Impact of the small RNA RyhB on growth, physiology and heterologous protein expression in Escherichia coli

The small noncoding RNA RyhB is a regulator of iron homeostasis in Escherichia coli. During iron limitation, it downregulates the expression of a number of iron-containing proteins, including enzymes of the tricarboxylic acid cycle and the respiratory chain. Because this infers a potential for RyhB to limit energy metabolism and biosynthetic capacity, the effect of knocking out ryhB on the physiology and heterologous protein productivity of E. coli has been analyzed. During iron limitation, induced either through insufficient extracellular supply or through overexpression of an iron-containing protein, ryhB mutants showed unaltered growth and substrate consumption. They did, however, exhibit significantly lowered acetate production rates. Plasmid-based expression of green fluorescent protein and the heterologous Vitreoscilla hemoglobin VHb was negatively affected by the ryhB knock-ou

Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli

The Vitreoscilla hemoglobin gene (vhb) is expressed under oxygen-limited conditions via an FNR-dependent mechanism. Furthermore, cAMP-CRP has been implicated in its regulation. Recently, VHb protein has been reported to protect a heterologous host from nitrosative stress. In this study we analyzed the regulation of the Vitreoscilla hemoglobin promoter (Pvhb) in Escherichia coli under nitrosative and oxidative stress conditions. Our results show unambiguously that expression of neither VHb nor chloramphenicol acetyltransferase under the control of Pvhb is induced under the experimental conditions used. Thus, a clear discrepancy between in vivo function, i.e. protection against nitrosative stress, and regulation of gene expression is obvious. The regulation of Pvhb reported here is in clear contrast to the expression pattern of flavohemoglobins from various microorganisms, which are generally induced by nitrosative stress. However, the length of Pvhb is only 146 bp and therefore, we cannot rule out that additional regulatory sequences may be located in the upstream region of Pvh

Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction

Present knowledge on plant non-symbiotic class-1 (Hb1) and truncated (TrHb) haemoglobin genes is almost entirely based on herbaceous species while the corresponding tree haemoglobin genes are not well known. The function of these genes has recently been linked with endosymbioses between plants and microbes. In this work, the coding sequences of hybrid aspen (Populus tremula×tremuloides) PttHb1 and PttTrHb were characterized, indicating that the key residues of haem and ligand binding of both genes were conserved in the deduced amino acid sequences. The expression of PttHb1 and PttTrHb was examined in parallel with that of the heterologous Vitreoscilla haemoglobin gene (vhb) during ectomycorrhiza/ectomycorrhizal (ECM) interaction. Both ECM fungi studied, Leccinum populinum and Xerocomus subtomentosus, enhanced root formation and subsequent growth of roots of all hybrid aspen lines, but only L. populinum was able to form mycorrhizas. Real-time PCR results show that the dual culture with the ECM fungus, with or without emergence of symbiotic structures, increased the expression of both PttHb1 and PttTrHb in the roots of non-transgenic hybrid aspens. PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, pointing to different functions for these genes during interaction with root growth-improving fungi. In contrast, ECM fungi were not able to enhance the expression of hybrid aspen endogenous haemoglobin genes in the VHb lines, which may be a consequence of the compensating action of heterologous haemoglobi

Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants

To date, haemoglobins (Hbs) have been shown to exist in all kingdoms of life. The least studied and understood groups are plant non-symbiotic haemoglobins (nsHbs) and the recently found plant truncated Hbs (trHbs). From a biotechnological point of view, the best characterized and almost exclusively applied Hb is the bacterial Vitreoscilla haemoglobin (VHb). In this review, the present state of knowledge of structural features and ligand binding kinetics of plant nsHbs and trHbs and their proposed roles as oxygen carriers, oxygen sensors, and for oxygen storage, in nitric oxide (NO) detoxification, and in peroxidase activity are described. Furthermore, in order to predict the functioning of plant Hbs, their characteristics will be compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb on plants are reviewed. Finally, the challenging future of plant Hb research is discusse

Bacterial hemoglobins and flavohemoglobins: Versatile proteins and their impact on microbiology and biotechnology

ISSN:0378-1097ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

Tissue Plasminogen Activator Coexpressed in Chinese Hamster Ovary Cells with α(2,6)-Sialyltransferase Contains NeuAcα(2,6)Galβ(1,4)Glc-N-AcR Linkages

Genetic alteration of the set of oligosaccharide biosynthesis enzymes expressed in a genetically engineered host cell line is a plausible strategy for manipulating the oligosaccharides on a cloned glycoprotein coexpressed in that cell line. This hypothesis was verified for the particular case of sialylation of recombinant human tissue plasminogen activator (tPA) expressed by an engineered Chinese hamster ovary (CHO) cell line. The gene for rat liver β-galactoside α(2,6)-sialyltransferase (2,6-ST) was cloned behind the MMTV promoter in the vector pMSG and transfected into a tPA-expressing CHO cell line. Selected and screened transfectants exhibited significantly greater surface fluorescence than controls in flow cytometric analyses of cells labeled with Sunabacus nigru agglutinin (SNA)-biotin and streptavidin-R-phycoerythrin; SNA specifically binds to NeuAcα(2,6)Gβ(1,4)Glc-N-AcR linkages, which are synthesized by 2,6-ST and which are not normally found on CHO cells. SNA blots of partially purified tPA from the culture supernatant demonstrated that tPA synthesized in the 2,6-ST transfectants possessed terminal NeuAcα(2,6)Gaβ(1,4)Glc-N-AcR linkages, while tPA from the original recombinant CHO cell line did not. Besides possibly allowing the production of glycoproteins in cell culture with glycosylation more closely resembling that in humans, extensions of this strategy have the potential to tailor the pharmacokinetics, targeting, and antigenic properties of cloned glycoproteins