Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli

In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.     

Sulfation in the Golgi lumen of Madin-Darby canine kidney cells is inhibited by brefeldin A and depends on a factor present in the cytoplasm and on Golgi membranes

Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.     

Geographic variation of the storing behaviour in the Coal tit Parus ater: role of winter residency and environmental conditions

Foraging and hoarding behaviour of the Coal Tit Parus ater were studied in two widely separated populations, in the Pyrenees and in Norway. The two populations differed in their degree of winter residence and environmental pressures. Location of foraging and hoarding sites were described in the Pyrenees during winter, and during autumn in Norway where no hoarding takes place during winter. Significant differences in the hoarding behaviour of Coal Tits between these areas were observed. In the Pyrenees, birds hoarded on inner and lower parts of the trees, using trunks and thick branches as caching sites, which are the substrates most heavily used in conditions of winter environmental stress. In Norway, birds hoarded items on outer and higher parts of the trees, mainly among needled twigs, which were the substrates used in normal foraging. Animal food was hoarded more frequently by the Norwegian population. Our results show that hoarding behaviour varies among populations and suggest that the differences recorded may be related to differences in competitive pressures and the prevailing environmental conditions. Furthermore, the absence of a safe long-term hoarding niche in the Norwegian population, due to interspecific competition, would make a strong sedentary habit difficult, offering a functional explanation for the differences observed in the degree of winter residence between Norwegian and Pyrenean Coal Tits.     

POPULATION DIVERGENCE IN SEXUAL ORNAMENTS: THE WHITE FOREHEAD PATCH OF NORWEGIAN PIED FLYCATCHERS IS SMALL AND UNSEXY

Models of sexual selection suggest that populations may easily diverge in male secondary sexual characters. Studies of a Spanish population of the pied flycatcher, Ficedula hypoleuca, and a Swedish population of the closely related collared flycatcher, F. albicollis, have indicated that the white forehead patch of males is a sexually selected trait. We studied the white forehead patch of male pied flycatchers (n = 487) in a Norwegian population over seven years. Males with large forehead patches were in general more brightly colored, but patch height was not correlated to body mass, body size, or parasite loads. Conditions during the nestling period did not seem to influence patch height as an adult. Patch height increased slightly from the first to the second year as adults, but then remained relatively constant at higher ages. Patch height was not related to survival. Year-to-year changes showed that males who increased in patch height also increased in body mass, suggesting that expression of the forehead patch may be partly condition dependent. However, changes in body mass explained only a small proportion of the variance in patch height between males. Thus, patch height would not be a good indicator of male quality. Furthermore, patch size was also not related to male ability to feed nestlings, indicating that females would not obtain direct benefits by choosing males with large patches. However, patch height could be a Fisher trait, but this requires heritability and there was no significant father-son resemblance in patch height. Comparisons of the males visited by each female during the mate sampling period indicated that chosen males did not have larger forehead patches than rejected males. Experimental manipulation of patch height did not affect male mating success. These results indicate that females do not use patch size as a mate choice cue. Finally, patch height did not predict the outcome of male contests for nestboxes, suggesting that the forehead patch is not an intrasexually selected cue of status. Norwegian pied flycatchers have smaller forehead patches than both Spanish pied flycatchers and Swedish collared flycatchers. We suggest that this pattern may be explained by the lack of sexual selection on the forehead patch in the Norwegian population as compared to the other populations, where the patch is apparently sexually selected. We discuss possible reasons for these population divergences, such as female choice on an alternative secondary sexual character (general plumage color) and speciation among Ficedula flycatchers.     

Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans.

In the leukemic macrophage cell-line THP-1, a fraction of the secreted matrix metalloproteinase 9 (MMP-9) is linked to the core protein of chondroitin sulfate proteoglycans (CSPG). Unlike the monomeric and homodimeric forms of MMP-9, the addition of exogenous CaCl2 to the proMMP-9/CSPG complex resulted in an active gelatinase due to the induction of an autocatalytic removal of the N-terminal prodomain. In addition, the MMP-9 was released from the CSPG through a process that appeared to be a stepwise truncation of both the CSPG core protein and a part of the C-terminal domain of the gelatinase. The calcium-induced activation and truncation of the MMP-9/CSPG complex was independent of the concentration of the complex, inhibited by the MMP inhibitors EDTA, 1,10-phenanthroline and TIMP-1, but not by general inhibitors of serine, thiol and acid proteinases. This indicated that the activation and truncation process was not due to a bimolecular reaction, but more likely an intramolecular reaction. The negatively charged chondroitin sulfate chains in the proteoglycan were not involved in this process. Other metal-containing compounds like amino-phenylmercuric acetate (APMA), NaCl, ZnCl2 and MgCl2 were not able to induce activation and truncation of the proMMP-9 in this heterodimer. On the contrary, APMA inhibited the calcium-induced process, whereas high concentrations of either MgCl2 or NaCl had no effect. Our results indicate that the interaction between the MMP-9 and the core protein of the CSPG was the causal factor in the calcium-induced activation and truncation of the gelatinase, and that this process was not due to a general electrostatic effect.     

DNA HETEROZYGOSITY AND GROWTH RATE IN THE ATLANTIC COD GADUS MORHUA (L)

Relationships between growth rate and the degree of individual heterozygosity at ten nuclear RFLP loci were examined in two populations of the Atlantic cod, Gadus morhua, from northern Norway. A highly significant positive correlation was observed between growth rate and DNA heterozygosity in one population (Balsfjord) but not in the other (Barents Sea). Our results provide support for an important prediction of the associative overdominance hypothesis that heterozygosity-fitness correlations can be detected at neutral genetic markers and suggest that environmental conditions might play a dominant role in the manifestation of the correlation.     

H2O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H₂O₂ at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H₂O₂ to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H₂O₂ (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.     

Bacteria in the small intestine of lichen-fed Norwegian reindeer (Rangifer tarandus tarandus)

It has been suggested that lactic acid-producing bacteria may protect the epithelium of the mammalian gastrointestinal tract from pathogenic micro-organisms. Consistent with this, bacteria isolated from the mucosa of the small intestine of five lichenfed, semi-domesticated reindeer included mainly Streptococcus spp. However, the population densities of bacteria associated with the mucosa and in the intestinal contents were generally low and there was a large amount of variation both between animals and with site of sampling. It therefore seems unlikely that Streptococcus spp. are essential for the function of the small intestine in captive reindeer, and their role here remains uncertain.     

Digestion of Herring by Indigenous Bacteria in the Minke Whale Forestomach

Northeastern Atlantic minke whales (Balaenoptera acutorostrata) have a multichambered stomach system which includes a nonglandular forestomach resembling that of ruminants. Bacteria from the forestomachs of herring-eating whales were enumerated and isolated in an anaerobic rumen-like culture medium (M8W medium). The total viable population of anaerobic bacteria ranged from 73 × 10⁷ to 145 × 10⁸/ml of forestomach fluid (n = 4). Lactobacillus spp. (19.7%), Streptococcus spp. (35.9%), and Ruminococcus spp. (12.8%) were the most common of the bacterial strains (n = 117) isolated by use of M8W medium from the forestomach fluid population of two minke whales. Most of the isolates stained gram positive (93.2%), 62.4% were cocci, and all strains were strictly anaerobic. The population of lipolytic bacteria in one animal, enumerated by use of a selective lipid medium, constituted 89.7% of the viable population. The total viable population of anaerobic bacteria in freshly caught and homogenized herring (Clupea harengus) ranged from 56.7 to 95.0 cells per gram of homogenized prey (n = 3) when M8W medium was used. Pediococcus spp. (30.6%) and Aerococcus spp. (25.0%) were most common of the bacterial strains (n = 72) isolated from the homogenized herring. Most of the bacterial strains were gram positive (80.6%), and 70.8% were cocci. Unlike the forestomach bacterial population, as many as 61.1% of the strains from the herring were facultatively anaerobic. All bacterial strains isolated from the prey had phenotypic patterns different from those of strains isolated from the dominant bacterial population in the forestomach, indicating that the forestomach microbiota is indigenous. Scanning electron microscopic examinations revealed large numbers of bacteria, surrounded by a glycocalyx, attached to partly digested food particles in the forestomach. These data support the hypothesis that symbiotic microbial digestion occurs in the forestomach and that the bacteria are indigenous to minke whales.