CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.     

Taura syndrome virus (TSV) in Thailand and its relationship to TSV in China and the Americas

The cultivation of exotic Penaeus vannamei in Thailand began on a very limited scale in the late 1990s, but a Thai government ban on the cultivation of P. monodon in freshwater areas in 2000 led many Thai shrimp farmers to shift to cultivation of P. vannamei. Alarmed by the possibility of Taura syndrome virus (TSV) introduction, the Thai Department of Fisheries required that imported stocks of P. vannamei be certified free of TSV by RT-PCR (Reverse Trasciption Polymerase Chain Reaction) testing. During the interval of allowed importation, over 150,000 broodstock shrimp were imported, 67% of these from China and Taiwan. Despite the safeguards, TSV outbreaks occurred and we confirmed the first outbreak by RT-PCR in early 2003. This resulted in a governmental ban on all shrimp broodstock imports from February 2003, but TSV outbreaks have continued, possibly due to original introductions or to the continued illegal importation of stocks. To determine the origin of the TSV in Thailand, the viral coat protein gene VP1 was amplified by RT-PCR from several shrimp specimens found positive for TSV by RT-PCR from January to November 2003. These included 7 samples from P. vannamei disease outbreaks in Thailand, 3 other non-diseased shrimp samples from Thailand and Burma and 6 samples including P. vannamei and P. japonicus from China. Comparison revealed that the Thai, Burmese and Chinese TSV types formed a clade distinct from a clade of TSV types from the Americas.     

Development of a Fetal Weight Chart Using Serial Trans-Abdominal Ultrasound in an East African Population: A Longitudinal Observational Study

Objective

To produce a fetal weight chart representative of a Tanzanian population, and compare it to weight charts from Sub-Saharan Africa and the developed world.

Methods

A longitudinal observational study in Northeastern Tanzania. Pregnant women were followed throughout pregnancy with serial trans-abdominal ultrasound. All pregnancies with pathology were excluded and a chart representing the optimal growth potential was developed using fetal weights and birth weights. The weight chart was compared to a chart from Congo, a chart representing a white population, and a chart representing a white population but adapted to the study population. The prevalence of SGA was assessed using all four charts.

Results

A total of 2193 weight measurements from 583 fetuses/newborns were included in the fetal weight chart. Our chart had lower percentiles than all the other charts. Most importantly, in the end of pregnancy, the 10^th percentiles deviated substantially causing an overestimation of the true prevalence of SGA newborns if our chart had not been used.

Conclusions

We developed a weight chart representative for a Tanzanian population and provide evidence for the necessity of developing regional specific weight charts for correct identification of SGA. Our weight chart is an important tool that can be used for clinical risk assessments of newborns and for evaluating the effect of intrauterine exposures on fetal and newborn weight.     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Synopsis of the International Gap Junction Conference in Elsinore, Denmark August 5-9, 2007

This synopsis covers the main results and conclusions from the platform presentations during the International Gap Junction Conference. More detailed information is provided in the mini reviews on controversial scientific issues, short reports of research results and conference abstracts published in this issue of Cell Communication and Adhesion.     

Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation

Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes , which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.     

Lactic acid bacteria as prime candidates for codon optimization

In species having a strong correlation of expressivity and codon bias it has been shown that heterologous expression can be optimized by changing codons of the introduced gene towards the set of codons that the host organism naturally uses in its highly expressed genes. Even though two lactic acid bacteria are fully sequenced, there are no reports on attempts of codon optimization in the literature. In this report it is demonstrated that codons used in highly expressed genes tend to differ from the codons in lowly expressed genes, and that there is a strong correlation of codon bias and empirical expressivity (codon adaptation index) in Lactococcus lactis and Lactobacillus plantarum. This strongly suggests that codon optimization strategies could be applied to expression systems with lactic acid bacteria as producer strains. A good example of a candidate for codon optimization is the mouse interleukin-2 gene, which in its natural form has an extremely low codon adaptation index for expression in Lc. lactis.     

The effect of varying nitrogen supplies on the content of amino acids in wheat grain

Summary The effect of nitrogen applications on the amino acid composition of wheat grain was studied in a pot experiment, in which spring wheat was supplied with increasing amounts of ammonium sulphate. The ensuing yield curve extended from very small N-applications to applications resulting in a toxic effect on the yield.The contents of dry matter, total N, 15 amino acids and ammonia were determined in samples taken from 9 representative treatments. The relationship between the yield of dry matter and the concentration in the dry matte rof the individual amino acids showed the same characteristic variations as those previously found for total N and for a number of plant nutrients.In accordance with the results cited in the literature, increasing applications of N caused an increase of glumatic acid and proline on a nitrogen basis, whereas lysine and arginine decreased.     

Loss of Hda activity stimulates replication initiation from I-box, but not R4 mutant origins in Escherichia coli

Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA^ATP specific sites (I-boxes, τ-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC . Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA^ATP containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA^ATP specific I-boxes. However, competition between I-box mutant and wild-type origins , revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA^ATP/DnaA^ADP ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.