Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)–KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.     

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin

Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.     

Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.     

Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communication

It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells.     

Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae

Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β’-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ’-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ’-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ’-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β’-NAC or α-NAC subunits as well as αβ’-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.