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Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the - 10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from - 1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses. (c) 2005 Elsevier B.V All rights reserved.

An open reading frame (ORF1) located upstream of the urease structural gene ureA in Rhizobium meliloti strain AK631 was cloned and characterized by DNA sequencing. Comparison of the amino acid sequence revealed partial homology with the urease accessory gene ureD of Klebsiella aerogenes and Proteus mirabilis. Mutational analysis of ORF1 showed that the gene is necessary for urease activity. Its function is still unknown.

Using an ureC-lacZ fusion, the expression of urease structural genes of the soil bacterium Rhizobium meliloti strain AK631 was studied in response to different nitrogen sources and nickel contents in the growth medium. The expression of urease genes is repressed by ammonia and is not inducible by urea. Urease activity depends on the nickel concentration of the medium. Nickel uptake is repressed in medium containing ammonia and is not affected by the genes located in the urease operon investigated.

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (> 200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.

The host plant genotype significantly influenced the nodule Lb content in all of the combinations examined between 5 alfalfa (Medicago sativa) cultivars and 5 Rhizobium meliloti strains inoculated, on the contrary not the bacterial genotype. The inoculation with the strains exhibiting an increased activity of uptake hydrogenase caused an elevation of the nodule Lb content. By IEF 4 major Lb components were found. Their proportions were changed throughout the nodule development.

By using a beta-glucanase from Bacillus as a model protein, we investigated whether the secretion competence based on the action of the kil gene can be improved using stronger promoters for the expression of the kil gene. Since the production of extracellular target proteins also depends on the promoter strengths of the target gene, we constructed four expression vectors with all possible combinations of a weak and a strong stationary-phase promoter for the kil gene, and a weak and a strong constitutive promoter, respectively, for the beta-glucanase gene. The results of batch fermentations showed that the use of stronger promoters generally decreased the cell density. However, a drastic increase of productivity of the cells to produce and secrete beta-glucanase resulted in a significantly higher activity of extracellular beta-glucanase. The yield of extracellular beta-glucanase can be increased (to 168 %) by using a strong promoter for the beta-glucanase alone. However, the increase was much higher when the weak promoter of the kil gene was replaced by a strong stationary-phase promoter (to 221 %). An even higher yield of extracellular beta-glucanase was reached when beta-glucanase was expressed by a strong promoter in addition indicating a combinatorial effect. This shows that the extracellular production of a recombinant target gene can be optimized by tuning the promoter strengths of components, the kil gene and the target gene.

We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known -10 consensus sequence, as well as the extended -10 region, and an A/T-rich region downstream of the -10 region were kept constant, whereas sequences from -37 to -14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the -10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme E sigma(s). The DNA sequence of these promoters differed significantly in the region from -25 to -16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses.

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.

Since Gram-negative bacteria are not able to transport proteins containing a leader sequence across the outer membrane, recombinant proteins cannot be secreted into the culture medium. On the other hand the extracellular production of overexpressed proteins is highly desirable, since downstream processing would be much easier. In recent years we developed an expression/secretion system for E. coli based on the membrane permeabilizing effect of the kil peptide of E. coli. Using this secretion system we demonstrate here the successful extracellular production of several important industrial enzymes. For some enzymes the results were confirmed bei experiments in bioreactors. Furthermore, a method was described to transfer the secretion system to other Gram-negative bacterial species. Using Klebsiella planticola and Acetobacter methanolicus it was shown that recombinant proteins can be produced extracellularly in an unexpectedly high level.

In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a beta-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn2+-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml(-1) PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher beta-glucanase concentration (65.6 kU ml(-1)), i.e. 1.5-fold compared to the internal adsorbent system.

Summary A rapid colour test is described which is selective for uptake hydrogenase (Hup+) activity under ex planta conditions. The test is based on decolonization of methylene blue. The decolonization time is a function of Hup activity. It is possible to select single colonies showing different Hup activities. A known Hup+ strain of R. leguminosarum is found to contain bacteria with both high and low Hup activities, respectively. The Hup phenotype of a given strain consequently represents a mean value determined by the proportion of cells with different Hup activities. Hup activity in R. leguminosarum was shown to vary under ex planta conditions and during symbiosis. It is shown that by selection for single colonies it is possible to obtain R. meliloti strain of enhanced uptake hydrogenase activity.

Summary Hup + derivatives of a Rhizobium meliloti strain were established by 3 approaches, first selection of Hup + colonies using a colour test and propagation, second transfer of the Rhizobium leguminosarum Sym plasmid pIJ1008 having hup genes and third transfer of the plasmid followed by additional selection with the colour test, respectively. Hydrogen uptake (Hup) activity under free living conditions was increased significantly to a similar extent in the first 2 approaches. After transfer of pIJ1008 Hup activity was further increased by additional selection with the colour test. The effect of Hup + and Hup − derivatives on symbiotic properties of alfalfa was compared. The Hup + strain selected only by the colour test showed its superiority in symbiosis with alfalfa compared with the original strain, e.g. acetylene reduction and plant dry matter were significantly increased. In contrast transfer of pIJ1008 to R. meliloti resulted in a decrease of acetylene reduction and plant dry matter, but the negative effects were eliminated by an additional selection with the colour test.

Transposon Tn7 was inserted into wide host range plasmid pSUP202 and used as a suicide plasmid vehicle for transposon mutagenesis in Rhizobium leguminosarum. Tn7 is transposed with high frequency into the self-transmissible plasmid pJB5JI without affecting the transfer, nodulation and nitrogen fixation functions. Tn7 transposition provides a useful tool for marking symbiotic plasmids.

Summary The investigation of fundamentals of the genetics of symbiotic nitrogen fixation has reached such a level that enables genetic manipulation of the bacteria involved in symbiosis. By this way it is possible to increase N 2 -fixation of rhizobia and to extend host specifity. The problems involved in the introduction of genetically manipulated strains of rhizobia are discussed. The symbiotic efficiency can also be increased by selection of suitable host plant genotypes.