Production, purification and characterization of serraticin A, a novel cold-active antimicrobial produced by Serratia proteamaculans 136.

AIM: This study focuses on the production, purification and characterization of serraticin A, a novel cold-active antimicrobial produced by Serratia proteamaculans 136. METHODS AND RESULTS: A Ser. proteamaculans strain producing a novel cold-active antimicrobial was isolated from Isla de los Estados, Argentina. Antimicrobial production was optimized in a BIOFLO 101 bioreactor under batch culture mode, with temperature, pH and dissolved oxygen controlled conditions. A purification protocol was developed including activated charcoal adsorption, solid-phase C18 extraction (SPE) and semi-preparative HPLC. The molecular weight was determined by LC/QTOF/MS/MS mass analysis. CONCLUSIONS: Serratia proteamaculans 136 produces a cold-active low molecular bacteriocin-like compound named serraticin A. In this work, it has been laboratory-scale produced, purified and partially characterized. Cross-immunity test revealed that serraticin A is very different from other well-known microcins assayed, with a wide inhibitory spectrum, showing an interesting biotechnology potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a cold-active compound with antimicrobial activity from Ser. proteamaculans. The work also highlights that cold environments could be a suitable source of micro-organisms with ability to produce cold-active biomolecules of biotechnological interest.

Antibody response and antigen-specific gamma-interferon profiles of vaccinated and unvaccinated pregnant sheep experimentally infected with Brucella melitensis.

It is well known that the immune response in sheep against Brucella melitensis is subject to individual variation, depending on diverse factors. It bears asking whether these factors (e.g. clinical disease, active infection, state of previous immunity), when affecting a group, can cause variation in the performance of different diagnostic tests. To clarify some of the circumstances in which this immune response can vary, we examine the immune-response profile of sheep protected against the clinical disease by prior vaccination with strain Rev. 1 in comparison with the profile of unprotected females showing the classical brucellosis symptoms. An experimental infection was provoked at midpregnancy under controlled conditions of both non-vaccinated (n=7) and previously Rev.1-vaccinated ewes (n=5). Their immune response was monitored from 7 to 9 weeks before abortion or normal birth to 30 weeks afterwards. Antibody response was assessed by classical tests (Rose Bengal test, complement fixation test (CFT)) in comparison with other diagnostic tests (indirect ELISA (iELISA), competitive ELISA (cELISA), fluorescence polarization assay (FPA), immunocapture test (ICT)). In addition, the cell-mediated immune response was indirectly evaluated by the in vitro antigen-specific release of gamma-interferon. The antibody levels and antigen-specific gamma-IFN profile of the non-vaccinated ewes having the disease and excreting the pathogen was notably high and differed significantly (P<0.05 or P<0.01) from those of vaccinated ewes that neither contracted brucellosis nor excreted the pathogen. In general, all the tests detect the infection in the non-vaccinated ewes with substantial effectiveness. It can be concluded that the high levels of circulating antibodies and of antigen-specific gamma-IFN are related to active Brucella infection. Similarly, the state of protection against the disease, but not necessarily against infection, due to a previous immunization with the Rev. 1 vaccination, appears to be responsible for a low level of detectable immune response. Nevertheless, the design of the study limits conclusions to pregnant ewes and cannot be extrapolated to non-pregnant ewes or rams. Likewise, the study provides no information on animals which are carriers of B. melitensis.

Construction of an integrative shuttle vector for Zymomonas mobilis.

An integrative shuttle vector, pZMOCP1, was constructed by ligating EcoRV digests of the plasmid cloning vector pBluescript and pZMP1, a cryptic plasmid of Zymomonas mobilis PROIMI A1. The 7.2-kb plasmid pZMOCP1 replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The vector was stably maintained in Z. mobilis after 200 generations without selective pressure.