Multiple co-regulatory elements and IHF are necessary for the control of fimB expression in response to sialic acid and N-acetylglucosamine in Escherichia coli K-12

Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region. Here it is demonstrated that NagC binding to a second site (O(NC2)), located 212 bp closer to fimB, also controls fimB expression, and that integration host factor (IHF), which binds midway between O(NC1) and O(NC2), facilitates NagC binding to its two operator sites. In contrast, IHF does not enhance the ability of NanR to activate fimB expression in the wild-type background. Neither sequences up to 820 bp upstream of O(NR), nor those 270 bp downstream of O(NC2), are required for activation by NanR and NagC. However, placing the NanR, IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression. These results support a refined model for how two potentially key indicators of host inflammation, Neu(5)Ac and GlcNAc, regulate type 1 fimbriation.     

Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli.

The expression of type 1 fimbriae in Escherichia coli is phase dependent, i.e. a cell is either completely fimbriated or bald. This phenomenon is due to the periodic inversion of a specific 300-bp DNA segment containing the promoter for the fimbrial subunit gene, fimA. The phase switch is controlled by the products of two regulatory genes, fimB and fimE, located upstream of fimA. The fimB and fimE proteins direct the phase switch into the 'on' and 'off' position, respectively. The DNA sequence of a 3000-bp region containing the two genes has been determined. The fimB and fimE proteins exhibit strong homology and have most likely originated by duplication of an ancestral gene. They are highly basic implying that they control the phase switch through interaction at the DNA level.     

FimE-catalyzed off-to-on inversion of the type 1 fimbrial phase switch and insertion sequence recruitment in an Escherichia coli K-12 fimB strain

We have investigated the capacity of a well-defined Escherichia coli fimB strain, AAEC350 (a derivative of MG1655), to express type 1 fimbriae under various growth conditions. The expression of type 1 fimbriae is phase-variable due to the inversion of a 314-bp DNA segment. Two tyrosine recombinases, FimB and FimE, mediate the inversion of the phase switch. FimB can carry out recombination in both directions, whereas the current evidence suggests that FimE-catalyzed switching is on-to-off only. We show here that AAEC350 is in fact capable of off-to-on phase switching and type 1 fimbrial expression under aerobic static growth conditions. The phase switching is mediated by FimE, and allows emerging fimbriate AAEC350 to outgrow their non-fimbriate counterparts by pellicle formation. Following inversion of the phase switch, this element can remain phase-locked in the on orientation due to integration of insertion sequence elements, viz. IS1 or IS5, at various positions in either the fimE gene or the phase switch.     

The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K-12

The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases. The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion. DNA inversion requires either fimB or fimE, site-specific recombinases that differ in both specificity and activity. Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON-to-OFF direction exclusively. In wild-type cells, fimE activity predominates and, hence, most bacteria are afimbriate. Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms. First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination. Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis-active sites that flank the fim switch also appear to be involved. Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression.     

CS3纤毛抗原表达调控机理的研究

ＣＳ３是某些肠毒素大肠杆菌菌体表面上的多聚物,它能使病原菌粘附于宿主的小肠上皮细胞上,是致病的重要因素．为了探索ＣＳ３菌毛抗原基因的表达调控机制,根据ＣＳ３亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着ｒｂｓ位点及原核启动子的－１０区和－３５区ＤＮＡ序列．采用基因重组技术将ＣＳ３结构基因上游１２０ｂｐ的ＤＮＡ片段亚克隆进缺乏启动子而只含报告基因ｌａｃＺ的质粒ｐＣＢ２６７中．凝胶滞留和启动报告基因表达的实验证明了ＣＳ３亚基结构基因具有自身的启动子（Ｐｓ）．将该启动子上游区域不同长度的核苷酸片段克隆进ｐＣＢ２６７中,报告基因表达结果表明ＣＳ３结构基因的表达受其上游区域的抑制．核苷酸序列分析发现,在Ｐｓ－３５区上游５５０ｂｐ和８４０ｂｐ处各存在一个富Ａ－Ｔ簇．结合原核启动子的一般作用规律推知,ＣＳ３的表达可能受ＤＮＡ结合蛋白型的正向调节因子的作用．用ＣＦＡ／１菌毛抗原基因的正向调节基因ｃｆａＤ对ＣＳ３基因进行的互补表达试验表明ｃｆａＤ基因不仅可消除上游区对Ｐｓ的抑制,而且可大幅度地提高Ｐｓ的启动能力．在分析表达调控的基础上获得ＣＳ３重组高效表达．同时提出了其表达调控模型．     

The enigma of the gene coding for ribosomal protein S12 in the chloroplasts of Nicotiana.

A 2.9 kbp region from within the inverted repeat of Nicotiana chloroplast DNA hybridized with a chloroplast DNA fragment from Euglena containing the complete rps12 gene coding for ribosomal protein S12. Nucleotide sequencing within this region revealed the existance of two rps12 coding stretches interrupted by 540 bp having class II intron structure. Joining and decoding the exon regions produced a sequence of 85 amino acids colinear and 81% homologous to the S12 protein of Euglena chloroplasts and E. coli, starting from amino acid residue 38 to the stop codon. Immediately upstream of codon 38, conserved intron sequences were located. However, the 5' 37 codon of Nicotiana chloroplast rps12 could not be identified by electron microscopy of RNA-DNA hybrids within a DNA region extending 4000 bp upstream of codon 38, nor by computer search of a completely sequenced region extending for more than 9000 bp upstream of this codon. In E. coli, alteration in rps12 codons 42 or 87 causes streptomycin resistance. However, the nucleotide sequence of the identified rps12 exons in two Nicotiana chloroplast mutants resistant to streptomycin were found to be identical to that of wild type.     

The fokI restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes

A DNA fragment that carried the genes coding for FokI endonuclease and methylase was cloned from the chromosomal DNA of Flavobacterium okeanokoites, and the coding regions were assigned to the nucleotide sequence by deletion analysis. The methylase gene was 1,941 base pairs (bp) long, corresponding to a protein of 647 amino acid residues (Mr = 75,622), and the endonuclease gene was 1,749 bp long, corresponding to a protein of 583 amino acid residues (Mr = 66,216). The assignment of the methylase gene was further confirmed by analysis of the N-terminal amino acid sequence. The endonuclease gene was downstream from the methylase gene in the same orientation, separated by 69 bp. The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the methylase gene, and the sequences adhering to the ribosome-binding sequence were identified in front of the respective genes. Analysis of the gene products expressed in E. coli cells by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weights of both enzymes coincided well with the values estimated from the nucleotide sequences, and that the monomeric forms were catalytically active. No significant similarity was found between the sequences of the two enzymes. Sequence comparison with other related enzymes indicated that FokI methylase contained two copies of a segment of tetra-amino acids which is characteristic of adenine-specific methylase.     

Cis-acting sequences sufficient for correct tissue and temporal specificity of larval serum protein 1 genes of Drosophila

We have constructed hybrid genes in which the coding region of the bacterial gene chloramphenicol acetyl transferase (CAT) has been linked to varying lengths of upstream sequences of Drosophila genes for larval serum sequence 1 (LSP1). These have been inserted into a P-element transformation vector and subsequently transferred into the germ-line of recipient flies. Transformants carrying the CAT gene linked to 1650 bp, 570 bp or 377 bp of upstream LSP1α sequences, or 745 bp or 471 bp of upstream β sequences express CAT with the same developmental and tissue specificity as the endogenous LSP1 genes. Constructs having only 66 bp of upstream LSP1β sequences, however, show extremely low levels of CAT expression in tissues and at developmental stages in which LSP1 is not expressed. We discuss the significance of short regions of homology between the DNA upstream of the α and β genes, which lie within the regions identified by the transformation experiments as being required for the cis-regulation of LSP1 synthesis.     

Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.     

Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes.

A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.