The ftsH Gene of the Wine Bacterium Oenococcus oeni Is Involved in Protection against Environmental Stress

The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.     

Lambda Xis Degradation In Vivo by Lon and FtsH

Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489–500, in A. D. Hershey, ed., The Bacteriophage Lambda, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation. Integration of lambda into the bacterial chromosome was delayed in the lon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.     

A new Escherichia coli gene,fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

The Helicobacter felis ftsH gene encoding an ATP-dependent metalloprotease can replace the Escherichia coli homologue for growth and phage λ lysogenization

Cloning and sequencing of an approximately 6.0-kb chromosomal DNA fragment from Helicobacter felis revealed five complete open reading frames. The deduced amino acid sequence of one ORF exhibited sequence similarity to the FtsH protein, an ATP-dependent metalloprotease, from various bacterial species. The encoded protein consists of 638 amino acid residues with a molecular mass of 70.2 kDa. The hydropathy profile of the FtsH protein predicted two N-terminal transmembrane regions that were confirmed experimentally. Insertion of ftsH into a new versatile expression vector resulted in overexpression of FtsH protein in Escherichia coli. In addition, the E. coli ftsH gene could be replaced by the H. felis homologue to allow reduced growth and tenfold increased lysogenization by temperate phage λ. Received: 6 November 1997 / Accepted: 22 January 1998     

Construction and Analyses of Mutant <Emphasis Type="Italic">ftsH</Emphasis> Alleles of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Involving the ATPase- and Zn-Binding Domains

The ftsH gene, present in all eubacterial species, is anchored in the cytoplasmic membrane and contains an ATP- and a Zn-binding domain that are both part of a metalloprotease activity. The Bacillus subtilis ftsH is not essential, but null mutants exhibit a pleiotropic phenotype including filamentous growth; hypersensitivity towards heat and salt stress and a failure to sporulate. To find out whether one or the other functional domain is involved in these different phenotypes, point mutations were introduced into the coding region for both domains leading to a replacement of conserved amino acid residues. The mutant alleles were fused to a xylose-inducible promoter and integrated ectopically into two different strains, one expressing the wild-type ftsH allele and the other carrying a ftsH knockout. While none of the strains exhibited a growth defect in rich medium at 37°C, those strains expressing only the mutant alleles did not resume growth after heat or salt stress challenge. Furthermore, none of the mutant alleles promoted sporulation. While only those purified mutant FtsH proteins with an intact Walker A box exhibited ATPase activity, all of them failed to degrade -casein.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Diverse genes of alkaliphilic Bacillus firmus OF4 that complement K+-uptake-deficient Escherichia coli include an ftsH homologue

Seven clones isolated from libraries of DNA from alkaliphilic Bacillus firmus OF4 restored the growth of a K⁺-uptake-deficient Escherichia coli mutant on only 10mM K⁺. None of the clones contained genes with apparent homology to known K⁺ transport systems in other organisms. Based on sequence homologies, the newly isolated alkaliphile loci included: ftsH; a dipeptide transport system; a gerC locus with hydrophobic open reading frames not found in the comparable locus of Bacillus subtilis; a sugar phosphotransferase enzyme; and a capBC homologue. The ftsH gene provided a new and striking example of a recognized property of extracellular and external regions of polytopic alkaliphile proteins: a significant paucity of basic amino acid residues relative to neutrophile counterparts. The alkaliphile ftsH gene was able to complement a mutant of E. coli with a temperature-sensitive ftsH gene product. Received: 5 August 1996 / Accepted: 14 October 1996     

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA.

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA^Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA^Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA₁^Leu and sequenced tRNA₃^Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA₃^Leu is highly preferred for the CUU codon; tRNA₄^Leu and tRNA₅^Leu both recognize UUA and UUG codons, with tRNA₄^Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX₄ (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX₄ leucine codon series is not paralleled in the UUX₄ leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.     

Membrane Proteases and Aminoglycoside Antibiotic Resistance

We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products.     

Synonymous codon usage in chloroplast genome of Coffea arabica

Synonymous codon usage of 53 protein coding genes in chloroplast genome of Coffea arabica was analyzed for the first time to find out the possible factors contributing codon bias. All preferred synonymous codons were found to use A/T ending codons as chloroplast genomes are rich in AT. No difference in preference for preferred codons was observed in any of the two strands, viz., leading and lagging strands. Complex correlations between total base compositions (A, T, G, C, GC) and silent base contents (A₃, T₃, G₃, C₃, GC₃) revealed that compositional constraints played crucial role in shaping the codon usage pattern of C. arabica chloroplast genome. ENC Vs GC₃ plot grouped majority of the analyzed genes on or just below the left side of the expected GC₃ curve indicating the influence of base compositional constraints in regulating codon usage. But some of the genes lie distantly below the continuous curve confirmed the influence of some other factors on the codon usage across those genes. Influence of compositional constraints was further confirmed by correspondence analysis as axis 1 and 3 had significant correlations with silent base contents. Correlation of ENC with axis 1, 4 and CAI with 1, 2 prognosticated the minor influence of selection in nature but exact separation of highly and lowly expressed genes could not be seen. From the present study, we concluded that mutational pressure combined with weak selection influenced the pattern of synonymous codon usage across the genes in the chloroplast genomes of C. arabica.     

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.     

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)–tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2⁻ mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.     

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA^Ser(CGA), an Arabidopsis intron-containing tRNA^Tyr(GTA) and an Arabidopsis intron-containing tRNA^Met(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA^Ser gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA^Tyr or tRNA^Met genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA^Met(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA^Tyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA^Tyr and tRNA^Met were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.     

Identification of the XorII methyltransferase gene and a vsr homolog from Xanthomonas oryzae pv. oryzae

We have changed the translation initiation codon of the COX2 mRNA of Saccharomyces cerevisiae from AUG to AUA, generating a mutation termed cox2-10. This mutation reduced translation of the COX2 mRNA at least five-fold without affecting the steady-state level of the mRNA, and produced a leaky nonrespiratory growth phenotype. To address the question of whether residual translation of the cox2-10 mRNA was initiating at the altered initiation codon or at the next AUG codon downstream (at position 14), we took advantage of the fact that the mature coxll protein is generated from the electrophoretically distinguishable coxII precursor by removal of the amino-terminal 15 residues, and that this processing can be blocked by a mutation in the nuclear gene PET2858. We constructed a pet2858, cox2-10 double mutant strain using a pet2858 allele from our mutant collection. The double mutant accumulated low levels of a polypeptide which comigrated with the coxII precursor protein, not the mature species, providing strong evidence that residual initiation was occurring at the mutant AUA codon. Residual translation of the mutant mRNA required the COX2 mRNA-specific activator PET111. Furthermore, growth of cox2-10 mutant strains was sensitive to alterations in PET111 gene dosage: the respiratory-defective growth phenotype was partially suppressed in haploid strains containing PET111 on a high-copy-number vector, but became more severe in diploid strains containing only one functional copy of PET111.     

Escherichia coli K-12 tolZ mutants tolerant to colicins E2, E3, D, Ia, and Ib: defect in generation of the electrochemical proton gradient

Spontaneous Escherichia coli K-12 mutants tolerant to colicin E3 were isolated, and on the basis of their tolerance patterns to 19 kinds of colicins, a new phenotypic class of tolZ mutants was found. The tolZ gene was located between min 77 and 78 on the E. coli K-12 genetic map. The tolZ mutants were tolerant to colicins E2, E3, D, Ia, and Ib, and showed an increased sensitivity to ampicillin, neomycin, and EDTA, but not to deoxycholate; they were able to grow on glucose minimal medium, but not on nonfermentable carbon sources (succinate, acetate, pyruvate, lactate, malate, etc.). The pleiotropic phenotype of the tolZ mutant was due to a single mutation. Both respiration and membrane ATPase activity of the tolZ mutant were normal. The tolZ mutant had a defect in the uptake of proline, glutamine, thiomethyl-beta-D-galactoside, and triphenylmethylphosphonium ion; these uptake systems are driven by an electrochemical proton gradient (delta-mu H+) or a membrane potential (delta psi). In contrast, the uptake of methionine and alpha-methyl-D-glucoside, which is not dependent on delta-mu H+ and delta psi, was normal in the tolZ mutant. Glucose 6-phosphate uptake at pH 5.5, which is driven by a transmembrane pH gradient, in the tolZ mutant was similar to the parent level. These results indicate that the tolZ mutant has a defect in the generation of delta-mu H+ and delta psi.     

The amino acid composition of proteins: a method of analysis.

Four quasiloglinear models are proposed for describing relationships between the amino acid composition of proteins and the structure of the genetic code. The models allow estimation of base frequencies in all three codon positions and can be used to investigate “interactions” between any two codon positions. The estimation procedure proposed by Ohta and Kimura (Genetics64 (1970), 387–395) is discussed and using two of the proposed quasiloglinear models an analysis of the amino acid composition of human cytochrome c is presented. The analysis suggests that of the six codons which code for leucine (CUU, CUC, CUA and CUG) do not occur in human cytochrome c.