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.     

Inhibitory effect of adenosine 3'',5''-phosphate on cell division of Escherichia coli K-12 mutant derivatives

Cell division of Escherichia coli K-12 strain PA3092 was inhibited by the addition of adenosine 3',5'-phosphate (cAMP), and the cellular morphology was changed from rods into filaments. Nucleoids in the filaments were regularly distributed and septum formation was perfectly inhibited. This inhibition of cell division by cAMP was reversed by the addition of guanosine 3',5'-monophosphate. To examine whether the inhibitory effect of cAMP on cell division in E. coli PA3092 was specific, its effect in several parental strains was investigated. Induction of cell filamentation by cAMP was observed in E. coli PA309 and P678, but not in E. coli W505, W1, Y10, or the wild-type strain. This result suggests that filamentation by cAMP in E. coli PA3092, PA309, and P678 was due to the mutagenesis by which E. coli P678 was derived from E. coli W595.     

Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli.

The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin.     

Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant.

A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.     

Dissociation of tsl-tif-Induced Filamentation and recA Protein Synthesis in Escherichia coli K-12

In Escherichia coli, expression of the tif-1 mutation (in the recA gene) induces the "SOS response" at 40 degrees C, including massive synthesis of the recA(tif) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the tsl-1 mutation (in the lexA gene) induces massive synthesis of the recA protein and cell filamentation at 42 degrees C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in tif and tsl strains at 42 degrees C is not due to the presence of a high concentration of recA protein since (i) no recA mutants (相似文献   

Effect of suppressors of SOS-mediated filamentation on sfiA operon expression in Escherichia coli

In Escherichia coli, the cell division block observed during the SOS response requires the product of the sfiA gene, whose expression is regulated negatively by the LexA repressor and positively by the RecA protease. We have studied the effect on sfiA expression of sfiA, sfiB, infA, and infB mutations, which are known to affect SOS-associated filamentation. To measure sfiA expression in the different strains, we first constructed a lambda transducing phage carrying an sfiA::lac operon fusion. Mutations at the sfiA locus (dominant and recessive) and the sfiB locus (recessive) had no effect on sfiA expression. The mutations tif (at the recA locus) and tsl (at the lexA locus) are known to induce filamentation and a high level of sfiA expression at 42 degrees C. The infB1 mutation, which suppresses filamentation in a tif tsl strain at 42 degrees C, reduced sfiA expression at 42 degrees C in tif tsl infB1 and tsl infB1 strains but not in a tif infB1 strain. The infA3 mutation, which suppresses tif-mediated filamentation, reduced induction of sfiA expression in a tif infA3 strain at 42 degrees C or after UV irradiation. The isolation and characterization of sfiA constitutive strains revealed only lexA-linked mutations in a sfiA-background, suggesting that LexA is the only readily eliminated repressor of the sfiA gene. Nevertheless, the infA and infB mutations could define elements involved in the regulation of sfiA expression.     

RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete yeast and opportunistic human pathogen of increasing clinical importance due to the increasing population of immunocompromised patients. To further investigate signal transduction cascades regulating fungal pathogenesis, we have identified the gene encoding a RAS homologue in this organism. The RAS1 gene was disrupted by transformation and homologous recombination. The resulting ras1 mutant strain was viable, but failed to grow at 37 degrees C, and exhibited significant defects in mating and agar adherence. The ras1 mutant strain was also avirulent in an animal model of cryptococcal meningitis. Reintroduction of the wild-type RAS1 gene complemented these ras1 mutant phenotypes and restored virulence in animals. A dominantly active RAS1 mutant allele, RAS1Q67L, induced a differentiation phenotype known as haploid fruiting, which involves filamentation, agar invasion and sporulation in response to nitrogen deprivation. The ras1 mutant mating defect was suppressed by overexpression of MAP kinase signalling elements and partially suppressed by exogenous cAMP. Additionally, cAMP also suppressed the agar adherence defect of the ras1 mutant. However, the ability of the ras1 mutant strain to grow at elevated temperature was not restored by cAMP or MAP kinase overexpression. Our findings support a model in which RAS1 signals in C. neoformans through cAMP-dependent, MAP kinase, and RAS-specific signalling cascades to regulate mating and filamentation, as well as growth at high temperature which is necessary for maintenance of infection.     

Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation.

We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene.     

Suppression of induction of SOS functions in an Escherichia coli tif-1 mutant by plasmid R100.1.

The tif-1 mutation in the recA gene of Escherichia coli caused, at 40 degrees C, lethal cell filamentation, induction of the recA protein, mutagenesis, and, in lambda lysogens, prophage induction. The presence of plasmid R100.1 in tif-1 strains suppressed tif-mediated cell filamentation and killing, recA protein induction, and prophage induction in lysogens. It also reduced mutagenesis in a tif-1 sfiA11(R100.1) strain. Plasmids F'lac, P1, and pMB9, in contrast, had little or no effect on tif-mediated induction of lambda. The presence of R100.1 did not inhibit the induction of the recA protein or of lambda by ultraviolet irradiation or mitomycin C treatment of tif-1(R100.1) or tif-1(lambda)(R100.1) strains.     

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp.

The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F- lambda- wild-type strain, is shown here to carry a cryptic mutation, ftsR1, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42 degrees C and the inability to grow in salt-free LB broth at 42 degrees C. The ftsR1 mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201 (Mecr) and alaS21 (Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a plac-relA' plasmid. These backgrounds also confer resistance in LB broth to the beta-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsR1 mutant (but not an isogenic ftsR+ strain) is sensitive to mecillinam in minimal glucose medium at 37 degrees C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsR1 mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37 degrees C but not the growth defect in salt-free LB broth at 42 degrees C. Genetic analysis indicated that the full phenotype of the ftsR1 mutant is due to a single mutation in the rpoB gene (90 min), coding for the beta subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.     

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.     

The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans

We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Galpha protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans.     

Control mechanism of the Escherichia coli K-12 cell cycle is triggered by the cyclic AMP-cyclic AMP receptor protein complex.

The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains. One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter. In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed. The other strains were constructed as double mutants (NC6707 cya sulB [ftsZ(Ts)] and TR3318 crp sulB [ftsZ(Ts)]). In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318. These results indicate that lateral wall synthesis in the E. coli cell cycle is triggered by the cAMP-cAMP receptor protein complex.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

Deletion of the Candida albicans G-protein-coupled receptor, encoded by orf19.1944 and its allele orf19.9499, produces mutants defective in filamentous growth

Filamentous growth of Candida albicans occurs in response to a variety of environmental signals. The C. albicans gene orf19.1944 and its allele orf19.9499 are identical and are predicted to encode an 823-residue, 7-transmembrane-domain protein that has all the expected features of a G-protein-coupled receptor. The protein is 20.9% identical to the Saccharomyces cerevisiae Gpr1p receptor that signals both glucose availability and nitrogen limitation. Deletion of both copies of the gene in C. albicans abolished filamentation by colonies embedded in rich media (YPS, YPGal, and YPGlu), whereas mutants carrying a single copy of the gene were indistinguishable from the parental strain under these conditions. On medium containing low concentrations of ammonia (SLAD and SLAM media), surface colonies of both the homozygous deletion mutants and the mutants carrying a single copy of the gene were defective in filamentation. Serum-induced germ tube formation was unaffected by deletion of this gene, as was filamentation of the mutants growing on the surface of solid Spider medium at 37 degrees C or embedded in solid Spider medium at 25 degrees C. The protein encoded by orf19.1944 and orf19.9499 has a role in filamentation by both surface and embedded colonies, presumably as a sensor of environmental cues.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA.

We have analysed an Escherichia coli temperature-sensitive mutant with altered messenger RNA stability, and it was found that: (1) the unstable fraction of pulse-labelled RNAs decayed with a half-life at 42 °C of about two minutes in the parent strain PA3092; the half-life was 11 to 12 minutes in the mutant HAK75. Puromycin enhanced the decay rate about twofold in both PA3092 and HAK75; the addition of chloramphenicol inhibited the degradation significantly in both strains. The rate of ribosomal RNA accumulation in the mutant cells at 42 °C did not differ from that in the wild-type cells. (2) Sedimentation analysis by sodium dodecyl sulphate/sucrose density-gradient centrifugation of bulk mRNA as well as tryptophan mRNA of the wild-type strain showed the expected rapid reduction in the size and level of those mRNA molecules at three minutes and five minutes respectively, after addition of rifampicin at 42 °C. In contrast, the cells of HAK75 retained almost full-length trp mRNA and bulk mRNA at 5 to 12 minutes after the addition of rifampicin at 42 °C, even though the total level of radioactivity in the mRNA fraction had decreased to about 60 to 75% of the initial activity. (3) Even though mRNA molecules were chemically protected at the non-permissive temperature in the mutant, the functional decay of both β-galactosidase and tryptophan synthetase occurred at a rate comparable to that in the parental strain. (4) We isolated temperature-resistant revertants from the mutant at a frequency of 5 × 10^?8, and these revertants (TR1 and TR2) had the normal decay rate of unstable RNA.