Detection of protein similarities using nucleotide sequence databases.

A simple procedure is described for finding similarities between proteins using nucleotide sequence databases. The approach is illustrated by several examples of previously unknown correspondences with important biological implications: Drosophila elongation factor Tu is shown to be encoded by two genes that are differently expressed during development; a cluster of three Drosophila genes likely encode maltases; a flesh-fly fat body protein resembles the hypothesized Drosophila alcohol dehydrogenase ancestral protein; an unknown protein encoded at the multifunctional E. coli hisT locus resembles aspartate beta-semialdehyde dehydrogenase; and the E. coli tyrR protein is related to nitrogen regulatory proteins. These and other matches were discovered using a personal computer of the type available in most laboratories collecting DNA sequence data. As relatively few sequences were sampled to find these matches, it is likely that much of the existing data has not been adequately examined.     

Intrinsic and extrinsic approaches for detecting genes in a bacterial genome.

The unannotated regions of the Escherichia coli genome DNA sequence from the EcoSeq6 database, totaling 1,278 'intergenic' sequences of the combined length of 359,279 basepairs, were analyzed using computer-assisted methods with the aim of identifying putative unknown genes. The proposed strategy for finding new genes includes two key elements: i) prediction of expressed open reading frames (ORFs) using the GeneMark method based on Markov chain models for coding and non-coding regions of Escherichia coli DNA, and ii) search for protein sequence similarities using programs based on the BLAST algorithm and programs for motif identification. A total of 354 putative expressed ORFs were predicted by GeneMark. Using the BLASTX and TBLASTN programs, it was shown that 208 ORFs located in the unannotated regions of the E. coli chromosome are significantly similar to other protein sequences. Identification of 182 ORFs as probable genes was supported by GeneMark and BLAST, comprising 51.4% of the GeneMark 'hits' and 87.5% of the BLAST 'hits'. 73 putative new genes, comprising 20.6% of the GeneMark predictions, belong to ancient conserved protein families that include both eubacterial and eukaryotic members. This value is close to the overall proportion of highly conserved sequences among eubacterial proteins, indicating that the majority of the putative expressed ORFs that are predicted by GeneMark, but have no significant BLAST hits, nevertheless are likely to be real genes. The majority of the putative genes identified by BLAST search have been described since the release of the EcoSeq6 database, but about 70 genes have not been detected so far. Among these new identifications are genes encoding proteins with a variety of predicted functions including dehydrogenases, kinases, several other metabolic enzymes, ATPases, rRNA methyltransferases, membrane proteins, and different types of regulatory proteins.     

Simple and efficient method for heterologous expression of clostridial proteins

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.     

Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium.

Analysis of the sequence of a 4.3-kb region downstream of rfaJ revealed four genes. The first two of these, which encode proteins of 27,441 and 32,890 Da, were identified as rfaY and rfaZ by homology of the derived protein sequences of their products to the products of similar genes of Salmonella typhimurium. The amino acid sequences of proteins RfaY and RfaZ showed, respectively, 70 and 72% identity. Genes 3 and 4 were identified as rfaK and rfaL on the basis of size and position, but the derived amino acid sequences of the products of these genes showed very little similarity (about 12% identity) between Escherichia coli K-12 and S. typhimurium. The next gene in the cluster, rfaC, encodes a product which also shows strong protein sequence homology between E. coli K-12 and S. typhimurium, as do the rfaF and rfaD genes which lie beyond it. Thus, the rfa gene cluster appears to consist of two blocks of genes which are conserved flanking a central region of two genes which are not conserved between these species. Although the RfaL protein sequence is not conserved, hydropathy plots of the two RfaL species are nearly identical and indicate that this is a typical integral membrane protein with 10 or more potential transmembrane domains. We noted the similarity of the structure of the rfa gene cluster to that of the rfb gene cluster, which has now been sequenced in several Salmonella serovars. The rfb cluster also contains a gene which lies within a central nonconserved region and encodes an integral membrane protein similar to protein RfaL. We speculate that protein RfaL may interact in a strain- or species-specific way with one or more Rfb proteins in the expression of surface O antigen.     

Gene products of Escherichia coli: sequence comparisons and common ancestries

Sequences of 1,862 chromosomally encoded Escherichia coli K12 proteins were examined to identify genes likely to have arisen by duplication of genes in an ancestral chromosome. The criteria for sequence relatedness were an alignment of at least 100 amino acid residues and a PAM distance (number of accepted point mutations per 100 residues separating two sequences) below 250. A total of 971 of the 1,862 proteins examined were found in 2,329 sequence-related pairs that met these criteria. Most proteins of the sequence-related pairs were related in cellular function, as judged by biochemical and/or physiological features. Many of the pairs of proteins could be grouped into sequence-related families. If such groupings were generated from ancestral genes by duplication and divergence events, through these sequence comparisons we can identify putative ancestral sequences of the present-day genes of E. coli and other organisms. The results suggest that the 971 paralogous genes could have been derived from only 204 ancestral genes. We have also shown that the process of duplication and divergence is not the exclusive mechanism of evolution of all E. coli genes. Indeed, the relationships among the sequences of multiple (in the sense of redundant) enzymes indicate that nearly half could have arisen either by convergent evolution or by lateral transfer. Therefore, not all functionally related genes need arise by duplication and divergence.      

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.     

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.     

Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.     

Comparative studies of ribosomal proteins and their genes from Methanococcus vannielii and other organisms

Using data from a partial protein sequence analysis of ribosomal proteins derived from the archaebacterium Methanococcus vannielii, oligonucleotide probes were synthesized. The probes enabled us to localize several ribosomal protein genes and to determine their nucleotide sequences. The amino acid sequences that were deduced from the genes correspond to proteins L12 and L10 from the rif operon, according to the genome organization in Escherichia coli, and to proteins L23 and L2, which have comparable locations, as in the Escherichia coli S10 operon. Various degrees of similarity were found when the four proteins were compared with the corresponding ribosomal proteins of prokaryotic or eukaryotic organisms. The highest sequence homology was found in counterparts from other archaebacteria, such as Halobacterium marismortui, Halobacterium halobium, or Sulfolobus. In general, the M. vannielii protein sequences were more related to the eukaryotic kingdom than to the Gram-positive or Gram-negative eubacteria. On the other hand, the organization of the ribosomal protein genes clearly follows the operon structure of the Escherichia coli genome and is different from the monocistronic eukaryotic gene arrangements. The protein coding regions were not interrupted by introns. Furthermore, the Shine-Dalgarno type sequences of methanogenic bacteria are homologous with those of eubacteria, and also their terminator regions are similar.     

Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits

The ripening of strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, is a complex developmental process that involves many changes in gene expression. To understand how these changes relate to the biochemistry and composition of the fruit the specific genes involved have been examined. A high-quality cDNA library prepared from ripe strawberry fruit was differentially screened for ripening-related clones using cDNA from ripe and white fruits. From 112 up-regulated clones obtained in the primary screen, 66 differentially expressed clones were isolated from the secondary screen. The partial sequences of these cDNAs were compared with database sequences and 26 families of non-redundant clones were identified. Northern analysis confirmed that all of these cDNAs were ripening-enhanced. The expression of many of their corresponding genes was negatively regulated in auxin-treated fruit. These sequences, several of which are novel to fruits, encode proteins involved in key metabolic events including anthocyanin biosynthesis, cell wall degradation, sucrose and lipid metabolism, protein synthesis and degradation, and respiration. These findings are discussed in relation to the role of these genes in determining fruit quality characteristics. Received: 19 January 1998 / Accepted: 5 February 1998     

Towards a classification of E. coli ribosomal proteins: a hypothetical 'small ribosome' as a primitive protein-synthesizing apparatus

Homologies were searched among the published primary sequences of 51 E. coli ribosomal proteins, partly by 'eye' and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered at str-stc region, rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications of very few (or only one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that 'a small ribosome' consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.     

The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.     

Collagen-like proteins in pathogenic E. coli strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.     

Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved.

The genes coding for the lactose permease and beta-galactosidase, two proteins involved in the metabolism of lactose by Lactobacillus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli. The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs). One of these ORFs codes for the lactose permease gene, and the other codes for the beta-galactosidase gene. The lactose permease gene is located in front of the beta-galactosidase gene, with 3 bp in the intergenic region. The two genes are probably transcribed as one operon. Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the beta-galactosidase gene. This promoter is similar to those found in E. coli with general characteristics of GC-rich organisms. In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L. bulgaricus genome, which is rich in GC (GC content of 54%). The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus. The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E. coli. Little homology was found between the lactose permease of L. bulgaricus and E. coli, but the residues which are involved in the binding and the transport of lactose are conserved. The carboxy terminus is similar to that of the enzyme III of several phosphoenolpyruvate-dependent phosphotransferase systems.