The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.     

Mutants suppressing novobiocin hypersensitivity of a mukB null mutation

The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.     

In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships

Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.     

Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG.

The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed.     

Amino acid replacement in a mutant lipoprotein of the Escherichia coli outer membrane.

The primary structure of a mutant lipoprotein of the outer membrane of Escherichia coli was investigated. This mutant was previously described as a mutant that forms a dimer of the lipoprotein by an S-S bridge (H. Suzuki et al., J. Bacteriol. 127:1494-1501, 1976). The amino acid analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of tryptic peptides, it was found that the arginine residue at position 57 was replaced with a cysteine residue. The amino terminal structure of the mutant lipoprotein was found to be glycerylcysteine, as in the case of the wild-type lipoprotein. The present results show that the mutation that was previously determined to map at 36.5 min on the E. coli chromosome occurred in the structure gene (lpp) for the lipoprotein. This was further confirmed by the fact that a merodiploid carrying both lpp+ and lpp produces not only the wild-type lipoprotein but also the mutant lipoprotein.     

Transmembrane signal transduction and osmoregulation in Escherichia coli: I. Analysis by site-directed mutagenesis of the amino acid residues involved in phosphotransfer between the two regulatory components, EnvZ and OmpR

Previously, the transfer of a phosphoryl group between the EnvZ and OmpR proteins, which are involved in expression of the ompF and ompC genes in response to the medium osmolarity, was demonstrated in vitro. In this study, the histidine (His) residue at position 243 of the EnvZ protein, and the aspartate (Asp) residues at positions 12 and 55 of the OmpR protein were changed, respectively, by means of site-directed mutagenesis. We characterized the mutant proteins in terms of not only their in vitro phosphotransfer reactions but also their in vivo osmoregulatory phenotypes. The mutant EnvZ protein was defective in its in vitro ability not only as to EnvZ-autophosphorylation but also OmpR-phosphorylation and OmpR-dephosphorylation. This particular mutant EnvZ protein seemed to exhibit null functions as to the in vivo osmoregulatory phenotype. The mutant OmpR protein with the amino acid change at position 12 was clearly phosphorylated in vitro, but at a very low rate as compared with the wild-type OmpR protein. In vitro phosphorylation of the mutant OmpR protein with the amino acid change at position 55 was more severely affected. This mutant OmpR protein appeared to exhibit null functions as to the in vivo osmoregulatory phenotype. These results suggest that the histidine residue at position 243 of the EnvZ protein and the aspartate residues at positions 12 and 55 of the OmpR protein are deeply involved in the phosphotransfer between the EnvZ and OmpR proteins.     

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.     

Carboxyl terminal region of the MukB protein in Escherichia coli is essential for DNA binding activity

Abstract The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity. We have isolated a mutation, mukB1013 , causing a substitution of valine at position 1379 to leucine. This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected. A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.     

Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature-Sensitive Phenotype of the Influenza Virus

A temperature-sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts-134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts-134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts-134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three-dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild-type both at permissive and nonpermissive temperatures.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

The penultimate tyrosine residue of the K99 fibrillar subunit is essential for stability of the protein and its interaction with the periplasmic carrier protein

The role of the penultimate and conserved tyrosine residue of the K99 major fibrillar subunit (FanC) in fibrillae biosynthesis and functioning was investigated. By using oligonucleotide-directed in vitro mutagenesis the TAT codon of tyrosine-158 of fanC was changed into a TAG stop codon. The mutant fanC gene encoded a truncated major subunit lacking the two carboxyl-terminal amino acid residues. Furthermore, the tyrosine residue (position 158) was replaced by a serine residue or by a glutamic acid residue. The effect of these mutations on the expression and binding capacity of K99 fibrillae was investigated by using an ELISA, an haemagglutination assay, Escherichia coli minicells and suppressor strains. All mutations completely blocked K99 fibrillae biosynthesis and haemagglutination activity. The mature form of the truncated mutant FanC polypeptide could not be detected in minicells, but its precursor was expressed at a normal level. The results showed that the penultimate tyrosine residue is essential for the expression of mature fibrillar subunits and suggested a function in the interaction with the periplasmic transport protein FanE.     

High expression of the second lysine decarboxylase gene, ldc, in Escherichia coli WC196 due to the recognition of the stop codon (TAG), at a position which corresponds to the 33th amino acid residue of sigma38, as a serine residue by the amber suppressor, supD

Escherichia coli WC196, which was obtained from the strain W3110 by nitrosoguanidine mutagenesis as an overproducer of lysine, produced approximately twenty times more cadaverine than did W3110, and had a twenty fold higher level of rpoS gene product, sigma38, than in W3110. Both WC196 and W3110 had a stop codon (TAG) in rpoS at position which corresponds to the 33th residue of sigma38 protein. In addition, WC196 but not W3110 had a mutation in the gene encoding Ser-tRNA (SerU), called, supD. Analysis of the amino acid sequence of a sigma38 preparation from WC196 showed that the 33th residue of sigma38 is a serine residue. The deltarpoS deltacadA mutant of E. coli W3110 harboring the plasmid containing rpoS, in which the TAG codon was converted to a TCG codon for serine-33 residue of sigma38, expressed a significant amount of Ldc and accumulated a large amount of sigma38. However, the deltarpoS deltacadA mutant of W3110 with the plasmid containing the intact rpoS from W3110 could synthesize neither sigma38 nor Ldc significantly.     

The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms.

Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores. However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.     

Val326 of Thermoactinomyces vulgaris R-47 amylase II modulates the preference for alpha-(1,4)- and alpha-(1,6)-glycosidic linkages

Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) catalyzes not only the hydrolysis of alpha-(1,4)- and alpha-(1,6)-glycosidic linkages but also transglycosylation. The subsite +1 structure of alpha-amylase family enzymes plays important roles in substrate specificity and transglycosylation activity. We focused on the amino acid residue at the 326th position based on information on the primary structure and crystal structure, and replaced Val with Ala, Ile, or Thr. The V326A mutant favored hydrolysis of the alpha-(1,4)-glycosidic linkage compared to the wild-type enzyme. In contrast, the V326I mutant favored hydrolysis of the alpha-(1,6)-glycosidic linkage and exhibited low transglycosylation activity. In the case of the V326T mutant, the hydrolytic activity was almost identical to that of the wild-type TVA II, and the transglycosylation activity was poor. These results suggest that the volume and the hydrophobicity of the amino acid residue at the 326th position modulate both the preference for glycosidic linkages and the transglycosylation activity.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

The reactivity of functional groups as a probe for investigating the topography of tobacco mosaic virus. The use of mutants with additional lysine residues in the coat protein

Several mutants of tobacco mosaic virus that contain additional lysine residues as a result of mutations in the coat protein were investigated. Mutant E66 has a lysine residue replacing asparagine at position 140 when compared with the wild-type vulgare and this lysine residue reacts readily in the intact virus with methyl picolinimidate. Mutant B13a has two new lysine residues in the coat protein, replacing a glutamine at position 9 and an asparagine at position 33, whereas mutant B13b has the single replacement of glutamine by lysine at position 9. The lysine residue at position 9 in mutants B13a and B13b also reacts readily with methyl picolinimidate in the intact virus but the lysine at position 33 in mutant B13a did not react under these conditions. However, when the isolated coat protein from mutant B13a was treated with methyl picolinimidate, the lysine residue at position 33 did become modified, showing that the loss in reactivity of this residue towards the imidoester in the intact virus is a result of the assembly of the protein subunit into the virus structure. These results are compatible with and extend previous studies on the sero-logical properties of mutants of tobacco mosaic virus and illustrate the value of methyl picolinimidate as a reagent for probing the accessibility of amino groups in proteins. When intact tobacco mosaic virus (vulgare) was treated with p-iodobenzenesulphonyl chloride, no reaction with the lysine residues at positions 33 or 68 in the virus subunit could be detected but complete modification of tyrosine-139 was achieved. This result also extends previous studies with other reagents. The usefulness of the differential reactivity of the lysine residues in tobacco mosaic virus and its mutants as a means of attaching heavy-atom labels at chemically defined positions for subsequent X-ray-diffraction analysis and the implications of these experiments for deciphering the folding of the peptide chain in the virus subunit are discussed.     

Site-directed mutagenesis of bacterial luciferase: Analysis of the ‘essential’ thiol

It has been appreciated for many years that the luciferase from the luminous marine bacterium Vibrio harveyi has a highly reactive cysteinyl residue which is protected from alkylation by binding of flavin. Alkylation of the reactive thiol, which resides in a hydrophobic pocket, leads to inactivation of the enzyme. To determine conclusively whether the reactive thiol is required for the catalytic mechanism, we have constructed a mutant by oligonucleotide directed site-specific mutagenesis in which the reactive cysteinyl residue, which resides at position 106 of the α subunit, has been replaced with a seryl residue. The resulting α106Ser luciferase retains full activity in the bioluminescence reaction, although the mutant enzyme has a ca 100-fold increase in the FMNH₂ dissociation constant. The α106Ser luciferase is still inactivated by N-ethylmaleimide, albeit at about 1/10 the rate of the wild-type (α106Cys) enzyme, demonstrating the existence of a second, less reactive, cysteinyl residue that was obscured in the wild-type enzyme by the highly reactive cysteinyl residue at position α106. An α106Ala variant luciferase was also active, but the α106Val mutant enzyme was about 50-fold less active than the wild type. All three variants (Ser, Ala and Val) appeared to have somewhat reduced affinities for the aldehyde substrate, the valine mutant being the most affected. It is interesting to note that the α106 mutant luciferases are much less subject to aldehyde substrate inhibition than is the wild-type V. harveyi luciferase, suggesting that the molecular mechanism of aldehyde substrate inhibition involves the Cys at α106.     

枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理,获得了耐NaCl浓度达14％的突变株,同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株,其胞内自由脯氨酸的含量随着盐浓度的提高显著增加,说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因,得到一个约2.3kb的DNA片段,序列分析表明该片段含有一完整的proB基因和部分proA基因,与野生菌株的proB基因相比,突变株proB基因中有3个碱基发生了改变,其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr),另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株,能够与其功能互补。同时对部分proA基因序列分析发现,其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列,其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。     

Functioning of a hybrid BRP-β-lactamase protein in the release of cloacin DF13 and lysis of Escherichia coli cells

The gene encoding a hybrid BRP-Bla protein consisting of the pCloDF13 encoded BRP signal sequence, 25 of the 28 amino acid residues of the mature bacteriocin release protein (BRP) and the mature portion of beta-lactamase (Bla) was subcloned in the expression vector pEB112. A similar construct was made using a mutant gene encoding a BRP-Bla protein in which the cysteine residue at the +1 position was changed into a glycine residue. The expression, processing, functioning and subcellular localization of the 'wild-type' and mutant hybrid protein at high-level expression conditions were studied. The 'wild-type' BRP-Bla protein was mainly found in the outer membranes and possessed all the activities of the BRP itself; the protein was able to bring about the release of cloacin DF13 and caused apparent cell-lysis after high-level synthesis. The mutant hybrid protein was predominantly located in the inner membranes, was inactive in the release of cloacin DF13, but caused apparent cell-lysis only after strong induction.     

Enhanced bacteriolytic activity of hen egg-white lysozyme due to conversion of Trp62 to other aromatic amino acid residues

Tryptophan at the 62nd position (Trp62) of hen egg-white lysozyme is an amino acid residue whose action is essential for its enzymatic activity. Its indole ring may possibly come into direct contact with sugar residues of the substrate, and thus contribute significantly to substrate binding. For further elucidation of its role in catalytic processes, this amino acid was converted to other aromatic residues, such as Tyr, Phe, and His, by site-directed mutagenesis. All the mutations were found to enhance the bacteriolytic activity but to decrease the hydrolytic activity toward an artificial substrate, glycol chitin. Such a change in substrate preference appears remarkable considering the smaller size of the aromatic residue on the mutant enzyme at the 62nd position.