DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.     

Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.     

Characterization and Nucleotide Sequence of the Cryptic Cel Operon of Escherichia Coli K12

Wild-type Escherichia coli are not able to utilize beta-glucoside sugars because the genes for utilization of these sugars are cryptic. Spontaneous mutations in the cel operon allow its expression and enable the organism to ferment cellobiose, arbutin and salicin. In this report we describe the structure and nucleotide sequence of the cel operon. The cel operon consists of five genes: celA, whose function is unknown; celB and celC which encode phosphoenolpyruvate-dependent phosphotransferase system enzyme IIcel and enzyme IIIcel, respectively, for the transport and phosphorylation of beta-glucoside sugars; celD, which encodes a negative regulatory protein; and celF, which encodes a phospho-beta-glucosidase that acts on phosphorylated cellobiose, arbutin and salicin. The mutationally activated cel operon is induced in the presence of its substrates, and is repressed in their absence. A comparison of proteins encoded by the cel operon with functionally equivalent proteins of the bgl operon, another cryptic E. coli gene system responsible for the catabolism of beta-glucoside sugars, revealed no significant homology between these two systems despite common functional characteristics. The celD and celF encoded repressor and phospho-beta-glucosidase proteins are homologous to the melibiose regulatory protein and to the melA encoded alpha-galactosidase of E. coli, respectively. Furthermore, the celC encoded PEP-dependent phosphotransferase system enzyme IIIcel is strikingly homologous to an enzyme IIIlac of the Gram-positive organism Staphylococcus aureus. We conclude that the genes for these two enzyme IIIs diverged much more recently than did their hosts, indicating that E. coli and S. aureus have undergone relatively recent exchange of chromosomal genes.     

Identification and mapping of self-assembling protein domains encoded by the Escherichia coli K-12 genome by use of lambda repressor fusions

Self-assembling proteins and protein fragments encoded by the Escherichia coli genome were identified from E. coli K-12 strain MG1655. Libraries of random DNA fragments cloned into a series of lambda repressor fusion vectors were subjected to selection for immunity to infection by phage lambda. Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232 E. coli genes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online at http://oligomers.tamu.edu/.     

A thermostable single-strand DNase from Methanococcus jannaschii related to the RecJ recombination and repair exonuclease from Escherichia coli

The RecJ protein of Escherichia coli plays an important role in a number of DNA repair and recombination pathways. RecJ catalyzes processive degradation of single-stranded DNA in a 5'-to-3' direction. Sequences highly related to those encoding RecJ can be found in most of the eubacterial genomes sequenced to date. From alignment of these sequences, seven conserved motifs are apparent. At least five of these motifs are shared among a large family of proteins in eubacteria, eukaryotes, and archaea, including the PPX1 polyphosphatase of yeast and Drosophila Prune. Archaeal genomes are particularly rich in such sequences, but it has not been clear whether any of the encoded proteins play a functional role similar to that of RecJ exonuclease. We have investigated three such proteins from Methanococcus jannaschii with the strongest overall sequence similarity to E. coli RecJ. Two of the genes, MJ0977 and MJ0831, partially complement a recJ mutant phenotype in E. coli. The expression of MJ0977 in E. coli resulted in high levels of a thermostable single-stranded DNase activity with properties similar to those of RecJ exonuclease. Despite overall weak sequence similarity between the MJ0977 product and RecJ, these nucleases are likely to have similar biological functions.     

Cloning and characterization of the hrpA gene in the terC region of Escherichia coli that is highly similar to the DEAH family RNA helicase genes of Saccharomyces cerevisiae.

During the course of systematic nucleotide sequence analysis of the terC region of E.coli K-12 by using the ordered lambda phage clones, we found the presence of a gene, termed hrpA, that showed a high degree of sequence similarity to the PRP2, PRP16 and PRP22 genes of Saccharomyces cerevisiae. The products of these yeast genes are known to play their roles in mRNA splicing, and belong to a group of proteins collectively called the DEAH family. The hrpA gene is the first example of a DEAH family gene in prokaryotes. The N-terminal region of the protein it encodes contains conserved sequence stretches characteristic of an RNA helicase. Its molecular mass is calculated to be 146 kDa. Previously, a 135 kDa protein was identified by Moir et al. [J. Bacteriol. (1992) 174, 2102-2110] in this region which is most likely identical to that encoded by hrpA. The C-terminal region of the hrpA gene product seems to contain an RNA binding motif weakly resembling that of ribosomal protein S1 of E.coli. Disruption of the hrpA gene suggested that it is not essential for the growth of E.coli.     

甲基营养菌MP688萄糖脱氢酶基因分离鉴定及性质研究

目的：鉴定甲基营养菌MP688中的葡萄糖脱氢酶基因。方法：对甲基营养菌MP688基因组序列进行比对和分析,找到与已知细菌葡萄糖脱氢酶同源性最高的基因序列mpq_2164,且该基因所编码蛋白经分析具有跨膜结构域。设计51物扩增mpq_2164和缺失跨膜区域序列的s-mpq_2164,将PCR产物克隆到表达载雄pET-15b上,在大肠杆菌BL21中完成异源重组表达,然后通过组氨酸标签镍柱亲和层析纯化,采用DCIP法测定葡萄糖脱氢酶的活力。结果：分离了甲基营养菌MP688中的葡糖糖脱氢酶基因,并实现了s-mpq_2164的高效异源重组表达;MPQ2164的氯基酸序列与已知的葡萄糖脱氢酶相似性很低,但酶活测定结果表明S-MPQ-2164具有很高的葡糖糖脱氢酶活性。结论：MPQ_2164是-个依赖于吡咯喹啉醌的葡萄糖脱氢酶,去掉跨膜结构域有利于该蛋白的异源嘉{大,     

Widespread protein sequence similarities: origins of Escherichia coli genes.

To learn more about the evolutionary origins of Escherichia coli genes, we surveyed systematically for extended sequence similarities among the 1,264 amino acid sequences encoded by chromosomal genes of E. coli K-12 in SwissProt release 26 by using the FASTA program and imposing the following criteria: (i) alignment of segments at least 100 amino acids long and (ii) at least 20% amino acid identity. Altogether, 624 extended alignments meeting the two criteria were identified, corresponding to 577 protein sequences (45.6% of the 1,264 E. coli protein sequences) that had an extended alignment with at least one other E. coli protein sequence. To exclude alignments of questionable biological significance, we imposed a high threshold on the number of gaps allowed in each of the 624 extended alignments, giving us a subset of 464 proteins. The population of 464 alignments has the following characteristics expressed as median values of the group: 254 amino acids in the alignment, representing 86% of the length of the protein, 33% of the amino acids in the alignment being identical, and 1.1 gaps introduced per 100 amino acids of alignment. Where functions are known, nearly all pairs consist of functionally related proteins. This implies that the sequence similarity we detected has biological meaning and did not arise by chance. That a major fraction of E. coli proteins form extended alignments strongly suggests the predominance of duplication and divergence of ancestral genes in the evolution of E. coli genes. The range of degrees of similarity shows that some genes originated more recently than others. There is no evidence of genome doubling in the past, since map distances between genes of sequence-related proteins show no coherent pattern of favored separations.     

Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.

The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal to tnaA and may be in the same operon. The pattern of codon usage in tnaA was intermediate between codon usage in four of the ribosomal protein structural genes and the structural genes for three of the tryptophan biosynthetic proteins.     

Mutagenic DNA repair in enterobacteria.

Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD' fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD' antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium umu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention.     

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.      

Functional analysis of <Emphasis Type="Italic">SlEZ1</Emphasis> a tomato <Emphasis Type="Italic">Enhancer of zeste</Emphasis> (<Emphasis Type="Italic">E(z))</Emphasis> gene demonstrates a role in flower development

The Enhancer of Zeste (E(z)) Polycomb group (PcG) proteins, which are encoded by a small gene family in Arabidopsis thaliana, have been shown to participate to the control of flowering and seed development. For the time being, little is known about the function of these proteins in other plants. In tomato E(z) proteins are encoded by at least two genes namely SlEZ1 and SlEZ2 while a third gene, SlEZ3, is likely to encode a truncated non-functional protein. The analysis of the corresponding mRNA demonstrates that these two genes are differentially regulated during plant and fruit development. We also show that SlEZ1 and SlEZ2 are targeted to the nuclei. These results together with protein sequence analysis makes it likely that both proteins are functional E(z) proteins. The characterisation of SlEZ1 RNAi lines suggests that although there might be some functional redundancy between SlEZ1 and SlEZ2 in most plant organs, the former protein is likely to play specific function in flower development.     

Chloroplast ribosomal protein L32 is encoded in the chloroplast genome

The 50 S subunit of chloroplast ribosomes was prepared from tobacco leaves. The proteins were fractionated and the N-terminal amino acid sequence of a 14 kDa protein was determined. This sequence matches the N-terminal sequence deduced from ORF55 located between ndhF and trnL on the small single-copy region of tobacco chloroplast DNA. The deduced protein shows homology to E. coli and B. stearothermophilus L32 proteins, and it has been named as CL32 and ORF55 as rpl32. The tobacco chloroplast genome therefore contains 21 different ribosomal protein genes.     

Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster.

To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes. Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress. Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex. A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D. melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa). Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family. We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here. Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent. When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-beta-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins.     

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.     

Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function

ABSTRACT: BACKGROUND: The human OXR1 gene belongs to a class of genes with conserved functions that protect cells from reactive oxygen species (ROS). The gene was found using a screen of a human cDNA library by its ability to suppress the spontaneous mutator phenotype of an E. coli mutH nth strain. The function of OXR1 is unknown. The human and yeast genes are induced by oxidative stress and targeted to the mitochondria; the yeast gene is required for resistance to hydrogen peroxide. Multiple spliced isoforms are expressed in a variety of human tissues, including brain. RESULTS: In this report, we use a papillation assay that measures spontaneous mutagenesis of an E. coli mutM mutY strain, a host defective for oxidative DNA repair. Papillation frequencies with this strain are dependent upon a G->T transversion in the lacZ gene (a mutation known to occur as a result of oxidative damage) and are suppressed by in vivo expression of human OXR1. N-terminal, C-terminal and internal deletions of the OXR1 gene were constructed and tested for suppression of the mutagenic phenotype of the mutM mutY strain. We find that the TLDc domain, encoded by the final four exons of the OXR1 gene, is not required for papillation suppression in E. coli. Instead, we show that the protein segment encoded by exon 8 of OXR1 is responsible for the suppression of oxidative damage in E. coli. CONCLUSION: The protein segment encoded by OXR1 exon 8 plays an important role in the anti-oxidative function of the human OXR1 protein. This result suggests that the TLDc domain, found in OXR1 exons 12-16 and common in many proteins with nuclear function, has an alternate (undefined) role other than oxidative repair.