[en] The dhlIVa gene coding for DehIVa was expressed in Escherichia coli and the protein was purified and crystallized using the hanging-drop method

[en] Highlights: • Average number of tRNA genes correlate with codon usage in highly expressed genes. • Changes in the number of tRNA genes do not correlate to codon usage. • Altered tRNA gene copy numbers do not correlate to changes of codon translation. • Our data indicates that tRNA gene copies adapt to codon usage, but not the contrary. It is widely believed that if a high number of genes are found for any tRNA in a rapidly replicating bacteria, then the cytoplasmic levels of that tRNA will be high and an open reading frame containing a higher frequency of the complementary codon will be translated faster. This idea is based on correlations between the number of tRNA genes, tRNA concentration and the frequency of codon usage observed in a limited number of strains as well as from the fact that artificially changing the number of tRNA genes alters translation efficiency and consequently the amount of properly folded protein synthesized. tRNA gene number may greatly vary in a genome due to duplications, deletions and lateral transfer which in turn would alter the levels and functionality of many proteins. Such changes are potentially deleterious for fitness and as a result it is expected that changes in tRNA gene numbers should be accompanied by a modification of the frequency of codon usage. In contrast to this model, when comparing the number of tRNA genes and the frequency of codon usage of several Salmonella enterica and Escherichia coli strains we found that changes in the number of tRNA genes are not correlated to changes in codon usage. Furthermore, these changes are not correlated with a change in the efficiency of codon translation. These results suggest that once a genome gains or loses tRNA genes, it responds by modulating the concentrations of tRNAs rather than modifying its frequency of codon usage.

[en] The ochre mutant oc9 of bacteriophage PHI X174 was irradiated with γ-rays and the revertants were assayed on unirradiated and UV-irradiated host bacteria carrying an amber suppressor. The yield of revertants (amber + wild type) was higher on UV-irradiated than on unirradiated bacteria, showing that γ-irradiated PHI X174 was subjected to W-mutagenesis. For oc9 γ-irradiated in the presence of oxygen the fraction of amber mutants among the revertants was lower when mutants were scored on UV-irradiated bacteria than when assayed on unirradiated indicator cells. The same fraction of ambers was obtained when mutants were assayed on unirradiated and UV-irradiated samples of a recA indicator strain. UV-irradiated PHI X174 showed a similar phenomenon. These results suggest that the specificity with regard to insertion of bases opposite radiation damage in PHI X174 DNA is different for host cells in which SOS repair has been induced and cellls in which SOS repair is not operative. (orig.)

[en] Highlights: • The 91,159 bp genome of E. coli phage D6 was sequenced. • D6 is a moderately distant relative of phage P1. • Bacterial sequence database harbors many unannotated P1-/D6-like plasmid prophages. • Phage SSU5 is not related to D6 or P1 but is almost certainly a temperate phage. • SSU5-like phages are present as circular plasmids in many Enterobacteriaceae genomes. The temperate Escherichia coli bacteriophage D6 can exist as a circular plasmid prophage, and we report here its 91,159 bp complete genome sequence. It is a distant relative of the well-studied phage P1, but it is sufficiently different that it typifies a previously undescribed tailed phage type or cluster. Examination of the database of bacterial genome sequences revealed that phage P1 and D6 prophage plasmids are common in the Enterobacteriales, and in addition, previously described Salmonella phage SSU5 represents a different type of temperate tailed phage with a circular plasmid prophage that is also very common in this host order. This analysis also discovered additional divergent clusters of putative circular plasmid prophages within the two larger P1 and SSU5 groups (superclusters) that inhabit the Enterobacteriales as well as bacteria in several other orders in the Gamma-proteobacteria class. Very few of these sequences are annotated as putative prophages.

[en] Highlights: • Expressing pglB from the genome creates a “plug and play” E. coli strain for homogeneous recombinant glycoprotein production. • This engineered strain can increase glycosylation efficiency and overall glycoprotein titres. • Parallel Reaction Monitoring can simultaneously confirm glycan addition and quantify absolute glycoprotein titres. Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.

[en] Some commercially important vinyl derivatives are produced by the decarboxylation of phenolic acids. Enzymatically, this process can be achieved by phenolic acid decarboxylases (PADs), which are able to act on phenolic acid substrates such as ferulic and p-coumaric acid. Although many microbial PADs have been characterized, little is known regarding their plant homologs. Transcriptome sequencing in the liverworts has identified seven putative PADs, which share a measure of sequence identity with microbial PADs, but are typically much longer proteins. Here, a PAD-encoding gene was isolated from the liverwort species Conocephalum japonicum. The 1197 nt CjPAD cDNA sequence was predicted to be translated into a 398 residue protein. When the gene was heterologously expressed in Escherichia coli, its product exhibited a high level of PAD activity when provided with either p-coumaric or ferulic acid as substrate, along with the conversion of caffeic acid and sinapic acid to their corresponding decarboxylated products. Both N- and C-terminal truncation derivatives were non-functional. The transient expression in tobacco of a GFP/CjPAD fusion gene demonstrated that the CjPAD protein is expressed in the cytoplasm. It is first time a PAD was characterized from plants and the present investigation provided a candidate gene for catalyzing the formation of volatile phenols.

[en] Highlights: • Rhodopsin of Dokdonia sp. is a highly specific light-dependent Na⁺ transporter. • H⁺-transport activity of Na⁺-rhodopsins is controlled by residue in position 253. • Replacement of a single atom confers a dual Na⁺/H⁺ specificity to Dokdonia rhodopsin. • The engineered rhodopsin photocycle was resolved on a submillisecond time scale. Bacterial Na⁺-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na⁺, but some can additionally transport H⁺. To determine the structural basis of cation promiscuity in Na⁺-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na⁺-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na⁺-rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na⁺-rhodopsin (NaR) was indeed found to transport both Na⁺ and H⁺ in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na⁺-deficient medium. The structural basis for the dependence of the Na⁺/H⁺ specificity in Na⁺-rhodopsin on residue 253 remains to be determined.

[en] Fresh, chilled chicken carcasses were irradiated at 2.5 kGy and stored at 40 degree C. At intervals samples were withdrawn for microbial, chemical and sensory evaluation. Result showed that combination of a 2.5 kGy irradiation dose and storage at 4 degree C were adequate for a radicidised chicken process. Immediately after irradiation, the microbial spoilage was reduced by at least 4 log cycles. The carcasses were qf excellent quality for at least 16 days of storage and were free from Salmonella and other food pathogens. Changes in chemical composition (moisture, fat, protein, ash and amino acids) and sensory quality of chicken carcasses irradiated at 2.5 kGy were not significant. Therefore the dose of 2.5 kGy should be the target for chilled chicken irradiation process

[en] Highlights: • A peptide of late embryogenesis abundant (LEA) protein was expressed in E. coli. • LEA peptide expression was induced with IPTG in a concentration-dependent manner. • LEA peptide expression improved E. coli growth under ultraviolet radiation. • LEA peptide could be used to genetically modify organisms for ultraviolet resistance. Ultraviolet (UV) radiation causes damage in all living organisms, including DNA damage that leads to cell death. Herein, we provide a new technique for UV radiation protection through intracellular short peptide expression. The late embryogenesis abundant (LEA) peptide, which functions as a shield that protects macromolecules from various abiotic stress, was obtained from the Polypedilum vanderplanki group 3 LEA protein. Recombinant Escherichia coli BL21 (DE3) expressing functional LEA short peptide in vivo were exposed to UVA and UVC radiation for 4, 6, and 8 h. E. coli transformants expressing the LEA peptide showed higher cell viability under both UVA and UVC treatment at all time points as compared with that of the control. Furthermore, the cells expressing LEA peptide showed a higher number of colony-forming units per dilution under UVA and UVC treatment. These results suggested that expression of the short peptide could be useful for the development of genetically modified organisms and in applications that require resilience of organisms to UV radiation.

[en] Highlights: • Ε.coli ΔSlyD/SlyX strains engineered using λ-red mediated chromosomal deletion. • Development of several bacterial strains extends the spectrum of target proteins. • Preparation of SlyD/SlyX-free target protein after one purification step only. • ΔSlyD/SlyX bacterial strains allow to obtain a fully functional protein. Binding of native bacterial protein SlyD to metal affinity matrices remains a major problem in affinity purification of His-tagged recombinant proteins from Escherichia coli cells. In this study, four novel E. coli strains that lack the expression of SlyD/SlyX, were engineered using λ-red mediated chromosomal deletion. The resultant mutant E. coli strains allow us to obtain SlyD-free proteins immediately after metal affinity chromatography, and eliminate additional purification processes. As a model protein, bispecific antibodies composed of anti-F4/80 V_HH module and anti-TNF V_HH module (MYSTI-2) were used. Using this protein we have shown that the SlyD/SlyX-deficient E. coli strains allow us to obtain a fully functional protein.