Whole genome analysis of non-optimal codon usage in secretory signal sequences of Streptomyces coelicolor

Non-optimal (rare) codons have been suggested to reduce translation rate and facilitate secretion in Escherichia coli. In this study, the complete genome analysis of non-optimal codon usage in secretory signal sequences and non-secretory sequences of Streptomyces coelicolor was performed. The result showed that there was a higher proportion of non-optimal codons in secretory signal sequences than in non-secretory sequences. The increased tendency was more obvious when tested with the experimental data of secretory proteins from proteomics analysis. Some non-optimal codons for Arg (AGA, CGU and CGA), Ile (AUA) and Lys (AAA) were significantly over presented in the secretary signal sequences. It may reveal that a balanced non-optimal codon usage was necessary for protein secretion and expression in Streptomyces.     

Experimental confirmation of a key role for non-optimal codons in protein export

Non-optimal codons are defined by low usage and low abundance of corresponding tRNA, and have an established role in translational pausing to allow the correct folding of proteins. Our previous work reported a striking abundance of non-optimal codons in the signal sequences of secretory proteins exported via the sec-dependent pathway in Escherichia coli. In the current study the signal sequence of maltose-binding protein (MBP) was altered so that non-optimal codons were substituted with the most optimal codon from their synonymous codon family. The expression of MBP from the optimized allele (malE-opt) was significantly less than wild-type malE. Expression of MBP from malE-opt was partially restored in a range of cytoplasmic and periplasmic protease deficient strains, confirming that reduced expression of MBP in malE-opt was due to its preferential degradation by cytoplasmic and periplasmic proteases. These data confirm a novel role for non-optimal codon usage in secretion by slowing the rate of translation across the N-terminal signal sequence to facilitate proper folding of the secreted protein.     

A novel method of analyzing proline synonymous codons in E. coli

Proline is a special imino acid in protein and the isomerization of the prolyl peptide bond has notable biological significance and influences the final structure of protein greatly, so the correlation between proline synonymous codon usage and local amino acid, the correlation between proline synonymous codon usage and the isomerization of the prolyl peptide bond were both investigated in the Escherichia coli genome by using a novel method based on information theory. The results show that in peptide chain, the residue at the first position C-terminal influences the usage of proline synonymous codon greatly and proline synonymous codons contain some factors influencing the isomerization of the prolyl peptide bond.     

Expression of a biologically-active conotoxin PrIIIE in Escherichia coli

Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Conus parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIIIE to small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an Escherichia coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5 mg/L of culture. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. This system may prove valuable for future structure-function studies.     

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains

Background

Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.

Results

The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage {"type":"entrez-protein","attrs":{"text":"P13374","term_id":"138227","term_text":"P13374"}}P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.

Conclusions

At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.     

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H,31P COSY, 1H,1H NOESY and 1H,13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.     

Stationary phase protein overproduction is a fundamental capability of Escherichia coli

Although Escherichia coli is well studied and various recombinant E. coli protein expression systems have been developed, people usually consider the rapid growing (log phase) culture of E. coli as optimum for production of proteins. However, here we demonstrate that at stationary phase three E. coli systems, BL21 (DE3)(pET), DH5alpha (pGEX) induced with lactose, and TG1 (pBV220) induced with heat shock could overexpress diversified genes, including three whose products are deleterious to the host cells, more stably and profitably than following the log phase induction protocol. Physical and patch-clamp assays indicated that characteristics of target proteins prepared from cultures of the two different growth phases coincide. These results not only provide a better strategy for recombinant protein preparation in E. coli, but also reveal that rapid rehabilitation from stresses and stationary phase protein overproduction are fundamental characters of E. coli.     

The Effect of Mutation and Selection on Codon Adaptation in Escherichia coli Bacteriophage

Studying phage codon adaptation is important not only for understanding the process of translation elongation, but also for reengineering phages for medical and industrial purposes. To evaluate the effect of mutation and selection on phage codon usage, we developed an index to measure selection imposed by host translation machinery, based on the difference in codon usage between all host genes and highly expressed host genes. We developed linear and nonlinear models to estimate the C→T mutation bias in different phage lineages and to evaluate the relative effect of mutation and host selection on phage codon usage. C→T-biased mutations occur more frequently in single-stranded DNA (ssDNA) phages than in double-stranded DNA (dsDNA) phages and affect not only synonymous codon usage, but also nonsynonymous substitutions at second codon positions, especially in ssDNA phages. The host translation machinery affects codon adaptation in both dsDNA and ssDNA phages, with a stronger effect on dsDNA phages than on ssDNA phages. Strand asymmetry with the associated local variation in mutation bias can significantly interfere with codon adaptation in both dsDNA and ssDNA phages.     

Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci

Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains).     

Structure of the cyclomodulin Cif from pathogenic Escherichia coli

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.     

Proteomic analysis of a multi-resistant clinical Escherichia coli isolate of unknown genomic background

Horizontal transfer of gene clusters occurs in Escherichia coli (E. coli), which could lead to evolution of new pathovars and improve survival fitness. However, this genetic event results in genomic plasticity which is a hindrance for proteomic characterization of strains with unknown genetic backgrounds. To characterize such isolate with many specific genetic variations we used the recently in-house designed MSMSpdbb software which merges protein databases from several sources of E. coli including type strains and other commensal and pathogenic isolates. We selected a multidrug resistant clinical isolate in order to check the capacity of our approach to identify selected protein markers. From the 1596 identified proteins, we found important virulence factors such as IutA, OmpA, TraT and selected enzymes conferring antibiotic resistance, such as CTX-M-15 (Extended-Spectrum Beta Lactamase - ESBL) and AAC(6′)-Ib-cr (to aminoglycoside + fluoroquinolone). In addition, we compared the protein identifications with E. coli gene annotation and found that 27% of the proteins identified in the present study corresponded to the pan-genome of E. coli species and are only present in a subset of strains. This demonstrates the ability of our approach to characterize the proteome of bacterial strains with complex genomic plasticity even without its genomic information.     

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.     

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 522/C1 and the international type strain from Escherichia coli O 178

The structure of the O-antigenic polysaccharide (PS) from the enteroaggregative Escherichia coli strain 522/C1 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy techniques were used to elucidate the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [ structure: see text]. Analysis of NMR data reveals that on average the PS consists of four repeating units and indicates that the biological repeating unit contains an N-acetylgalactosamine residue at its reducing end. Serotyping of the E. coli strain 522/C1 showed it to be E. coli O 178:H7. Determination of the structure of the O-antigen PS of the international type strain from E. coli O 178:H7 showed that the two polysaccharides have identical repeating units. In addition, this pentasaccharide repeating unit is identical to that of the capsular polysaccharide from E. coli O9:K 38, which also contains O-acetyl groups.     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

Quantitative export of a reporter protein,GFP, by the twin-arginine translocation pathway in Escherichia coli

The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm. However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes. Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100%. These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.