The hydH/G Genes from Escherichia coli code for a zinc and lead responsive two-component regulatory system.

The hydH/G genes from Escherichia coli code for a two-component regulatory system that has been implicated in the regulation of hydrogenase 3 formation. In a detailed study of the function of HydH/G employing hycA'-'lacZ reporter gene fusions, it was shown that HydH/G indeed led to a stimulation of activation of the hycA promoter responsible for hydrogenase 3 synthesis but only when hydG is overexpressed from a plasmid in a strain lacking FhlA. Since the stimulation was not observed with an fdhF'-'lacZ fusion, and since it was independent from a functional hydH gene product, it must be considered as unspecific cross-talk. An extensive search for the actual physiological signal of HydH/G showed that the system responds to high concentrations of zinc or lead in the medium. Expression of zraP, a gene inversely oriented to hydH/G whose product seems to be involved in acquisition of tolerance to high Zn(2+) concentrations, is stimulated by high Zn(2+) and Pb(2+) concentrations and this stimulation requires both HydH and HydG. Purified HydG in the presence of phosphoryl donors binds to a region within the zraP-hydHG intergenic region that is characterised by two inverted repeats separated by a 14 bp spacer. Putative -12/-24 sigma(54)-dependent promoter motifs are present upstream of both the zraP and the hydHG transcriptional units; in accordance, transcription of zraP is strictly dependent on the presence of a functional rpoN gene. The expression of hydH/G is autoregulated: high Zn(2+) and Pb(2+) concentrations lead to a significant increase of the HydG protein content which took place only in a hydH(+) genetic background. Since HydH binds to membranes tightly, it is assumed that the HydH/G system senses high periplasmic Zn(2+) and Pb(2+) concentrations and contributes to metal tolerance by activating the expression of zraP. The redesignation of hydH/G as zraS/R is suggested.

Analysis of the domain structure and the DNA binding site of the transcriptional activator FhlA.

FhlA is the transcriptional activator of the genes coding for the formate hydrogen lyase system in Escherichia coli. It is activated by the binding of formate and induces transcription by sigma54 RNA polymerase after binding to specific upstream activating sequences (UAS). Sequence comparison had shown that FhlA exhibits a structure composed of three domains, which is typical for sigma54-dependent regulators. By analyzing the N-terminal domain of FhlA of E. coli (amino acids 1-378; FhlA-N) and the rest of the protein (amino acids 379-693; FhlA-C) as separate proteins in vivo and in vitro the functions of the different domains of FhlA were elucidated. The FhlA-C domain is active in ATP hydrolysis and activation of transcription and its activity is neither influenced by the presence of formate nor of the antiactivator HycA. However, it is stimulated in the presence of the FhlA-specific UAS, indicating that this region of FhlA is responsible for DNA binding. FhlA-N is not active itself but able to reduce the activity of full-length FhlA in trans, probably by formation of nonfunctional heterooligomers. The DNA binding site of FhlA was analyzed by hydroxyradical footprinting. Each UAS consists of two binding sites of 16 bp separated by a spacer region. A consensus sequence could be deduced and a model is presented and supported by in vivo data in which a FhlA tetramer binds to the UAS on one side of the DNA helix. Performing an extensive screening we could show that the FhlA regulatory system is conserved in different species of the family Enterobacteriaceae. The analysis of orthologs of FhlA revealed that they are able to functionally replace the E. coli enzyme.

Solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae K40.

This paper reports some physicochemical properties of the capsular polysaccharide produced by Klebsiella pneumoniae serotype K40 (K40-CPS) in aqueous solution. The polymer has a linear hexasaccharide repeating unit containing one glucuronic acid residue as the only ionizable group. Potentiometric, viscometric, chiro-optical and rheological measurements have been carried out over a range of ionic strength, pH and temperature, with the aim of characterizing the conformational state of the polysaccharide in aqueous solution. All the data reported indicate that the K40-CPS does not undergo a cooperative conformational transition under the investigated experimental conditions. Furthermore, the viscosity data and the viscoelastic spectra suggest that the K40-CPS is rather flexible and adopts a random coil conformation in solution.

Use of collagen hydrolysate as a complex nitrogen source for the synthesis of penicillin by Penicillium chrysogenum.

Optimal conditions for both biomass formation and penicillin synthesis by a strain of Penicillium chrysogenum were determined when using a collagen-derived nitrogen source. Preliminary investigations were carried out in shaken flask cultures employing a planned experimental program termed the Graeco-Latin square technique (Auden et al., 1967). It was initially determined that up to 30% of a conventional complex nitrogen source such as cottonseed meal could be replaced by the collagen-derived nitrogen source without decreasing the productivity with respect to the penicillin yield. In the pilot scale experiments using a 30 l stirred tank type of bioreactor, higher penicillin yields were obtained when 70% of the conventional complex nitrogen source in the form of cottonseed meal was replaced by the collagen hydrolysate. Furthermore, the maximum rate of penicillin synthesis continued for over a longer period when using collagen hydrolysate as a complex nitrogen source. Penicillin synthesis rates were determined using a linear regression.