Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.     

CS3抗原基因的表达及纤毛装配元件的研究

在肠毒素大肠杆菌(ETEC)的已知定居因子中,CS3是临床分离株中最常见的抗原之一。为了研究CS3纤毛装配的基本元件,绘制了CS3亚基结构基因和辅助蛋白编码区的限制酶谱。通过亚克隆的亚基基因和不同辅助蛋白基因之间的互补性表达结果,确定了CS3纤毛装配所需要的辅助蛋白的DNA功能片段。微细胞分析结果显示,CS3基因的有效表达和纤毛装配至少需要6条蛋白多肽,分子量分别为(×10~3)15、17、24、27、48和90。除了15×10~3/17×10~3的蛋白多肽为CS3亚基外,其余的蛋白多肽参与CS3亚基的转运及纤毛的装配。根据以上结果初步确定了上述相关基因的相对位置。     

Colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli: molecular cloning of the CS3 determinant

The genes for the cell surface associated antigen CS3, produced by CFA/II type enterotoxigenic Escherichia coli, have been cloned in the plasmid vector pBR322 to produce a family of recombinant plasmids. These plasmids contain a series of HindIII fragments of which a fragment of 4.6 kb is common to all those expressing CS3. One of these plasmids, pPM474, has been subjected to mutagenesis with Tn1725 and deletions generated using Bal31. This has defined a minimum region of 3.75 kb necessary for the production of CS3 on the cell surface and implying genetic complexity as has been observed with other fimbrial antigens. Analysis of the plasmid encoded proteins in E. coli K-12 minicells has confirmed this complexity.     

Cloning of the locus encoding synthesis of an outer membrane protein (Omp2) of the calcium dependence plasmid of Yersinia pestis (Lehmann, Neumann)

The genetic locus of Yersinia pestis encoding synthesis of a 46-kDa heat-inducible outer membrane protein (Omp2) was cloned into pBR322 plasmid. The Omp2 was shown to be analogous to previously described YopH and Yop2b proteins. The fifth HindIII fragment of 48-MDa calcium dependence plasmid pCad358 mediates production of 31- and 28-kDa proteins, irrespective of orientation of the insertion. A 31-kDa polypeptide seems to correspond to the YopJ described elsewhere. The maps of BamHI and HindIII of pCad358 region studied differed from those described for pCD1 plasmid of Y. pestis KIM. The products encoded by genes from the fragment cloned in the Pgm+ background give rise to considerable growth of Y. pestis within mouse peritoneal macrophages but were not sufficient to cause lethal infectious process.     

Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium.

The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIII fragment revealed an open reading frame which could encode a protein of 407 amino acids with an Mr of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane beta-sheet structures, implying a possible role for the protein in translocation of hydrophilic O-antigen-containing materials. Salmonella strains of groups A, B, and D1 contained rfc-homologous DNA, but strains of groups C1, C2, C3, D2, and E2 did not.     

Identification of the initiation site of poliovirus polyprotein synthesis.

The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVP00. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VP0, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of the tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of the poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.     

A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-p-Dioxin Pathway Genes in Sphingomonas sp. Strain RW1

The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively. dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp. strain RW1 thus suggests itself.     

Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66

The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence. The DNA sequences of the three 1 kb repeats were determined. Two copies (left and middle repeats) were identical: (1009 by in length) and the right repeat was 1012 bp long and differed at 63 positions. The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight. The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes. The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.     

Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66

The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence. The DNA sequences of the three 1 kb repeats were determined. Two copies (left and middle repeats) were identical: (1009 by in length) and the right repeat was 1012 bp long and differed at 63 positions. The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight. The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes. The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.     

Characterization of the yellow-pigment genes of Erwinia herbicola

A 6.7 kb DNA fragment containing the pigment genes of Erwinia herbicola Eho13 has been cloned into Escherichia coli. These genes were chromosomally encoded in E. herbicola. The entire DNA fragment could be divided into at least three regions. Deletions in Region I resulted in a non-pigmented phenotype, a deletion in Region II resulted in a pink/yellow phenotype, deletions in Region III resulted in either a pink or a non-pigmented phenotype. Tn1000 insertions in the same regions, however, gave different phenotypes. Insertions in Region II produced a pink phenotype. Insertions in Region III resulted in either a light-yellow or a non-pigmented phenotype. Minicell studies showed that the 6.7 kb DNA fragment encoded at least five proteins (50 kDa, 42 kDa, 36 kDa, 35 kDa and 34 kDa). A 2.7 kb HindIII deletion in Region I caused the disappearance of these proteins, suggesting that this 2.7 kb fragment may play a regulatory role in pigment synthesis. Our results also showed that a 4.1 kb EcoRV fragment consisted of Region I and a part of Region II complemented a pink/yellow clone of Eho10 (pHL545), suggesting that the pigments of Eho13 and Eho10 were probably similar or identical.     

High-level ribosomal frameshifting directs the synthesis of IS150 gene products.

IS150 contains two tandem, out-of-phase, overlapping genes, ins150A and ins150B, which are controlled by the same promoter. These genes encode proteins of 19 and 31 kD, respectively. A third protein of 49 kD is a transframe gene product consisting of domains encoded by both genes. Specific -1 ribosomal frameshifting is responsible for the synthesis of the large protein. Expression of ins150B also involves frameshifting. The IS150 frameshifting signals operate with a remarkably high efficiency, causing about one third of the ribosomes to switch frame. All of the signals required for this process are encoded in a 83-bp segment of the element. The heptanucleotide A AAA AAG and a potential stem-loop-forming sequence mark the frameshifting site. Similar sequence elements are found in -1 frameshifting regions of bacterial and retroviral genes. A mutation within the stem-loop sequence reduces the rate of frameshifting by about 80%. Artificial transposons carrying this mutation transpose at a normal frequency, but form cointegrates at a approximately 100-fold reduced rate.     

Analysis of the cya locus of Escherichia coli

A 9500-bp DNA segment containing the adenylate cyclase gene (cya) of Escherichia coli has been isolated and analyzed. Four large proteins are encoded within this fragment - the adenylate cyclase protein (92 kDal), two proteins of unknown function (37 and 32 kDal), and a part of the uvrD-coded protein. Various truncated adenylate cyclase proteins, made from cya genes having as much as 60% of their carboxy-terminal end deleted, are sufficient to complement cya- hosts. When these truncated cya genes are present on a multicopy plasmid in a cya- host, the synthesis of beta-galactosidase is still regulated by glucose. The "maxicell" technique was used to visualize the four proteins encoded by this region and some of the truncated adenylate cyclase proteins.     

Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli.

Intracellular amplification of the Escherichia coli RecB and RecC proteins does not result in an increase in Exonuclease V activity unless the level of a third protein, encoded between the recB and argA genes, is also amplified. Nucleotide sequence analysis of this region reveals a 1,824 nucleotide open reading frame which would encode a protein of 608 amino acids with a calculated molecular weight of 66,973. This is assumed to be the structural gene for the alpha subunit of Exonuclease V, recently designated recD. The proposed initiation codon of the recD gene overlaps the termination codon of the upstream recB gene by one nucleotide, suggesting that these genes may form an operon. The deduced amino acid sequence of the RecD protein contains a region which is homologous to highly conserved sequences in adenine nucleotide binding proteins.