene expression of Bacillus subtilis exposed to sub-lethal levels of 37 different antibiotics. Using unsupervised analysis (dendrogram) of the matrix of normalized gene-gene correlations, we identified the operons as they form distinct clusters of genes in the sorted correlation matrix. Applying dimension-reduction algorithm (Principal Component Analysis, PCA) to the matrices of normalized correlations reveals functional motifs. The genes are placed in a reduced 3-dimensional space of the three leading PCA eigen-vectors according to their corresponding eigen-values. We found that the organization of the genes in the reduced PCA space recovers motifs of the operon internal structure, such as the order of the genes along the genome, gene separation by non-coding segments, and translational start and end regions. In addition to the intra-operon structure, it is also possible to predict inter-operon relationships, operons sharing functional regulation factors, and more. In particular, we demonstrate the above in the context of the competence and sporulation pathways.

Conclusions/Significance

We demonstrated that by analyzing gene-gene correlation from gene-expression data it is possible to identify operons and to predict unknown internal structure of operons and gene-networks regulation.     

Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum

The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.     

Use of the most probable number technique to quantify soil-borne plant pathogens

Bioassays using serial soil dilutions and most probable number (MPN) estimations have been used by various authors to quantify inoculum of soil-borne plant pathogens. The requirements of a reliable bioassay are discussed; they include a good choice of dilution series and reproducible growing conditions. Sources of computer programs for analysis of the data are listed. The importance of testing the fit to the mathematical model used is illustrated and emphasised. Factors affecting the size and stability of the standard errors and of the inherent bias of the most probable number estimate are discussed. Equations are presented for calculating the expected standard errors, approximate confidence limits and least significant differences for different dilution factors and numbers of replicates. The benefits of using uneven replication are illustrated. Mathematical considerations show that the technique should enable differences of an order of magnitude to be detected and MPNs should be quoted with a maximum of two significant figures. Dilution ratios as large as 10 should be avoided. Statistical and biological difficulties, especially in standardising growing conditions when soil moisture is critical, indicate that results should normally be regarded as relative, rather than absolute, measurements of inoculum.     

Variety of sporulation phenotypes resulting from mutations in a single regulatory locus, spoIIA, in Bacillus subtilis

Closely linked mutations in either of the two putative genes of the sporulation locus spoIIA can affect, in quite diverse ways, spore incidence, the production of alkaline phosphatase and DNAase, and the stability of the cells in sporulation medium. It is concluded that the locus has a regulatory function affecting the activation or induction of at least two, and possibly more, sporulation-associated operons.     

Computational analysis of Ciona intestinalis operons

Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ～15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ～20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like C. elegans, operons in Ciona are comprised of a variety of genes that are not necessarily related in function. The genes in only 50% of the operons appear to be co-regulated, suggesting that more complex gene regulatory mechanisms are likely operating.     

Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine

Exogenous leucine affects the expression of a number of different operons in Escherichia coli. For at least some of these operons, the leucine-related effect is mediated by a protein called Lrp (Leucine-responsive regulatory protein). The purification of Lrp to near homogeneity is described. Lrp is a moderately abundant, basic protein composed of two subunits of molecular mass 18.8 kDa each. In addition, the corresponding protein was purified from a strain having a mutation within the gene that encodes Lrp (lrp). This mutation (lrp-1) causes high constitutive expression of ilvIH, one of the operons controlled by Lrp (Platko, J. V., Willins, D.A., and Calvo, J.M. (1990) J. Bacteriol. 172, 4563-4570). The Lrp-1 and Lrp proteins have similar physical properties, but they show some differences in the characteristics with which they bind DNA upstream of the ilvIH promoter. The nucleotide sequences of the lrp and lrp-1 genes differ by only a single nucleotide, a C to G change that would substitute a Glu for an Asp at amino acid 114. Lrp has some amino acid sequence similarity to AsnC, a protein that regulates asnA expression (Kolling, R., and Lother, H. (1985) J. Bacteriol. 164, 310-315).     

Properties of purified sporlets produced by spoII mutants of Bacillus subtilis.

A number of abortively disporic spoII mutants of Bacillus subtilis released their forespore compartments (termed stage II sporlets) after mother cell lysis during sporulation in nutrient exhaustion or resuspension media. Stage II sporlets were viable and contained levels of ATP and a number of enzymes similar to those in cells 2 to 3 h after sporulation. However, stage II sporlets carried out essentially no macromolecular synthesis, a result suggesting that they were in a quiescent state. The nucleoid of these quiescent stage II sporlets was significantly condensed relative to that in the original vegetative cells, as was previously found to take place 1 to 2 h after initiation of sporulation (B. Setlow, N. Magill, P. Febbroriello, L. Nakhimousky, D. E. Koppel, and P. Setlow, J. Bacteriol. 173:6270-6278, 1991). Stage II sporlets may be a useful model system for analysis of forespore properties early in stage II of sporulation.