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

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth propertie

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

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

ISSN:0378-1097ISSN:1574-6968ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

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

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

Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli

A novel cross-regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphemcol acetyltransferase (CAT) as a model system, several factors determining performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT product10n were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with pr10r simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10-fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2-fold increase in CAT accumulation was achieved by extending cell growth in a fed-batch process

Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli

A novel cross-regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphemcol acetyltransferase (CAT) as a model system, several factors determining performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT product10n were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with pr10r simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10-fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2-fold increase in CAT accumulation was achieved by extending cell growth in a fed-batch process