The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele,encoding TonBQ160K,which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3′ end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

T7 Protein Synthesis in F-Factor-Containing Cells: Evidence for an Episomally Induced Impairment of Translation and Its Relation to an Alteration in Membrane Permeability

T7 infection of F-factor-containing PIFA⁺,B⁺ cells is abortive. In spite of the presence of mRNA for all three classes of T7 proteins, only the earliest of the T7 proteins are synthesized. A crucial question is whether the failure of T7 to develop in PIFA⁺,B⁺ cells is the result of an inability to translate the late classes of T7 mRNA or, as has been recently suggested (Britton, and Haselkorn, 1975; Condit, 1975), whether it is the result of a more generalized alteration in membrane permeability. We have examined the effects of the wild-type PIFA⁺,B⁺ episome and two episomal mutations (pifA⁻ and pifB⁻) on in vitro translation and membrane permeability. In vivo the episomal mutations allow partial or complete T7 development to occur. We demonstrate that cell-free protein-synthesizing systems from T7-infected PIFA⁺,B⁺ cells show a three- to fivefold decrease in the rate of translation of both natural and synthetic mRNA. In addition, ribosomes from T7-infected PIFA⁺,B⁺ cells are defective in their ability to bind Fmet tRNA_f in response to natural mRNA. By contrast, cell-free extracts from T7-infected pifA⁻ (PIFA⁻,B⁺) cells retain the ability to bind Fmet tRNA_f and to translate natural and synthetic mRNA at normal rates. The defective T7-infected PIFA⁺,B⁺ ribosomes can be restored to full activity by a trypsin-sensitive fraction from uninfected PIFA⁺,B⁺ or T7-infected PIFA⁻,B⁺ cells. Despite the differences in translational capacity of these extracts, both T7-infected PIFA⁺,B⁺ and PIFA⁻,B⁺ cells display the same permeability lesions as measured by the loss of ATP from the cells into the supernatant. Mutation of the episome to pfiB⁻ prevents the loss of ATP from the cells after T7 infection.     

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele, encoding TonBQ160K, which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3 end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

Host dependenet inactivation by IS2 of induced E.coli penicillin amidase gene cloned on multicopy plasmids

Structural instability of the cloned penicillin acylase gene (pac) from E.coli ATCC11105 was studied under various physiological conditions. Structural changes which adversely affect the expression of penicillin acylase gene were selected for only under conditions in which the gene was active and fully induced. In E.coli strain YMC the predominant mutations were the insertions of the IS2 element at various sites within the 700 bp proximal portion of the pac gene. The results indicated that the induction of the plasmid cloned gene was the main factor which rendered it a target for inactivation by insertions of the host encoded IS2 elements. The mutational events were host specific and they were not influenced by mutual positions and orientations of key marker genes on the plasmid.     

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10^?3–10^?5 per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA⁺ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.     

Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2

The broad host range plasmid RK2 has previously been found to contain three separate regions of the genome involved in replication and maintenance in Escherichia coli (C. M. Thomas, R. Meyer, D. R. Helinski, 1980, J. Bacteriol.141, 213–222). They include the origin of replication (ori_RK2) and the trfA region which encodes a trans-acting function required for replication. The third region (trfB), although not essential for replication, supplies a function involved in the maintenance of plasmid RK2. Using the maxicell system of labeling plasmid-specific proteins, we have identified all of the proteins encoded by two miniplasmid derivatives of RK2 which contain only the regions ori_RK2, trfA, and trfB. To determine which region specifies each protein, RK2/mini-ColE1 hybrid plasmids were used which contain various restriction fragments of the mini-RK2 replicon. The trfA region appears to encode three proteins designated A1 (39,000 MW), A2 (31,000 MW), and A3 (14,000 MW). Analysis of proteins synthesized by plasmids containing deleted forms of the trfA region indicates that the A2 protein is the essential trfA-encoded replication protein of plasmid RK2. The proteins A1 and A3 may be the products specified by the genes tra3 (involved in transmissibility) and kilB1 (involved in host-cell viability) which also map in the trfA region. The trfB region specifies two proteins designated B1 (36,000 MW) and B2 (30,000 MW). These may be the products of the two kil-override (kor) genes located in the trfB region which have been implicated in plasmid maintenance.     

The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat — unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg₆₈ (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln₅₃ to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu₆₈ mutations in rps4. Instead, six of 15 contained a Gln₇₃ (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln₇₃ to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.     

Molecular cloning and amplification of the gene for thymidylate synthetase of E. coli

The thyA gene of Escherichia coli, which directs the synthesis of the enzyme thymidylate synthetase, has been subcloned from a recombinant λ phage (Hickson et al., 1982) into the multicopy plasmid pBR325 to give the plasmid pPE245. To identify the thyA gene product, the transposon Tn1000 was inserted into pPE245 and derivative plasmids isolated that were no longer able to complement thyA mutations. When proteins synthesised by these plasmids and by pPE245 were labelled and analysed on SDS-polyacrylamide gels a protein of 33000 M_r, presumably the thyA⁺ gene product was absent whenever the thyA gene was inactivated. On assaying cell extracts prepared from cells harbouring pPE245 for thymidylate synthetase, the level of this enzyme was found to be elevated by a factor of at least 25.     

Plasmid mini-F encoded proteins

Summary Proteins specified by the mini-F plasmid (EcoRI restriction fragment f5) were labeled in Escherichia coli minicells and analyzed by SDS-PAGE. Four mini-F encoded proteins could be identified, having molecular weights of 44,000 (A), 36,000 (B), 34,000 (C), and 25,300 (D) daltons. The absence of certain proteins in deleted derivatives of mini-F, generated by treatment with various restriction endonucleases, allowed mapping of the proteins. The A protein maps between F-coordinates 45.7 and 47.9 kb. The gene locus for the B protein is located between 47.2 and 49.3 kb. The C protein maps on a BamHI fragment bordered by F-coordinates 41.5 and 42.8 kb, and finally the D protein maps between 42.8 and 43.8 kb. In addition our data confirm that there are two incompatibility loci on the mini-F genome, located between 45.7 and 47.2 kb (incA) and 44.0 and 45.7 kb (incB).We suggest that (i) the C and D proteins are positive control elements, interacting with origin I and origin II, respectively, (ii) that the incB locus is involved in plasmid partitioning, and (iii) that the A protein encoded by the incA locus is a negative control element.     

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene: the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage λ⁺, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA^? mutant strains and will provide a means of transferring ssb^? mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.     

Genetic Analysis of the CDI Pathway from Burkholderia pseudomallei 1026b

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDI_II ^Bp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDI_II ^Bp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDI_II ^Bp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CT_II ^Bp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiA_II ^Bp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDI_II ^Bp1026b, they provide no protection against the CDI^E264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.     

Guanylate cyclases and associated activator proteins in retinal disease

Two isoforms of guanylate cyclase, GC1 and GC2 encoded by GUCY2D and GUCY2F, are responsible for the replenishment of cGMP in photoreceptors after exposure to light. Both are required for the normal kinetics of photoreceptor sensitivity and recovery, although disease mutations are restricted to GUCY2D. Recessive mutations in this gene cause the severe early-onset blinding disorder Leber congenital amaurosis whereas dominant mutations result in a later onset less severe cone–rod dystrophy. Cyclase activity is regulated by Ca²⁺ which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. No recessive mutations in either of these genes have been reported. Dominant missense mutations are largely confined to the Ca²⁺-binding EF hands of the proteins. In a similar fashion to the disease mechanism for the dominant GUCY2D mutations, these mutations generally alter the sensitivity of the cyclase to inhibition as Ca²⁺ levels rise following a light flash.     

Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

Iron control of the Vibrio fischeri luminescence system in Escherichia coli

Iron influences liminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB ⁺ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICD ABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di (o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB ⁺ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of -galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8–9 fold each for tonB, 2–3 fold each for tonB ⁺) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.     

Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product

Summary The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB ⁺ allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB ⁺), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the lkyB gene product was a membrane protein of molecular weight 42,000.     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

Proteomic Analysis of Salt-Responsive Proteins in the Leaves of Mangrove Kandelia candel during Short-Term Stress

Salt stress is a major abiotic stress that limits crop productivity in many regions of the world. A comparative proteomic approach to identify salt stress-responsive proteins and to understand the molecular mechanisms was carried out in the woody halophyte Kandelia candel. Four-leaf-old K. candel seedlings were exposed to 150 (control), 300, 450, and 600 mM NaCl for 3 days. Proteins extracted from the leaves of K. candel seedlings were separated by two-dimensional gel electrophoresis (2-DE). More than 900 protein spots were detected on each gel, and 53 differentially expressed protein spots were located with at least two-fold differences in abundance on 2-DE maps, of which 48 were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). The results showed that K. candel could withstand up to 450 mM NaCl stress by up-regulating proteins that are mainly involved in photosynthesis, respiration and energy metabolism, Na⁺ compartmentalization, protein folding and assembly, and signal transduction. Physiological data, including superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities, hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂ ⁻) contents, as well as Na⁺ content and K⁺/Na⁺ ratios all correlated well with our proteomic results. This study provides new global insights into woody halophyte salt stress responses. Identification of differentially expressed proteins promotes better understanding of the molecular basis for salt stress reduction in K. candel.     

A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.     

Cloning and characterization of the exbB-exbD-tonB locus of Pasteurella haemolytica A1

A recombinant plasmid (pMG1) carrying Pasteurella haemolytica A1 DNA which complements a tonB mutation of Escherichia coli has been isolated. E. coli tonB metE which carries pMG1 exhibits growth kinetics in the presence of vitamin B₁₂ similar to that of the wild-type host. In addition, the complemented E. coli is susceptible to killing by bacteriophage φ80 and colicin B. Analysis of the nucleotide sequence in the complementing DNA showed that it codes for three genes in the order of exbB-exbD-tonB. This genetic organization has been reported in Haemophilus influenzae, H. ducreyi, Pseudomonas putida and Vibrio cholerae, and may represent a separate lineage of evolution from that of the Enterobacteriaceae in which tonB is unlinked with the accessory genes exbB and exbD. A comparison of the DNA flanking the exbB-exbD-tonB locus in P. haemolytica A1 and H. influenzae showed that the flanking regions are completely different between the two organisms.