RecA-promoted, RecFOR-independent progressive disassembly of replisomes stalled by helicase inactivation.

In all organisms, replication impairment is a recognized source of genomic instability, raising an increasing interest in the fate of inactivated replication forks. We used Escherichia coli strains with a temperature-inactivated replicative helicase (DnaB) and in vivo single-molecule microscopy to quantify the detailed molecular processing of stalled replication forks. After helicase inactivation, RecA binds to blocked replication forks and is essential for the rapid release of hPol III. The entire holoenzyme is disrupted little by little, with some components lost in few minutes, while others are stable in 70% of cells for at least 1 hr. Although replisome dissociation is delayed in a recA mutant, it is not affected by RecF or RecO inactivation. RecFOR are required for full RecA filaments formation, and we propose that polymerase clearance can be catalyzed by short, RecFOR-independent RecA filaments. Our results identify a function for the universally conserved, central recombination protein RecA.

Within-litter covariance of allele-specific MHC heterozygosity, coccidian endoparasite load and growth is modulated by sibling differences in starting mass.

Although littermates in altricial mammals usually experience highly similar environmental conditions during early life, considerable differences in growth and health can emerge among them. In a study on subadults of a European rabbit (Oryctolagus cuniculus) population with low MHC polymorphism, we tested whether litter-sibling differences in endoparasitic coccidia load and body mass at the end of the vegetation period were associated with within-litter differences in starting body mass (measured around 2 weeks prior to weaning) and in immune-genetic (MHC class II DRB) constitution. We hypothesized that siblings with a lighter starting mass might be more susceptible to endoparasite infections and thus, negative effects of a more unfavourable MHC constitution might be particularly pronounced in such individuals. Within-litter comparisons revealed that animals with a lighter starting mass reached a relatively lower body mass in autumn. Furthermore, there were indications for an allele-specific heterozygote advantage, as animals with heterozygous combinations of the allele Orcu-DRB*4 had relatively lower hepatic coccidia loads than their littermates with certain homozygous allele combinations. Consistent with our hypothesis, significantly higher hepatic coccidia loads and tendentially lower autumn body masses in homozygous compared to heterozygous individuals for the allele Orcu-DRB*4 were evident in initially lighter but not in heavier siblings, suggesting synergistic effects between an unfavourable MHC constitution and a light starting mass. Taken together, these effects might lead to notable differences in fitness among litter siblings, as a low body mass and a high endoparasite burden are key factors limiting young rabbits' survival during winter.

The role of planktonic Flavobacteria in processing algal organic matter in coastal East Antarctica revealed using metagenomics and metaproteomics.

Heterotrophic marine bacteria play key roles in remineralizing organic matter generated from primary production. However, far more is known about which groups are dominant than about the cellular processes they perform in order to become dominant. In the Southern Ocean, eukaryotic phytoplankton are the dominant primary producers. In this study we used metagenomics and metaproteomics to determine how the dominant bacterial and archaeal plankton processed bloom material. We examined the microbial community composition in 14 metagenomes and found that the relative abundance of Flavobacteria (dominated by Polaribacter) was positively correlated with chlorophyll a fluorescence, and the relative abundance of SAR11 was inversely correlated with both fluorescence and Flavobacteria abundance. By performing metaproteomics on the sample with the highest relative abundance of Flavobacteria (Newcomb Bay, East Antarctica) we defined how Flavobacteria attach to and degrade diverse complex organic material, how they make labile compounds available to Alphaproteobacteria (especially SAR11) and Gammaproteobacteria, and how these heterotrophic Proteobacteria target and utilize these nutrients. The presence of methylotrophic proteins for archaea and bacteria also indicated the importance of metabolic specialists. Overall, the study provides functional data for the microbial mechanisms of nutrient cycling at the surface of the coastal Southern Ocean.

Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.

A metaproteomic assessment of winter and summer bacterioplankton from Antarctic Peninsula coastal surface waters.

A metaproteomic survey of surface coastal waters near Palmer Station on the Antarctic Peninsula, West Antarctica, was performed, revealing marked differences in the functional capacity of summer and winter communities of bacterioplankton. Proteins from Flavobacteria were more abundant in the summer metaproteome, whereas winter was characterized by proteins from ammonia-oxidizing Marine Group I Crenarchaeota. Proteins prevalent in both seasons were from SAR11 and Rhodobacterales clades of Alphaproteobacteria, as well as many lineages of Gammaproteobacteria. The metaproteome data were used to elucidate the main metabolic and energy generation pathways and transport processes occurring at the microbial level in each season. In summer, autotrophic carbon assimilation appears to be driven by oxygenic photoautotrophy, consistent with high light availability and intensity. In contrast, during the dark polar winter, the metaproteome supported the occurrence of chemolithoautotrophy via the 3-hydroxypropionate/4-hydroxybutyrate cycle and the reverse tricarboxylic acid cycle of ammonia-oxidizing archaea and nitrite-oxidizing bacteria, respectively. Proteins involved in nitrification were also detected in the metaproteome. Taurine appears to be an important source of carbon and nitrogen for heterotrophs (especially SAR11), with transporters and enzymes for taurine uptake and degradation abundant in the metaproteome. Divergent heterotrophic strategies for Alphaproteobacteria and Flavobacteria were indicated by the metaproteome data, with Alphaproteobacteria capturing (by high-affinity transport) and processing labile solutes, and Flavobacteria expressing outer membrane receptors for particle adhesion to facilitate the exploitation of non-labile substrates. TonB-dependent receptors from Gammaproteobacteria and Flavobacteria (particularly in summer) were abundant, indicating that scavenging of substrates was likely an important strategy for these clades of Southern Ocean bacteria. This study provides the first insight into differences in functional processes occurring between summer and winter microbial communities in coastal Antarctic waters, and particularly highlights the important role that 'dark' carbon fixation has in winter.