Inhibition of lateral wall elongation by mecillinam stimulates cell division in certain cell division conditional mutants of Escherichia coli.

The effect of mecillinam, a beta-lactam antibiotic that specifically binds penicillin-binding protein 2 of Escherichia coli, causes transition from rod to coccal shape, and inhibits cell division in sensitive cells, has been tested on three different E. coli temperature-sensitive cell division mutants. At the nonpermissive temperature, the antibiotic allows an increase in cell number for strains BUG6 and AX655 but not for AX621. In strain AX655, the cell division stimulation was observed only if the antibiotic was added immediately after shifting to the nonpermissive temperature, whereas in BUG6, the rise in cell number was observed also when mecillinam was added after 90 min of incubation at the nonpermissive temperature. In all cases, cell division began occurring 30 min after addition of the antibiotic. Mecillinam had no effect on division of dnaA, dnaB temperature-sensitive mutants or on division of BUG6 derivatives made resistant to this antibiotic. Other beta-lactam antibiotics such as penicillin, ampicillin, cephalexin, and piperacillin and non beta-lactam antibiotics such as fosfomycin, teichomycin, and vancomycin that inhibit cell wall synthesis did not show any effect on cell division for any of the mutants. The response of the three cell division mutants to mecillinam is interpreted in terms of a recently proposed model for shape regulation in bacteria.     

Accumulation of cyclic GMP in filaments of Escherichia coli BUG6

Experiments with Escherichia coli BUG6, a temperature-sensitive cell division mutant, have shown that at the restrictive temperature (42 degrees C) the loss of cell division potential (filamentation) was accompanied by an unusual increase in intracellular cyclic GMP (cGMP). At the permissive temperature (30 degrees C), cell division proceeded normally, and cGMP did not accumulate. Increasing the osmotic strength of the medium with NaCl suppressed filamentation in BUG6 at 42 degrees C and also suppressed the temperature-sensitive accumulation of cGMP. The addition of nalidixic acid to BUG6 at 30 degrees C induced filamentation but failed to cause cGMP accumulation. A similar accumulation of cGMP has not been observed in other E. coli strains.     

Pole cap formation in Escherichia coli following induction of the maltose-binding protein

After induction with maltose, 30–40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity.After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps.We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

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.     

Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start.

The dicA1 mutation, located in the replication termination region of Escherichia coli at 34.9 min, confers a temperature-sensitive, division defective phenotype to its hosts. Previous analysis had suggested that dicA codes for a repressor of a nearby division inhibition gene dicB. We show now that gene dicB is part of a complex operon. Five open reading frames (ORFs 1 to 5) preceeded by a promoter sensitive to dicA repression are found within a 1500 bp segment, and are organized into two clusters separated by a long untranslated region. Evidence for expression of these ORFs was obtained from in vitro or in vivo translation of plasmid-coded genes. IPTG-dependent cell filamentation was obtained when either the entire or the C-terminal part of the fourth ORF was placed under control of the lac promoter. In both cases, a 7 KD protein corresponding to translation from an in-frame ATG of ORF4 (dicB) was made. We propose that this C-terminal protein is the division inhibitor synthesized in dicA1 mutants.     

Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg.

The obg gene is part of the spo0B sporulation operon and codes for a GTP-binding protein which is essential for growth. A temperature-sensitive mutant in the obg gene was isolated and found to be the result of two closely linked missense mutations in the amino domain of Obg. Temperature shift experiments revealed that the mutant was able to continue cell division for 2 to 3 generations at the nonpermissive temperature. Such experiments carried out during sporulation showed that Obg was necessary for the transition from vegetative growth to stage 0 or stage II of sporulation, but sporulation subsequent to these stages was unaffected at the nonpermissive temperature. Spores of the temperature-sensitive mutant germinated normally at the nonpermissive temperature but failed to outgrow. The primary consequence of the obg mutation may be an alteration in initiation of chromosome replication.     

Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene.

The Fts proteins play an important role in the control of cell division in Escherichia coli. These proteins, which possibly form a functional complex, are encoded by genes that form an operon. In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids. Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C. When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions. On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor. The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature. The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed. Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation.     

Characterization of mutations affecting the Escherichia coli essential GTPase era that suppress two temperature-sensitive dnaG alleles.

Two suppressor mutations of the temperature-sensitive DNA primase mutant dnaG2903 have been characterized. The gene responsible for suppression, era, encodes an essential GTPase of Escherichia coli. One mutation, rnc-15, is an insertion of an IS1 element within the leader region of the rnc operon and causes a polar defect on the downstream genes of the operon. A previously described polar mutation, rnc-40, was also able to suppress dnaG2903. The other mutation, era-1, causes a single amino acid substitution (P17R) in the G1 region of the GTP-binding domain of Era. Analysis of the GTPase activity of the Era-1 mutant protein showed a four- to five-fold decrease in the ability to convert GTP to GDP. Thus, lowered expression of wild-type Era caused by the polar mutations and reduced GTPase activity caused by the era-1 mutation suppresses dnaG2903 as well as a second dnaG allele, parB. Phenotypic analysis of the era-1 mutant at 25 degrees C showed that 10% of the cells contain four segregated nucleoids, indicative of a delay in cell division. Possible mechanisms of suppression of dnaG and roles for Era are discussed.     

The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system.

Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes. A second site mutation in malI abolishes the constitutive expression. The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J. Reidl, K. R?misch, M. Ehrmann, and W. Boos, J. Bacteriol. 171:4888-4899, 1989). We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY. MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE). When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose. Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX. MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion. We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system. The second gene in the operon, malY, encodes a protein of 43,500 daltons. Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences. The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant. The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose. This is not the case in a malT(Con) strain that expresses the mal genes constitutively. We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis.     

Involvement of cyclic AMP and its receptor protein in filamentation of an Escherichia coli fic mutant

Cyclic AMP (cAMP) inhibited septum formation in Escherichia coli PA3092 and induced cell filamentation at elevated temperatures. This phenomenon was first observed in E. coli PA3092 and is due to a temperature-sensitive mutation. We tentatively named this mutation fic (filamentation induced by cAMP). The fic gene was located near rpsL (formerly strA) on the E. coli K-12 map. the inhibitory effect of cAMP on cell division and filamentation in a fic mutant was not observed in a crp mutant. When cAMP was removed from the culture medium, filaments were divided into rods as the intracellular cAMP level decreased. These results suggest that the cAMP-cAMP receptor protein complex causes filamentation in the fic mutant, E. coli PA3092.     

Nucleotide sequence and insertional inactivation of a Bacillus subtilis gene that affects cell division, sporulation, and temperature sensitivity.

Located at 135 degrees on the Bacillus subtilis genetic map are several genes suspected to be involved in cell division and sporulation. Previously isolated mutations mapping at 135 degrees include the tms-12 mutation and mutations in the B. subtilis homologs of the Escherichia coli cell division genes ftsA and ftsZ. Previously, we cloned and sequenced the B. subtilis ftsA and ftsZ genes that are present on an 11-kilobase-pair EcoRI fragment and found that the gene products and organization of these two genes are conserved between the two bacterial species. We have since found that the mutation in the temperature-sensitive filamenting tms-12 mutant maps upstream of the ftsA gene on the same 11-kilobase-pair EcoRI fragment in a gene we designated dds. Sequence analysis of the dds gene and four other open reading frames upstream of ftsA revealed no significant homology to other known genes. It was found that the dds gene is not absolutely essential for viability since the dds gene could be insertionally inactivated. The dds null mutants grew slowly, were filamentous, and exhibited a reduced level of sporulation. Additionally, these mutants were extremely temperature sensitive and were unable to form colonies at 37 degrees C. Another insertion, which resulted in the elimination of 103 C-terminal residues, resulted in a temperature-sensitive phenotype less severe than that in the dds null mutant and similar to that in the known tms-12 mutant. The tms-12 mutation was cloned and sequenced, revealing a nonsense codon that was predicted to result in an amber fragment that was about 65% of the wild-type size (elimination of 93 C-terminal residues).     

The E. coli divE mutation, which differentially inhibits synthesis of certain proteins, is in tRNASer1.

The temperature-sensitive divE mutant of Escherichia coli cannot synthesize certain membrane and cytoplasmic proteins at a non-permissive temperature. Growth of the mutant cells is arrested at a specific stage of the cell cycle when exposed to the non-permissive conditions, suggesting that the divE mutant possesses a defect in cell division control. From sequence determination of a cloned 1.35-kbp DNA fragment that complements the temperature-sensitive divE42 mutation, we characterized two genes in the segment ; one for tRNASer1 and the other for a 23 500 dalton protein. In parallel experiments we cloned the homologous 1.35-kbp DNA fragment from the divE42 mutant and determined its entire nucleotide sequence. Comparison of the two sequences showed that the mutation site is located not in the protein gene, but in the tRNA gene, where A10 is replaced by G10 in the D-stem. Lambda transducing phages carrying the subcloned tRNASer1 gene complemented the divE42 mutation, thereby confirming the conclusion obtained from sequence analyses of the fragments. This finding indicates that tRNASer1 is specifically involved in regulation of cell cycle-specific protein synthesis, coupled with an important step in the process of cell division, or that usage of serine tRNA is functionally specific for the biosynthesis of certain proteins.