Per Nygaard

Essential Bacillus subtilis genes

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285 degrees on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

Location on the chromosome of Escherichia coli of genes governing purine metabolism

SummaryGenes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was contransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.

Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-α-1-pyrophosphate (PRPP): Determination of cellular PRPP pools and assay of PRPP synthetase activity

Abstract Conditions are described where 5-phosphoribosyl-α-1-pyrophosphate (PRPP) can be determined by thin-layer chromatographic methods commonly used for the determination of nucleoside triphosphate pools in 32P-labeled bacteria. A two-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 μmol per gram dry weight bacteria) can be determined. In a uni-dimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 μmol per gram dry weight bacteria. This uni-dimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [γ-32P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of 32P labeling. The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase have been applied to the analysis of some mutants of Salmonella typhimurium and have provided results that agree well with the results obtained by conventional methods of PRPP analysis.

Definition of the Bacillus subtilis PurR operator using genetic and bioinformatic tools and expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO.

The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subject to control by the purR gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the purA and purR genes. A consensus sequence for the binding of PurR, named the PurBox, has been suggested (M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900-3906, 1998). To determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the B. subtilis genome sequence and found several candidate PurBoxes. By the use of transcriptional lacZ fusions, five selected genes or operons (glyA, yumD, yebB, xpt-pbuX, and yqhZ-folD), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR. Using a machine-learning algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two PurBoxes in their upstream control regions. The two boxes are divergently oriented, forming a palindromic sequence with the inverted repeats separated by 16 or 17 nucleotides. A computerized search revealed one additional PurR-regulated gene, ytiP. The significance of the tandem PurBox motifs was demonstrated in vivo by deletion analysis and site-directed mutagenesis of the two PurBox sequences located upstream of glyA. All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism. Functional analysis showed that yebB encodes the previously characterized hypoxanthine-guanine permease PbuG and that ytiP encodes another guanine-hypoxanthine permease and is now named pbuO. yumD encodes a GMP reductase and is now named guaC.

Regulation of levels of purine biosynthetic enzymes in Bacillus subtilis : effects of changing purine nucleotide pools

The genes encoding the enzymes of IMP biosynthesis in Bacillus subtilis constitute the pur operon, whereas the genes encoding GMP biosynthetic enzymes, guaA (GMP synthetase) and guaB (IMP dehydrogenase), and the purA gene encoding adenylosuccinate (sAMP) synthetase all occur as single units. The purB gene encodes an enzyme involved in both IMP and AMP biosynthesis and is located in the pur operon. The levels of purine biosynthetic enzymes (except for GMP synthetase) were repressed in cells grown in the presence of purine compounds. Transcription of the pur operon is regulated negatively by adenine and guanine compounds. Our results suggest that ATP and guanine (or hypoxanthine) act as low molecular mass repressors. The level of IMP dehydrogenase was repressed by guanosine, but not in the presence of adenine, and was negatively correlated with the GTP/ATP pools ratio. The level of sAMP synthetase was repressed by adenine and increased by guanosine, and was positively correlated with the GTP/ATP pools ratio. It appears that the mode of regulating purine biosynthetic enzyme levels coincides with the cellular need for the individual enzymes.

Role of Guanosine Kinase in the Utilization of Guanosine for Nucleotide Synthesis in Escherichia coli

Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated by plating cells on medium with guanosine as the sole purine source. These mutants had altered guanosine kinase activity and the mutations were mapped in the gene encoding guanosine kinase, gsk. Some of the mutants had acquired an additional genetic lesion in the purine de novo biosynthetic pathway, namely a purF, a purL or a purM mutation. A revised map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE.

Nucleosides as a carbon source in Bacillus subtilis : characterization of the drm-pupG operon

In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.

Functional analysis of the Bacillus subtilis purT gene encoding formate-dependent glycinamide ribonucleotide transformylase.

The purT gene from Bacillus subtilis encoding the formate-dependent glycinamide ribonucleotide transformylase T was cloned by functional complementation of an Escherichia coli purN purT double mutant. The nucleotide sequence revealed an open reading frame of 384 amino acids. The purT amino acid sequence showed similarity to the enzyme phosphoribosylaminoimidazole carboxylase encoded by the purK gene but not to the N10-formyltetrahydrofolate-dependent glycinamide ribonucleotide transformylase N enzyme encoded by the purN gene. The glycinamide ribonucleotide transformylase T level was repressed in cells grown in rich medium compared to minimal-medium-grown cells. However, when the culture entered the stationary-growth phase the enzyme level increased in rich medium and decreased in minimal medium. By comparing the deduced amino acid sequence of the B. subtilis purT gene product with translated nucleotide sequences in various databanks, evidence for the existence of putative purT genes in the Gram-negative bacteria Pasteurella haemolytica and Pseudomonas aeruginosa was obtained.

Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in GDEase activity, a collection of mutant strains was screened for strains unable to use guanine as a nitrogen source. The strain BFA1819 (yknA) showed the expected phenotype and no GDEase activity could be detected in this strain. A new name for yknA, namely gde, is proposed. The gde gene encodes a 156 amino acid polypeptide and was preceded by a promoter sequence that is recognized by the sigma(A) form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular mass inducer molecule. The level of GDEase was found to be subjected to global nitrogen control exerted by the GlnA/TnrA-dependent signalling pathway. The two regulatory proteins of this pathway, TnrA and GlnR, indirectly and positively affected gde expression. This is the first instance of a gene whose expression is positively regulated by GlnR. The GDEase amino acid sequence shows no homology with the mammalian enzyme. In agreement with this are the different physiological roles for the two enzymes.