Adenylate cyclase mutations rescue the degP temperature-sensitive phenotype and induce the sigma E and Cpx extracytoplasmic stress regulons in Escherichia coli

Inactivation of the gene encoding the periplasmic protease DegP confers a high-temperature-sensitive phenotype in Escherichia coli. We have previously demonstrated that a degP mutant of E. coli strain CBM (W3110 pldA1) is not temperature sensitive and showed that this was most likely due to constitutive activation of the sigma E and Cpx extracytoplasmic stress regulons in the parent strain. In this study, further characterization of this strain revealed a previously unknown cryptic mutation that rescued the degP temperature-sensitive phenotype by inducing the extracytoplasmic stress regulons. We identified the cryptic mutation as an 11-bp deletion of nucleotides 1884 to 1894 of the adenylate cyclase-encoding cyaA gene (cyaAΔ11). The mechanism in which cyaAΔ11 induces the sigma E and Cpx regulons involves decreased activity of the mutant adenylate cyclase. Addition of exogenous cyclic AMP (cAMP) to the growth medium of a cyaAΔ11 mutant strain that contains a Cpx- and sigma E-inducible degP-lacZ reporter fusion decreased β-galactosidase expression to levels observed in a cyaA⁺ strain. We also found that a cyaA null mutant displayed even higher levels of extracytoplasmic stress regulon activation compared to a cyaAΔ11 mutant. Thus, we conclude that the lowered concentration of cAMP in cyaA mutants induces both sigma E and Cpx extracytoplasmic stress regulons and thereby rescues the degP temperature-sensitive phenotype.     

Involvement and necessity of the Cpx regulon in the event of aberrant β‐barrel outer membrane protein assembly

The Cpx and σ^E regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σ^E pathway monitors the biogenesis of β‐barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β‐barrel OMP mis‐assembly, by utilizing mutants expressing either a defective β‐barrel OMP assembly machinery (Bam) or assembly defective β‐barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σ^E pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β‐barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β‐barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly‐defective β‐barrel OMP species. Together, these results showed that both the Cpx and σ^E regulons are required to reduce envelope stress caused by aberrant β‐barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.     

High relative content of lysophospholipids of Helicobacter pylori mediates increased risk for ulcer disease

Helicobacter pylori phospholipase A (OMPLA) degrades bacterial membrane phospholipids to lysophospholipids. High levels of lysophospholipids are associated with higher hemolytic activity, increased release of urease and vacA and better adherence to epithelial cells in vitro. The phospholipase A gene (pldA) displays phase variation due to a slippage in a homopolymeric tract. The aim of this study was to determine if the relative amount of lysophospholipids in the cell wall is associated with ulcer disease, and to further investigate the significance of pldA phase variation. H. pylori isolates of 40 patients were examined. The relative lysophospholipid content of each isolate was determined and the pldA gene was sequenced. The study indicated that H. pylori can regulate its OMPLA activity by phase variation in the pldA gene or by protein level regulation among phase variants in the pldA 'ON' status. We found a significant difference between the relative amount of lysophospholipids of the ulcer group and the non-ulcer group (p=0.022). When the lysophospholipid/phospholipid ratios were compared with outcome, the OR for ulcer disease was 9.0 (95% CI 1.6-49.4; p=0.014). Isolates with a high OMPLA activity are significantly associated with patients with ulcer disease.     

The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease.

The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

In silico evolutionary analysis of Helicobacter pylori outer membrane phospholipase A (OMPLA)

ABSTRACT: BACKGROUND: In the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA) is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK) gene, and a possible origin via horizontal gene transfer (HGT) will be evaluated through both at intra- and inter-species evolutionary analyses. RESULTS: In this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G) and HK (GTR + I + G) sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found. CONCLUSIONS: Our findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences indicated that the transfer occurred long ago. Our results indicate that the bacterium is preserving the function of OMPLA, although some sites are still being evolutionarily optimized.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

The Cpx two-component signal transduction pathway is activated in Escherichia coli mutant strains lacking phosphatidylethanolamine.

The CpxA-CpxR two-component signal transduction pathway of Escherichia coli was studied in a mutant (pss-93) lacking phosphatidylethanolamine (PE). Several properties of this mutant are comparable to phenotypes of cpxA point mutants, indicating that this two-component pathway is activated in PE-deficient cells. In contrast to point mutants, cpx operon null mutants have a wild-type phenotype. By use of this information, a cpx operon null allele was introduced into a pss-93 mutant. Certain altered properties of PE-deficient mutants, which were consistent with activation of the Cpx pathway, returned to the wild-type phenotype, namely, active accumulation of proline and thiomethyl-beta-D-galactopyranoside was partially restored to wild-type levels, increased resistance to amikacin returned to wild-type sensitivity, and high levels of degP expression returned to repressed wild-type levels. Elevated levels of acetyl phosphate and nlpE gene product can result in activation of the Cpx pathway. However, inactivation of the nlpE gene or mutations eliminating the ability to make acetyl phosphate did not alter the high level of degP expression in pss-93 mutants. We propose that the lack of PE results in an alteration in cell envelope structure or physical properties, leading to direct activation of the Cpx pathway.     

Escherichia coli K-12 mutants in which viability is dependent on recA function.

A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E. coli genetic map. In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation. The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon. The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids. The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.     

Reversal of the ΔdegP phenotypes by a novel rpoE allele of Escherichia coli

RseA sequesters RpoE (σ(E)) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σ(E) to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σ(E) regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σ(E) levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σ(E)-dependent RybB::LacZ construct showed only a weak activation of the σ(E) pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σ(E) and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrA(L222Q), it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σ(E)-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σ(E)-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σ(E) may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σ(E) levels.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER Xii. the Genetic and Developmental Effects of Dominant Lethals on Chromosome 3

Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22 degrees C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.     

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli , has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non-permissive temperature) and at 37°C (semi-permissive temperature), but not at 28°C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.     

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli.

The mukB gene encodes a protein involved in chromosome partitioning in Escherichia coli. To study the function of this protein, we isolated from the temperature-sensitive mukB null mutant and characterized 56 suppressor mutants which could grow at 42 degrees C. Ten of the mutants also showed cold-sensitive growth at 22 degrees C. Using one of the cold-sensitive mutants as host, the wild type of the suppressor gene was cloned. The cloned suppressor gene complemented all of the 56 suppressor mutations. DNA sequencing revealed the presence of an open reading frame of 723 bp which could encode a protein of 25,953 Da. The gene product was indeed detected. The previously undiscovered gene, named smbA (suppressor of mukB), is located at 4 min on the E. coli chromosome, between the tsf and frr genes. The smbA gene is essential for cell proliferation in the range from 22 to 42 degrees C. Cells which lacked the SmbA protein ceased macromolecular synthesis. The smbA mutants are sensitive to a detergent, sodium dodecyl sulfate, and they show a novel morphological phenotype under nonpermissive conditions, suggesting a defect in specific membrane sites.     

Mutations in the Ion gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase

We have isolated spontaneous temperature-resistant revertants of a temperature-sensitive mutation (rpoD800) in the sigma subunit of E. coli K12 RNA polymerase. These revertants still contained the rpoD800 allele. They were mucoid, and sensitive to ultraviolet light and the radiomimetic agent nitrofurantoin, which are characteristics of lon mutants. One revertant, Tr29, was mapped to the lon region of the chromosome. Lon- rpoD800 double mutants were constructed, and were phenotypically indistinguishable from the spontaneous temperature-resistant revertant. It is the degradation-deficient property of lon mutants that is responsible for the suppression of the temperature-sensitive phenotype. We show that the rpoD800 sigma polypeptide is a substrate for the ion proteolytic system, and that mutations in lon decrease the rate of mutant sigma degradation. The rate of synthesis of mutant sigma was also affected in lon- strains. The net effect of lon-mutations was to increase the concentration of mutant sigma. We conclude that the temperature-sensitive phenotype results from insufficient concentration, rather than altered function, of the mutant protein.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

Cpx signal transduction is influenced by a conserved N-terminal domain in the novel inhibitor CpxP and the periplasmic protease DegP

In Escherichia coli, envelope stress can be overcome by three different envelope stress responses: the sigma(E) stress response and the Bae and Cpx two-component systems. The Cpx envelope stress response is controlled by the sensor kinase CpxA, the response regulator CpxR, and the novel periplasmic protein CpxP. CpxP mediates feedback inhibition of the Cpx pathway through a hypothetical interaction with the sensing domain of CpxA. No informative homologues of CpxP are known, and thus it is unclear how CpxP exerts this inhibition. Here, we identified six cpxP loss-of-function mutations using a CpxP-beta-lactamase (CpxP'-'Bla) translational fusion construct. These loss-of-function mutations identified a highly conserved, predicted alpha-helix in the N-terminal domain of CpxP that affects both the function and the stability of the protein. In the course of this study, we also found that CpxP'-'Bla stability is differentially controlled by the periplasmic protease DegP in response to inducing cues and that mutation of degP diminishes Cpx pathway activity. We propose that the N-terminal alpha-helix is an important functional domain for inhibition of the Cpx pathway and that CpxP is subject to DegP-dependent proteolysis.