Biosynthesis of Sugar Nucleotides Found in the Exopolysaccharide of Myxococcus xanthus

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Chapter 3: Using the NarX-DifA Chimeric Protein to Help Elucidate the Point of Regulation of Exopolysaccharide Production by the Dif Chemotaxis Pathway in Myxococcus xanthus

ABSTRACT

Myxococcus xanthus is a Gram negative soil bacterium which does not use glucose or other sugars as a primary carbon source. However, this organism produces extracellular polysaccharides (EPS) which are essential for social motility. Social (S) motility involves the movement of large groups of cells and is required for development and movement on soft (0.4%) agar. EPS are one of the cellular structures required for this motility. Currently, the chemotaxis-like Dif (defective in fruiting/fibrils) pathway is known to regulate the production of EPS, but the exact point of regulation is unknown. In this study, we demonstrated that EPS production could be restored to the EPS negative pgi mutant by glucose supplementation. Along with the ability of another mutant strain to activate the Dif pathway in the presence of nitrate, the location of regulation by the Dif pathway was explored. The results, although not conclusive, point to the Dif pathway regulating EPS production downstream of sugar monomer production.

INTRODUCTION

Myxococcus xanthus is a Gram negative bacterium commonly found in the soil (16). These bacteria display unique multicellular behaviors including the wolf-pack-hunting behavior in which large groups of cells “attack” and consume other bacteria, and development during which large groups of cells aggregate and differentiate into fruiting bodies consisting of environmentally resistant myxospores (14, 16, 35). Social (S) motility, one of two motility systems in M. xanthus, is required for the movement of large groups of these cells (30).
S motility requires two cellular structures: type IV pili (TFP) and extracellular polysaccharides (EPS) (35). The production of EPS is known to be regulated by the chemotaxis-like Dif (defective in fruiting/fibrils) pathway (77). However, it is unknown exactly how the Dif pathway regulates the process of EPS production. Previously, DifA, the signaling protein in the Dif pathway, was combined with NarX, a nitrate sensor (40). In a difA mutant containing the resulting chimeric protein, NafA, EPS production is regulated by the presence of nitrate in the media (73). This study also showed that DifC and DifE are essential for the propagation of the signal from DifA.
xanthus does not use glucose or other sugars as a primary carbon source (8, 12, 27, 67). Interestingly, this organism produces exopolysaccharide containing glucose and at least seven other monosaccharides (see Chapter 2). In this study we show the EPS negative pgi (phosphor-glucose isomerase) mutant which lacks the enzymes required to produce glucose-6-phosphate through gluconeogensis is able to produce EPS when grown in media containing glucose. Using the pgi mutant with NafA, an experiment to narrow the possible reactions regulated by the Dif pathway was conceived. The NafA protein was expressed in a pgi difA double deletion mutant. Theoretically, this mutant strain (YZ1017) can only produce EPS in the presence of both nitrate and glucose. So, if the strain is introduced to these substances in sequence, it may be possible to discover whether the Dif pathway regulates EPS production upstream or downstream of monosaccharide production. The results of this experiment were unfortunately inconclusive with some indications that the Dif pathway likely does not regulate EPS production upstream of sugar biosynthesis.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Unless otherwise noted, all strains are grown on CYE plates with 1.5% agar or in CYE broth. Plates and cultures are incubated at 32°C in a stationary or shaking incubator, respectively. M. xanthus strains used in this study are listed in Table 3-1. Kanamycin was added to media at 100 g/ml for selection purposes when appropriate.
Construction of mutants. The construction of pgi (YZ1001) is described in Chapter 2 of this document. Briefly, an in-frame deletion of the pgi gene was made by using PCR to amplify portions of the genome directly upstream and downstream of pgi and an overlapping PCR was used to fuse these pieces (53). The resulting fragment was then ligated into pBJ113 (31) and the resulting plasmid, pLC1001, was transformed into wild type M. xanthus, DK1622. Colonies were selected using a modified positive-negative kanamycin/galactose (KG) cassette (66) and confirmed using triple primer PCR as described in Chapter 2.
YZ1001 was transformed by electroporation with the plasmid pWB116 to produce pgi difA (YZ1011). The plasmid pXQ719 (73)was then transformed into this strain to produce YZ1017 ( pgi difA/Pdif-nafA; Kanr).
Motility Assays. Methodology for this experiment is previously described (3, 55). Briefly, 5 µL of an approximately 5 x 109 cells/mL MOPS (morpholinepropanesulfonic acid) buffer (10 mM MOPS [pH 7.6], 2 mM MgSO4) solution was spotted onto the center of an agar plate containing either 1.5% or 0.4% agar. The plates were then allowed to grow at 32°C for two to five days respectively before being viewed both macroscopically and microscopically to assess colony spreading and morphology, both at the edge and overall.
Agglutination Assay. One of the most sensitive ways to detect EPS production is an assay to examine the cellular cohesion known as an agglutination assay. The methods were similar to those used by Wu et al (72). Briefly, cultures are grown overnight and then resuspended to a cell density of approximately 2.8 x 108 cells/mL in Casitone-Tris (CTT) broth. The optical density at 600 nm of these samples is then measured at various time points for two hours. These measurements were then plotted based on relative absorbance which was calculated by dividing the optical density at each time point by the initial optical density for the given strain.
Modified Agglutination Assay. Cells are grown in 5 mL CYE broth overnight shaking at 37ëC. These cells are then inoculated into 50 mL CTT broth at an approximate optical density at 600 nm of 0.1 and allowed to grow overnight shaking at 37ëC to an optical density at 600 nm range of 0.3 to 0.6. The cells are collected by centrifugation, washed twice with CTT and resuspended to an optical density at 600 nm of 0.5 in 5 ml CTT in tubes containing a final concentration of 100 µM nitrate, 50 mM glucose, or nothing. These tubes are then incubated for 2 hours shaking at 37ëC. At the end of 2 hours, the cells were collected, washed three times with CTT and resuspended in 5 ml of CTT with glucose, nitrate, or both. These solutions were then incubated another 2 hours at 37ëC, shaking. After the final incubation period, an agglutination assay was conducted to detect the presence of EPS.

RESULTS

Recovery of the pgi mutantin the presence of glucose. YZ1001 ( pgi) is a mutant which displays an EPS negative phenotype. However, when this strain is grown on media containing glucose, it is able to recover a phenotype similar to that of wild type (Figure 3-1). YZ1001 becomes motile on soft agar and forms fruiting bodies on starvation media when glucose is supplemented. Both of these phenotypes indicate that when glucose is present, YZ1001 is able to produce EPS.
Construction of a pgi difA mutant containing NafA. A mutant with both the pgi and difA genes deleted was constructed as described in the Materials and Methods. The plasmid which contains nafA was then introduced into this double mutant to generate YZ1017. YZ1017 displayed phenotypes indicating that EPS was being produced only in the presence of glucose and nitrate (data not shown).
Combining two recoverable mutations in one strain creates a unique “switch”system. If glucose and nitrate are added to YZ1017 in sequence, nitrate followed by glucose or vice versa, the inherent mutations in the strain would allow these compounds to act as “switches” turning portions of the EPS production process on and off (Figure 3-2). An agglutination assay can then be used to determine the conditions under which the cells produce the most EPS. Since cells will theoretically stockpile intermediates to some extent, one series of exposures should produce more EPS than the other.
When this experiment was performed, the results were somewhat unclear. The sample which was exposed first to glucose and then to nitrate agglutinated better than the opposite sequence of exposures, nitrate followed by glucose. However, the glucose-nitrate sample did not agglutinate as well as the positive control which was exposed to both glucose and nitrate simultaneously (Figure 3-3).

DISCUSSION

Tentatively, looking at sample D (nitrate followed by glucose) displayed in Figure 3-3, the lack of agglutination of this sample is preliminary evidence that the Dif pathway does not regulate EPS production upstream of glucose production and therefore all monosacchride production. On the other hand, the sample E (glucose followed by nitrate) does not agglutinate similarly to the positive control C (glucose and nitrate simultaneously) indicating that sample E does not produce EPS in amounts similar to the positive control. Unexpectedly, sample A (nitrate only), a negative control, displayed better agglutination than samples B (glucose only), a second negative control, and D (nitrate followed by glucose).
A few scenarios to explain the effect caused by the addition of nitrate alone can be hypothesized at this point. YZ1017 ( pgi difA/Pdif-nafA) may not contain a completely stable mutation. Also, the “stockpiles” of intermediates formed during a given incubation may not be large enough to produce EPS at wild type levels. Lastly, M. xanthus could be affected by nitrate in such a way that the effect is not readily apparent on solid media but becomes a factor in liquid media. The last theory is unlikely as an difA/Pdif-nafA M. xanthus strain has been tested in liquid medium with nitrate without any of the effects shown here (73). The first theory is also unlikely as all of the mutations introduced into the strain are stable when present alone and are on the genome in locations well removed from one another. The theory that the cell is not “stockpiling” enough intermediates seems the most likely explanation for the reduced level of agglutination compared to wild type. Energetically this explanation is viable because the cell would not waste energy producing monosaccharides that are not being immediately used in cellular processes.

Chapter 1: Introduction and Review of Literature 
A. Myxobacteria
B. Myxococcus xanthus.
C. EPS in bacteria
D. LPS
E. Research Questions.
Chapter 2: Biosynthesis of Sugar Nucleotides Found in the Exopolysaccharide of Myxococcus xanthus
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Chapter 3: Using the NarX-DifA Chimeric Protein to Help Elucidate the Point of Regulation of Exopolysaccharide Production by the Dif Chemotaxis Pathway in Myxococcus xanthus 
ABSTRACT
INTRODUCTION .
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
GET THE COMPLETE PROJECT
BIOSYNTHESIS OF NUCLEOTIDE SUGAR MONOMERS FOR EXOPOLYSACCHARIDE PRODUCTION IN MYXOCOCCUS XANTHUS

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