Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane.

The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein. Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorporate the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body.     

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5' end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.     

Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.     

N-terminal amino acid sequence of the Borrelia burgdorferi flagellin

Abstract The 41 kDa flagellar protein of Borrelia burgdorferi appears to be an immunodominant antigen producing an early and strong response in most, if not all, individuals during infection in humans. It would represent a very good antigen for serodiagnosis of Lyme disease, if its crossreactivity with flagella of other bacteria was low. To gain information on this point we isolated the B. burgdorferi flagellin by preparative two-dimensional electrophoresis for N-terminal amino acid analysis. By comparing the N-terminal amino acid sequences of flagellar proteins from other eubacteria we found that the first six out of twenty nine amino acids were identical to the Treponema pallidum and Treponema phagedenis 'class B' flagellins. All 29 N-terminal residues exhibited a moderate inter-genus homology (44–55%), in contrast to the high degree (67–95%) of inter-species conservation of the treponemal 'class B' flagellar N-terminal sequences. There was little similarity to other flagellins except the B. subtilis flagellar protein.     

Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in Bacillus brevis.

The gene (bdb) for protein thiol-disulfide oxidoreductase cloned from Bacillus brevis was found to encode a polypeptide consisting of 117 amino acid residues with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of disulfide oxidoreductases such as DsbA, protein disulfide isomerase, and thioredoxin. The deduced amino acid sequence showed significant homology with those of several bacterial thioredoxins. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B. brevis was enzymatically active in both reduction and oxidization of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.     

Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides a bacterium with a unidirectional, stop-start flagellum

Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation tops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli , a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli . MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB , Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a σ⁵⁴- type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of he R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.     

The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export.

flaFIX, the structural gene for the periplasmic P ring of the flagellar basal body of Salmonella typhimurium, was cloned. Two gene products with apparent molecular weights of 38,000 and 40,000 were identified by minicell analysis. Data from pulse-chase and membrane fractionation experiments and data on the inhibitory effect of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone all indicated that the 40-kilodalton protein was a precursor form which, after export across the cytoplasmic membrane accompanied by cleavage of a signal peptide, gave rise to the mature protein in the periplasm. The N-terminal amino acid sequence of the FlaFIX protein, predicted from the DNA sequence, conformed well to known signal peptide sequences. The results indicate that the P-ring protein of the basal body (unlike flagellin and possible some other external flagellar components) crosses the cytoplasmic membrane in a conventional signal peptide-dependent manner.     

Analysis of the Escherichia coli K-12 cysB gene and its product using the method of gene fusion

The amino-terminal structure and the essential functional region of the cysB gene product of Escherichia coli K-12 were analyzed by the method of gene fusion. The translational start codon of the cysB gene was located by determining the amino-terminal sequence of a hybrid protein containing the first 31 amino acid residues of the CysB protein at the amino terminus of beta-galactosidase(LacZ protein). The fact that two other CysB'-'LacZ hybrid polypeptides expressed a normal CysB activity indicated that the functional region of the CysB protein was located within the first 215 amino acid residues of the total 324 amino acids deduced from the nucleotide sequence.     

Cloning and sequencing of the central region of the flagellin gene from the Gram-positive bacterium Clostridium tyrobutyricum ATCC 25755

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum fiagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.     

Analysis and expression of the Borrelia burgdorferi P/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190-234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIS.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.     

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen

Abstract The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyne disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum .     

Unique sequences in region VI of the flagellin gene of Salmonella typhi

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.     

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Flagellar transcriptional activators FlbB and FlaI: gene sequences and 5' consensus sequences of operons under FlbB and FlaI control

The regulation of the expression of the operons in the flagella-chemotaxis regulon in Escherichia coli has been shown to be a highly ordered cascade which closely parallels the assembly of the flagellar structure and the chemotaxis machinery (T. Iino, Annu. Rev. Genet. 11:161-182, 1977; Y. Komeda, J. Bacteriol. 168: 1315-1318). The master operon, flbB, has been sequenced, and one of its gene products (FlaI) has been identified. On the basis of the deduced amino acid sequence, the FlbB protein has similarity to an alternate sigma factor which is responsible for expression of flagella in Bacillus subtilis. In addition, we have sequenced the 5' regions of a number of flagellar operons and compared these sequences with the 5' region of flagellar operons directly and indirectly under FlbB and FlaI control. We found both a consensus sequence which has been shown to be in all other flagellar operons (J. D. Helmann and M. J. Chamberlin, Proc. Natl. Acad. Sci. USA 84:6422-6424) and a derivative consensus sequence, which is found only in the 5' region of operons directly under FlbB and FlaI control.     

Characterization of cDNA clones for the human c-yes gene.

Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family.