The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism.     

Matrilineal Cohesion and Social Networks in Macaca fuscata

In a provisioned troop of Japanese macaques (Macaca fuscata) in Arashiyama, Japan, greater adherence to Kawamura's rules of matrilineal rank inheritance and youngest ascendancy occurred among high-ranking females versus low-ranking females. Accordingly, high-ranking females formed more clustered hierarchies and low-ranking females had more dispersed hierarchies. A proximate explanation for this finding may be related to differences in how females maintain their social networks. To determine whether the clustering in the hierarchy was reflected in patterns of social cohesiveness, I compared network sizes of coalition and grooming partners for females in each third of the hierarchy. I calculated the proportion of available partners that were coalition and grooming partners within each category of relatedness (0.5 r 0.004 and r = 0). High-ranking females formed coalitions with a large proportion of their close relatives and a small proportion of their distant relatives; middle-ranking females supported an intermediate proportion of their close relatives and a small proportion of their distant relatives; and, low-ranking females formed coalitions with very few available close and distant relatives. High-ranking females groomed nearly all available close relatives and an intermediate proportion of distant relatives, whilst middle- and low-ranking females groomed a large proportion of available close relatives and a very small proportion of distant relatives. Thus, levels of clustering within the hierarchy appeared to reflect levels of social cohesion, in terms of grooming and coalition formation.     

New substrates for beta-lactam-recognizing enzymes: aryl malonamates

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.     

Immune and Stress Responses Covary with Melanin-Based Coloration in the Barn Swallow

Eumelanin and pheomelanin are the main endogenous pigments in animals and melanin-based coloration has multiple functions. Melanization is associated with major life-history traits, including immune and stress response, possibly because of pleiotropic effects of genes that control melanogenesis. The net effects on pheo- versus eumelanization and other life-history traits may depend on the antagonistic effects of the genes that trigger the biosynthesis of either melanin form. Covariation between melanin-based pigmentation and fitness traits enforced by pleiotropic genes has major evolutionary implications particularly for socio-sexual communication. However, evidence from non-model organisms in the wild is limited to very few species. Here, we tested the hypothesis that melanin-based coloration of barn swallow (Hirundo rustica) throat and belly feathers covaries with acquired immunity and activation of the hypothalamic–pituitary–adrenal (HPA) axis, as gauged by corticosterone plasma levels. Individuals of both sexes with darker brownish belly feathers had weaker humoral immune response, while darker males had higher circulating corticosterone levels only when parental workload was experimentally reduced. Because color of belly feathers depends on both eu- and pheomelanin, and its darkness decreases with an increase in the concentration of eu- relative to pheomelanin, these results are consistent with our expectation that relatively more eu- than pheomelanized individuals have better immune response and smaller activation of the HPA-axis. Covariation of immune and stress response arose for belly but not throat feather color, suggesting that any function of color as a signal of individual quality or of alternative life-history strategies depends on plumage region.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.