Insertion of an E-coli lacZ gene in Acetobacter xylinus for the production of cellulose in whey

A mini-Tn10:lacZ: kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in beta-galactosidase activity when grown in lactose medium. beta-Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and beta-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l(-1) after 4 days incubation. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies. (c) 2005 Elsevier Ltd. All rights reserved.