A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

Localised mutagenesis of the fts YEX operon conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients

Summary After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.     

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ³², was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis

Summary The oligopeptide permease is encoded by at least four genes which are transcribed as a single operon. We cloned and characterized this operon from Salmonella typhimurium, as well as the flanking genes, tonB, ana and a new gene, cwd, which affects cell wall synthesis. We correlated the physical map of opp DNA with a detailed genetic map of the opp operon and the individual opp genes were accurately located with respect to various restriction sites by Southern blotting. The region of the chromosome near opp was found to be highly unstable with deletions arising at a high frequency. The operon also contains hot-spots for IS1 and IS5 insertions.     

Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

Characterization of three genes in the dam-containing operon of Escherichia coli

The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS. We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa. Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode. The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle. In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe. Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph. The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions. Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division. We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph. Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.     

The immunoglobulin <Emphasis Type="Italic">IGHD</Emphasis> gene locus in C57BL/6 mice

Four immunoglobulin heavy chain diversity (IGHD) gene subgroups (DFL16, DSP2, DQ52, and DST4) have been identified previously in BALB/c mice. Although the locations of most IGHD genes have been established based on restriction map and Southern blot analysis, a complete mouse IGHD gene locus map at the sequence level is still not available. In addition, a previous restriction fragment length polymorphism study suggested that significant difference in the IGHD gene locus exists between C57BL/6 and BALB/c mice. The author has now analyzed the C57BL/6 mouse genomic data and established a complete map of the IGHD gene locus. All four IGHD subgroups previously identified in BALB/c mice were found to be present in C57BL/6 mice. However, unlike the BALB/c mice, which have at least 13 IGHD genes, the C57BL/6 genome contains only ten IGHD genes, which include one DFL16, six DSP2, one DQ52, and two DST4 genes. There are also differences in the coding regions of the DST4 and DQ52 genes between the two mouse strains.     

The sporulation capable (sca) mutation of Saccharomyces cerevisiae is an allele of the SIR2 gene

Summary We have used the special properties of the spo13-1 mutation in order to study the regulation of yeast meiosis by the mating type loci. We have found that both the rme1-1 mutation and the sca mutation allow haploid meiosis in spo13-1 strains. Therefore, haploid meiosis is regulated in the same manner as diploid meiosis. Unlike rme1-1, the sca mutation allows meiosis through derepression of the silent mating type cassettes; sca strains can sporulate only because they express both MAT a and MAT information. We have found further that sca is an allele of SIR2, one of the genes involved in repression of the silent cassettes. Therefore, the RME1 gene is the only known candidate for a master negative regulator through which the MAT locus controls meiosis.     

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant

Summary We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485–510 kb) whereas sipC and sipD both map to 82 min (3850–3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.     

Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii

Heat shock proteins are ubiquitous and highly conserved. Recently they have become implicated in the import of proteins into organelles. All the heat shock genes characterized to date, however, are known or assumed to be encoded in the nuclear genome even if the corresponding protein can be localised in the mitochondrion or chloroplast. In contrast, we identify here an hsp70 gene in the unicellular chromophytic alga Pavlova lutherii which is located on the chloroplast genome. Localisation of this gene to the chloroplast chromosome is confirmed by Southern blot analysis and pulse-field gel electrophoresis which also reveals that the length of the P. lutherii chloroplast chromosome is 115 kb. We compare the predicted protein of this hsp70 gene with that of maize and of the analogous proteins in the prokaryotic organisms Escherichia coli and Synechocystis PCC6803. The greatest identity is found with the cyanobacterium Synechocystis PCC6803.     

sub, a host mutation that specifically allows growth of replication-deficient gene B mutants of coliphage P2

Summary Bacteriophage P2 normally requires the products of its early genes A and B for lytic growth in its host, Escherichia coli C. A host mutation, sub-1, which allows P2 to grow without a functional B gene product is described. The sub-1 mutation is recessive and maps at approximately 10 min on the E. coli genetic map.     

Heat Stress-Induced Life Span Extension in Yeast

The yeastSaccharomyces cerevisiaehas a limited life span that can be measured by the number of times individual cells divide. Several genetic manipulations have been shown to prolong the yeast life span. However, environmental effects that extend longevity have been largely ignored. We have found that mild, nonlethal heat stress extended yeast life span when it was administered transiently early in life. The increased longevity was due to a reduction in the mortality rate that persisted over many cell divisions (generations) but was not permanent. The genesRAS1andRAS2were necessary to observe this effect of heat stress. TheRAS2gene is consistently required for maintenance of life span when heat stress is chronic or in its extension when heat stress is transient or absent altogether.RAS1,on the other hand, appears to have a role in signaling life extension induced by transient, mild heat stress, which is distinct from its life-span-curtailing effect in the absence of stress and its lack of involvement in the response to chronic heat stress. This distinction between theRASgenes may be partially related to their different effects on growth-promoting genes and stress-responsive genes. Theras2mutation clearly hindered resumption of growth and recovery from stress, while theras1mutation did not. TheHSP104gene, which is largely responsible for induced thermotolerance in yeast, was necessary for life extension induced by transient heat stress. An interaction between mitochondrial petite mutations and heat stress was found, suggesting that mitochondria may be necessary for life extension by transient heat stress. The results raise the possibility that theRASgenes and mitochondria may play a role in the epigenetic inheritance of reduced mortality rate afforded by transient, mild heat stress.     

Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation

Summary The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than I I min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.