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Analysis of the codon bias in E. coli sequences.

Fifty-three gene sequences from E. coli containing 18,288 reading frame triplets have been characterized according to the nature and level of average codon preference. The distribution of average preferences is bimodal, with approximately half the genes using an average of only 36 codons, and the remainder just 42 codons. There is a high correlation between the level of codon bias, the tRNA population and the abundance of protein product, indicating biased patterns are exploited by the cell for the production of widely different levels of gene product. This relationship is especially striking in genes involved in the production of components for transcription and translation. Overall, the genes for these processes generate some five-fold more protein than the average in the genome, and use about five fewer codons. The very high codon bias found in the RNA polymerase gene thus provides a simple, autogenous mechanism for the coordinate synthesis of these components and RNA polymerase. A surprisingly high level of codon probability is also found in triplets of the complement of coding sequences. This is apparently due to the evolutionary dispersion of coding sequences and/or the requirement for increased levels of secondary structure in messenger RNAs.

Base Sequence↗

Distribution and evolution of sequence characteristics in the E. coli genome.

The mean (G + C) composition (51.0%) and standard deviation (+/- 3.8%) of published DNA sequences accounting for 10% of the E. coli genome is in excellent agreement with the principal overall distribution determined by high resolution melting. While differences in base and neighbor characteristics are small and uniform throughout all regions of the genome, it is found that the (G + C) content of sequences varies in segmented fashion within boundaries corresponding to coding (53% G + C) and noncoding (46% G + C) regions; with variances in the latter being six-fold greater than in coding regions. The variance in different regions shows a strong negative dependence on (G + C) content of the region, reflecting the condition that A-T and G-C base pairs are preferred neighbors of A-T and C-G pairs, respectively; with the bias increasing with decreasing (G + C) content. Neighbor analysis indicates the most extreme positive biases occur in AA, TT, GC and CG throughout all regions, but particularly in noncoding regions. Extraordinary numbers of oligomeric strings of (A)n, etc., are the further consequence of this bias. These and other characteristics point to the existence of inherent biases in neighbor frequencies levied during replication or repair, and which reflect, in turn, neighbor influences during mutation. The bias in codon usage noted by Grantham and others is seen here as due, in part, to the adaptation of coding sequences to this microenvironment through selection among synonymous codons so as to preserve inherent neighbor biases.

Base Composition↗

Molecular cloning of mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K): expression of calbindin-D28K RNAs in rodent brain and kidney.

We report the isolation of a cloned cDNA for the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K; CaBP28K) by immunological screening of a lambda gt11 bacterial expression library. The library contained cDNAs copied from poly(A)RNA of adult mouse cerebellum. We confirmed the identity of the CaBP28K cDNA by comparing its DNA sequence with that of chick CaBP28K cDNA. In the coding region, 79% of the mouse cDNA sequence was identical to the reported sequence of CaBP28K cDNA derived from chicken intestine. Rat brain and kidney each contain three species of poly(A)RNA that hybridize to CaBP28K cDNA--a major species of 1.9 kb, and rarer components of 2.8 kb and 3.2 kb. All three RNAs appear to be transcribed from a single gene. The ratios of these CaBP28K RNAs were the same in brain and kidney. In the cerebellum, in situ hybridization reveals that CaBP28K RNAs are confined to Purkinje neurons.

Animals↗

Small DNA deletions creating avirulence in Streptococcus pyogenes.

The M protein is the antigen on the surface of group A streptococci that allows these bacteria to resist phagocytosis. DNA encoding the M12 protein was cloned into Escherichia coli and used as an isotopically labeled hybridization probe to compare genomic DNA's isolated from M+ and M- isogenic cultures in an effort to elucidate the genetic basis of this variation. DNA's from two spontaneous, independent M- variants contained small (approximately 50 base pairs) deletions which were mapped to identical restriction fragments within or adjacent to the M protein coding sequence. Taken together with the pleiotropic nature of these deletions, this suggests that they define a regulatory switch.

Antigens, Bacterial↗

Effects of deletion of the gene for the development-specific protein S on differentiation in Myxococcus xanthus.

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.

Bacterial Proteins↗

Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis.

A novel penicillin-binding protein (PBP 5*) with D,D-carboxypeptidase activity is synthesized by Bacillus subtilis, beginning at about stage III of sporulation. The complete gene (dacB) for this protein was cloned by immunoscreening of an expression vector library and then sequenced. The identity of dacB was verified not only by the size and cross-reactivity of its product but also by the presence of the nucleotide sequence that coded for the independently determined NH2 terminus of PBP 5*. Analysis of its complete amino acid sequence confirmed the hypothesis that this PBP is related to other active-site serine D,D-peptidases involved in bacterial cell wall metabolism. PBP 5* had the active-site domains common to all PBPs, as well as a cleavable amino-terminal signal peptide and a carboxy-terminal membrane anchor that are typical features of low-molecular-weight PBPs. Mature PBP 5* was 355 amino acids long, and its mass was calculated to be 40,057 daltons. What is unique about this PBP is that it is developmentally regulated. Analysis of the sequence provided support for the hypothesis that the sporulation specificity and mother cell-specific expression of dacB can be attributed to recognition of the gene by a sporulation-specific sigma factor. There was a good match of the putative promoter of dacB with the sequence recognized by sigma factor E (sigma E), the subunit of RNA polymerase that is responsible for early mother cell-specific gene expression during sporulation. Analysis of PBP 5* production by various spo mutants also suggested that dacB expression is on a sigma E-dependent pathway.

Amino Acid Sequence↗

Cloning and physical mapping of the EcoRI fragments of the giant linear plasmid SCP1.

A cosmid library was constructed for the 350-kb giant linear plasmid SCP1 and aligned on a successive linear map. Only a 0.8-kb gap has remained uncloned in the terminal inverted repeats close to both ends. Partial digestion of the aligned cosmids with EcoRI and hybridization with the flanking fragments of the vector enabled physical mapping of all of the EcoRI fragments. On this map, the methylenomycin biosynthetic gene cluster, the insertion sequence IS466, and the sapCDE genes coding for spore-associated proteins were localized.

Cloning, Molecular↗

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity.

1. Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. 2. Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end. Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. 3. The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay. The full-length replicase is active. The aminoterminal truncated enzyme had 0.02% activity of the intact one. 4. This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.

Amino Acid Sequence↗

[The upstream sequence of cholera toxin B subunit gene: effect on CTB expression].

In this work, we have studied the effect of cholera toxin A structure gene on the expression of the distal ctxB gene by the methods of deletion and frame-shift mutation. The results showed that: The expression level of Plasmid pUC19CTB, which was constructed by cloning the XbaI-EcoRI restriction fragment into pUC19 and ctxA gene was out-frame with lacZ' gene, is about 30 micrograms/ml; If a frame shift mutation was introduced at XbaI site of pUC19CTB so that the cholera toxin A gene was inframe with lacZ' and could be translated, the expression level of ctxB was decreased to 12 micrograms/ml; When A further deletion from XbaI to ClaI of cholera toxin A gene (about 550bp) was made and ctxA was outframe with LacZ', ctxB expression was decreased two fold compared to pUC19CTB; If the ctxA was inframe with LacZ' so ctxA gene could be translated, the expression level of CTB is much lower than the plasmid outframe with lacZ'. These observations could not be explained by the current knowledge about genetical regulation of cholera toxin operon. The promoter we found located in the cholera toxin A subunit gene, which is responsible for the expression of cholera toxin B subunit, may answer the question why the 550bp non-coding sequence could enhance the expression of cholera toxin B subunit.

Base Sequence↗

The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin.

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identified so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis.

Acyltransferases↗

A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima.

We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B). The deduced protein is composed of 636 aa with a calculated molecular mass of 72415 Da. It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE). Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV. This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability. No gyrA-like gene has been found near top2B. A gene coding for a transaminase B-like protein was found in the upstream region of top2B.

Amino Acid Sequence↗

DNA microarray-based genome comparison of a pathogenic and a nonpathogenic strain of Xylella fastidiosa delineates genes important for bacterial virulence.

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

Adhesins, Bacterial↗

Avoidance of host recognition by alterations in the repetitive and C-terminal regions of AvrXa7, a type III effector of Xanthomonas oryzae pv. oryzae.

avrXa7 is a member of the avrBs3/pthA gene family. The gene is a critical type III effector in several strains of Xanthomonas oryzae pv. oryzae (virulence activity), and in the presence of the Xa7 host gene for resistance, controls the elicitation of resistance in rice (avirulence activity). The ability of strains containing avrXa7 to adapt to the presence of Xa7 in the host population is dependent, in part, on the genetic plasticity of avrXa7. The potential for the conversion of avrXa7 to a virulence effector without Xa7-dependent elicitor activity was examined. Internal reorganization of avrXa7 by artificially deleting a portion of the central repetitive region resulted in gene pthXo4, which retained virulence activity and lost Xa7-dependent avirulence activity. Similarly, spontaneous rearrangements between repetitive regions of avrXa7 during bacterial culture gave rise to gene pthXo5, which also had virulence activity without Xa7-dependent avirulence activity. pthXo5 appeared to be the result of recombination between avrXa7 and a related gene in the genome. Loss of avirulence activity and retention of virulence activity also resulted from replacement of a portion of the C-terminal coding region of avrXa7 with the corresponding sequence from avrBs3. The results demonstrated the potential for a critical virulence effector to lose avirulence activity while retaining effector function. The results also demonstrated that features of both repetitive and nonrepetitive C-terminal regions of AvrXa7 are involved in avirulence specificity.

Amino Acid Sequence↗

Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi.

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

Amino Acid Sequence↗

Aristolochene synthase. Isolation, characterization, and bacterial expression of a sesquiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti.

Aristolochene is the likely precursor of the sesquiterpenoid toxins produced by a number of filamentous fungi. One of these, PR-toxin, is produced by Penicillium roqueforti. We report here the isolation of a gene (Ari1) coding for the sesquiterpene cyclase, aristolochene synthase (AS), from P. roqueforti. Nucleotide sequence analysis of genomic and cDNA clones revealed that the Ari1 gene contains two introns. A Protein A/AS fusion enzyme was expressed in Escherichia coli and shown to have sesquiterpene cyclase activity. Analysis of the Protein A/AS fusion enzyme reaction mixtures by TLC and gas chromatography/mass spectrometry identified aristolochene as a major product. The Ari1 gene encodes a polypeptide of molecular weight 39,200. Expression of Ari1 occurs in stationary phase cultures of P. roqueforti and appears to be transcriptionally regulated.

Amino Acid Sequence↗

Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways to valine, isoleucine, and leucine. It is the target of two structurally unrelated classes of herbicides, the sulfonylureas and the imidazolinones. Genomic clones encoding ALS have been isolated from the higher plants Arabidopsis thaliana and Nicotiana tabacum, using a yeast ALS gene as a heterologous hybridization probe. Clones were positively identified by the homology of their deduced amino acid sequences with those of yeast and bacterial ALS isozymes. The tobacco and Arabidopsis ALS genes have approximately 70% nucleotide homology, and encode mature proteins which are approximately 85% homologous. Little homology is seen between the amino acid sequences of the presumptive N-terminal chloroplast transit peptides. Both plant genes lack introns. The tobacco ALS gene was isolated from a line of tobacco which is resistant to the sulfonylurea herbicides due to an alteration in ALS. The tobacco gene which was isolated codes for an ALS that is sensitive to the herbicides, as assayed by transformation of the gene into sensitive tobacco cells.

Journal Article↗