Matthias Rose

Complete DNA sequence of yeast chromosome II

In the framework of the EU genome‐sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37‐45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT‐rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT‐rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose‐specific phosphoenolpyruvatesugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI ) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsI form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling‐circle replication

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus. This gene is located at 55° on the chromosome and belongs to a putative operon together with a ligase gene (lig ) and two unknown genes named pcrB and yerH. As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling‐circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading‐strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli. PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep− phenotype on E. coli. Altogether, these results show that PcrA is an helicase used for plasmid rolling‐circle replication and suggest that it is also involved in UV repair.

Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence

PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus. It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins. Expression of the plcR gene is autoregulated and activated at the onset of stationary phase. Here, we used mini‐Tn10 transposition to generate a library of B. thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene. Three mutant strains were identified carrying distinct mini‐Tn10 insertions. The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B. thuringiensis strain in which the plcR gene had been disrupted. The insertion sites of the three mini‐Tn10 transposons mapped in a five‐gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization. By analogy, the five B. thuringiensis genes were designated oppA, B, C, D and F. In vitro disruption of the B. thuringiensis oppB gene reproduced the effect of the mini‐Tn10 insertions (i.e. the loss of haemolytic activity) and reduced the virulence of the strain against insects. These phenotypes are similar to those of a ΔplcR mutant. Opp is required for the import of small peptides into the cell. Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum‐sensing effector. The opp mutations impaired the sporulation efficiency of B. thuringiensis when the cells were cultured in LB medium. Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B. subtilis. However, analysis of plcR expression in ΔoppB, Δspo0A and ΔoppBΔspo0A mutants indicates that Opp is required for plcR expression via a Spo0A‐independent mechanism.

Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin

PIcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PIcR are: pIcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putative S-layer like surface protein and a putative extracellular RNase. By analysing 37.1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.

ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100‐type Clp ATPases is their amino‐terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two σA‐type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.

Ubc8p functions in catabolite degradation of fructose‐1,6‐bisphosphatase in yeast

The key gluconeogenic enzyme fructose‐1,6‐bisphosphatase (FBPase) is synthesized when cells of the yeast Saccharomyces cerevisiae are grown on a non‐fermentable carbon source. After shifting the cells to glucose‐containing medium, in a process called catabolite degradation, FBPase is selectively and rapidly broken down. We have isolated gid mutants, which are defective in this glucose‐induced degradation process. When complementing the defect in catabolite degradation of FBPase in gid3‐1 mutant cells with a yeast genomic library, we identified the GID3 gene and found it to be identical to UBC8 encoding the ubiquitin‐conjugating enzyme Ubc8p. The in vivo function of Ubc8p (Gid3p) has remained a mystery so far. Here we demonstrate the involvement of Ubc8p in the glucose‐induced ubiquitylation of FBPase as a prerequisite for catabolite degradation of the enzyme via the proteasome. Like FBPase, Ubc8p is found in the cytoplasmic fraction of the cell. We demonstrate cytoplasmic degradation of FBPase.

Proteins of Newly Isolated Mutants and the Amino-terminal Proline Are Essential for Ubiquitin-Proteasome-catalyzed Catabolite Degradation of Fructose-1,6-bisphosphatase of Saccharomyces cerevisiae

Addition of glucose to cells of the yeastSaccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1,gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphospha-tase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Site-directed mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.

New genes in the 170° region of the Bacillus subtilis genome encode DNA gyrase subunits, a thioredoxin, a xylanase and an amino acid transporter

A DNA contig of 26.2 kb covering the 170 degrees region of the Bacillus subtilis strain 168 genome was isolated and sequenced. For DNA isolation, suitable restriction sites at the end of previously known genes were chosen to amplify adjacent unknown DNA regions by inverse PCR. On the basis of the DNA sequence, 26 ORFs were identified of which eglS and ccdA, as well as part of citB and tkt have been described previously. Here we report the complete sequences of the aconitase (citB) and transketolase (tkt) genes. Of the other proteins encoded on the 26.2 kb fragment, eight revealed similarities to previously described proteins. These included a pair of newly identified DNA gyrase subunits A (grlA) and B (grlB), a sodium/proton-dependent alanine carrier (alsT), a member of the thioredoxin family (TlpA), an endo-1,4-beta-xylanase (xynD) and a response regulator protein. Comparison of the physical and the genetic maps revealed several differences. According to its flanking sequences the lexA (dinR) gene which was previously mapped at 162 degrees was found to be adjacent to yneA localized at 170 degrees. Genes citB and eglS were located the opposite way round and closer together than expected from the genetic map (citB at 173 degrees and eglS at 170 degrees). The prkA gene, which was mapped at 169 degrees, was not present on the respective fragment. Sequence comparison actually showed that prkA is located close to 70 degrees on the B. subtilis genome.

Molecular analysis of the yeast SER1 gene encoding 3-phosphoserine aminotransferase: regulation by general control and serine repression

Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxypyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43 401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression.