The use of a radial basis neural network and genetic algorithm for improving the efficiency of laccase-mediated dye decolourization.

A radial basis function neural network (RBF) and genetic algorithm (GA) were applied to improve the efficiency of the oxidative decolourization of the recalcitrant dye Reactive Black 5 (RB 5) by a technical laccase (Trametes spp.) and the natural mediator acetosyringone (ACS). The decolourization of RB 5 in aqueous solution was studied with a 3(4) factorial design including different levels of laccase (2, 100, 200 U L(-1)), acetosyringone (5, 50, 100 µM), pH value (3, 4.5, 6) and incubation time (10, 20, 30 min). The generated RBF network was mathematically evaluated by several statistical indices and revealed better results than a classical quadratic response surface (RS) model. The experimental data showed that within 10 min of incubation time a complete decolourization (>90%) was achieved by using the highest amount of laccase (200 U L(-1)) and acetosyringone (100 µM) at pH 6. By applying the RBF-GA methodology, the efficiency of the laccase-mediated decolourization was improved by minimising the required amount of laccase and acetosyringone by 25% and 21.7% respectively. Complete decolourization (>90%) was obtained within 10 min at the GA-optimised process conditions of laccase (150 U L(-1)) and acetosyringone (78.3 µM) at pH 5.67. These results illustrate that the RBF-GA methodology could be a powerful technique during scale-up studies.

Trehalose is not relevant for in vivo activity of sigmaS-containing RNA polymerase in Escherichia coli.

The sigmaS- and sigma70-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on sigmaS. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of EsigmaS and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649-3654, 1997). However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various sigmaS-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of sigmaS-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.

The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the sigmaS subunit of RNA polymerase in Escherichia coli.

The hfq-encoded RNA-binding protein HF-I has long been known as a host factor for phage Qbeta RNA replication and has recently been shown to be essential for translation of rpoS, which encodes the sigmaS subunit of RNA polymerase. Here we demonstrate that an hfq null mutant does not synthesize glycogen, is starvation and multiple stress sensitive, and exhibits strongly reduced expression of representative sigmaS-regulated genes. These phenotypes are consistent with strongly reduced sigmaS levels in the hfq mutant. However, the analysis of global protein synthesis patterns on two-dimensional O'Farrell gels indicates that approximately 40% of the more than 30 proteins whose syntheses are altered in the hfq null mutant are not affected by an rpoS mutation. We conclude that HF-I is a global regulator involved in the regulation of expression of sigmaS and sigmaS-independent genes.

Heat shock regulation of sigmaS turnover: a role for DnaK and relationship between stress responses mediated by sigmaS and sigma32 in Escherichia coli.

The cellular level of the rpoS-encoded sigmaS subunit of RNA polymerase increases in response to various stress situations that include starvation, high osmolarity, and shift to acid pH, and these different stress signals differentially affect rpoS translation and/or sigmaS stability. Here we demonstrate that sigmaS is also induced by heat shock and that this induction is exclusively due to an interference with sigmaS turnover. Some sigmaS-dependent genes exhibit similar heat shock induction, whereas others are not induced probably because they need additional regulatory factors that might not be present under conditions of heat shock or exponential growth. Despite its induction, sigmaS does not seem to contribute to heat adaptation but may induce cross-protection against different stresses. While sigmaS is not involved in the regulation of the heat shock sigma factor sigma32, the heat shock protein DnaK has a positive role in the posttranscriptional control of sigmaS. The present evidence suggests that DnaK is involved in the transduction of two of the signals that result in reduced sigmaS turnover, i.e., heat shock and carbon starvation. Heat shock induction of sigmaS also clearly indicates that a cessation of growth or even a reduction of the growth rate is not a prerequisite for the induction of sigmaS and sigmaS-dependent genes and underscores the importance of sigmaS as a general stress sigma factor.

The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli.

The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.

Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli.

The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth. Osmotic induction of rpoS can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine. Stimulation of rpoS translation and a change in the half-life of sigma(s) from 3 to 50 min both contribute to osmotic induction. Experiments with lacZ fusions inserted at different positions within the rpoS gene indicate that an element required for sigma(s) degradation is encoded between nucleotides 379 and 742 of the rpoS coding sequence.

The response regulator RssB controls stability of the sigma(S) subunit of RNA polymerase in Escherichia coli.

The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post-transcriptional growth phase-related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C-terminal output domain and is the first known response regulator involved in the control of protein turnover.

Role for the histone-like protein H-NS in growth phase-dependent and osmotic regulation of sigma S and many sigma S-dependent genes in Escherichia coli.

The sigma S subunit of RNA polymerase (encoded by the rpoS gene) is the master regulator in a complex regulatory network that controls stationary-phase induction and osmotic regulation of many genes in Escherichia coli. Here we demonstrate that the histone-like protein H-NS is also a component of this network, in which it functions as a global inhibitor of gene expression during the exponential phase of growth. On two-dimensional gels, at least 22 sigma S-controlled proteins show increased expression in an hns mutant. H-NS also inhibits the expression of sigma S itself by a mechanism that acts at the posttranscriptional level. Our results indicate that relief of repression by H-NS plays a role in stationary-phase induction as well as in hyperosmotic induction of rpoS translation. Whereas certain sigma S-dependent genes (e.g., osmY) are only indirectly regulated by H-NS via its role in the control of sigma S expression, others are also H-NS-regulated in a sigma S-independent manner. (For this latter class of genes, rpoS hns double mutants show higher levels of expression than mutants deficient in rpoS alone.) In addition, we demonstrate that the slow-growth phenotype of hns mutants is suppressed in hns rpoS double mutants and that many second-site suppressor mutants that spontaneously arise from hns strains carry lesions that affect the expression of sigma S.