Shear Stress: Shear-o-meter

A. Spy Promoter

A common drawback in high-density bioreactor runs is the shear stress imparted by the impeller blades to the cultivating E coli cells. This inflicts damage to the bacterial cell wall and exposes its cell membrane and thus makes the cell osmotically fragile and prone to lysis. However, E coli cells already have a built in protective mechanism in which they can detect presence of heterogenous unfolded proteins from the sheared outer membrane in the periplasmic space and activate the expression of particular proteins to solve the problem. One of this protein is the Spy (Spheroplast protein Y), which was first discovered exclusively in cells which were being subjected to sphaeroplasting stress. Further investigation has revealed the function of Spy protein to be quite similar to chaperones and it represses protein aggregation from sphaeroplasting by aiding protein folding.

The spy transcription activation mechanism has been studied and it has been indicated that the spy promoter sequence contains 2 phosphorylated BaeR (Bacterial Adaptative rEsponse) binding sites and 1 phosphorylated CpxR (Conjugative Plasmid eXpression) binding site and transcription is initiated under sigma factor 70 (RpoD). For the conjugative plasmid expression pathway, the CpxA-CpxR dimer plays the role of the signal transduction system. The CpxA (a histidine kinase) is the sensor unit which auto-phosphorylates a conserved histidine residue when proteins designated for the outer membrane or secretion (e.g. pili or curli fibres) are mounting in the periplasm. The phosphate from the CpxA is then transferred to a conserved aspartate residue on CpxR which becomes activated as a DNA binding protein and acts as a transcriptional regulator for various genes. The genes activated are mostly chaperones and proteases involved in the refolding or degradation of proteins in the periplasm. Studies carried out by Price and Ravio (2009) indicates that the level of consensus with the proposed sequence for the CpxR binding site and its orientation seems to play a minor role when determining the strength of induction by CpxR for a particular promoter. However the location of the binding site seems to be of more importance instead.

The bacterial adaptive response pathway was identified later and is also initiated by a two-component signal transduction system. BaeS-R dimer is also activated in a similar way as the CpxA-R dimer by stimuli like PapG overexpression, spheroplast formation and indole. BaeS-R activation pathway is less well characterised and Bae-R is known to activate only 4 operons so far (spy, mdtA, arcD and ycaC) with no known negative regulatory mechanism. Analysis of the upstream region of spy, mdtA and arcD, by Nishino et al. (2005), has led to the identification of a 18 bp long binding sequence for BaeR. Further experiments on the spy promoter, by Yamamoto et al. (2008), have identified the promoter sequence TCTNCANAA as the BaeR-box. Further transcription activator for the Spy locus like RcsB (Regulator of colanic acid Capsule Synthesis) are still being identified.

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Experiments, by Yamamoto et al. (2008), in which spy promoter fragments have been exposed to purified BaeR and acetyl phosphate have revealed a promoter-distal site between -162 and -137 and a promoter-proximal site between -109 and -79. The BaeR-box TCTNCANAA is present as a direct repeat in the promoter-distal site. The promoter-proximal site includes one single copy of the BaeR-box.</br> GTAAANNNNNGTAAA has been proposed as the CpxR binding site. (Wulf et al. 2002).

A. DegP Promoter

The DegP protein expressed in E coli is a periplasmic serine protease necessary for survival at very high temperatures. The main role of the protein is to degrade abnormal and excess proteins present in the periplasmic space. DegP has also been demonstrated to possess chaperone activity, which is even retained in proteolytically inactive mutant isoforms. The DegP promoter sequence has been characterised and is known to include 2 binding sites for phosphorylated CpxR (Conjugative Plasmid eXpression) and transcription from this promoter is therefore activated in a very similar way to that of the Spy promoter. However, the DegP promoter binds to sigma E factor instead of sigma factor 70 (as in the case of the spy promoter) and therefore expression from this promoter is dependent on the growth phase of the cell and cannot be activated by the presence of an excess of phosphorylated CpxR alone. Hence the degP promoter activity is only displayed during thr stationary phase of the cell.

ANOTHER PICTURE

Hypoxic Stress: Oximter

A. Nark & mNark Promoter

Another common issue faced in industrial grade bioreactor fermentations is the presence of dead spots inside the fermenter vessel where oxygen is not reaching in sufficient amount. Even though electronic probes can determine the amount of dissolved oxygen in the culture broth, in large volumes the presence of hypoxic regions is inevitable and cannot be detected by oxygen probes alone. Experiments by Kolesnikow et al. (1992) have showed that NarK protein expression in E coli is upregulated 100-fold under anaerobic conditions and a further 8-fold in the presence of nitrate. Therefore the Nark promoter can essentially used as a sensory tool for detecting hypoxic regions in a bioreactor.

The nark promoter sequence has been studied and is now knows to contain 2 Fnr (fumarate and nitrate reductase), 1 Fis, 4 NarL and 1 IHF binding sites that initiates transcription from the promoter by sigma factor 70. The chieft protein in this sensory pathway is Fnr which is always present in the cell in a fairly large and constant quantity. In the presence of oxygen inside the cell, the [4Fe-4S]2+ cluster of Fnr monomer is oxidized to a a [2Fe-2S]2+ cluster and the protein therefore remain in their inactive monomer state. But in the absence of oxygen, the [4Fe-4S]2+ cluster is retained and the Fnr protein dimerises and can then bind to DNA sequences and act as a transcription factor in order to upregulate the expression of various genes.

The optimal and conserved Fnr binding sequence has been determined as TTGAT-4N-ATCAA. (Spiro and Guest, 1990). But the distal Fnr binding site in the E coli NarK promoter is slightly different from the consensus region and therefore has a lower affinity for Fnr. For this reason, a modified NarK promoter sequence was deisgned in which the distal binding site was re-written to the optimal sequence and also some of the excess nucleotides were removed. As a result, the final mNark promoter sequence is shorter and has a better affinity for activated Fnr and is therefore has a stronger activity under anaerobic condition.

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LVA Tag

Fluorescent proteins (e.g. GFP, YFP, etc.) have been widely used since their discovery as reporter proteins to monitor various cellular processes. However there is one major disadvantage of these proteins as they are quite stable and has a longer life. As a result the fluorescent readings measured from a cell sample may not be quite accurate due to the presence of residual proteins which was expressed at a previous time point for sample collection. As a result, this property renders them unsuitable for monitoring and quantifying rapid changes of gene expression. However researchers have demonstrated that the addition of protease tag (amino acid sequences) to these proteins targets them for degradation by proteases and therefore shorten their half-life. Among all the protease tags identified so far, the LVA tag has proven to be the most efficient in making GFP (the most commonly used varied of fluorescent protein) unstable. This LVA tag consists of a short peptide sequence (AANDENYALVA) and is added to the C-terminal end of a GFP, which is then targeted for degradation by tail-specific proteases like Tsp protease in the periplasmic space and ClpXP and ClpAP in the cytoplasm of E coli.