Flagellar motility is an important form of movement in bacteria. In this way microbes in suspension are mobilised and may travel to locations more beneficial for their sustained growth. To determine which direction to travel, microbes follow chemical gradients, this is chemotaxis. Chemotaxis is the recognition of gradients and the alteration of locomotion in response to this, with the intention of moving up or down a gradient. Methyl-accepting chemotaxis proteins (MCPs) are located on the cell's surface and act as receptors for specific attractants. If the chemical target attaches to the MCPs then the CheAY two-component system is initiated. This results in a change of tumbling frequency, which in turn leads to net movement towards the attractant.
The Euprymna scolopes-Vibrio fischeri symbiosis is transmitted horizontally i.e. from the environment not from the maternal line. For this to work there must be a method of attraction to the squid to allow rapid colonization. The V. fischeri genome has previously been sequenced and this lead to the identification of 43 predicted MCPs. To better understand these, we can use mutant strains and compare the behaviour to the wild type. In this study 12 MCP mutants were discovered and a further 7 plasmid integration mutants were produced and their characterization was attempted.
In a plate based chemotaxis assay the mutant strains as well as the wild type V. fischeri were tested for responses to glucose, serine, N-acetylglucosamine (GlcNAc), N,N'-diacetyl-chitoboise [(GlcNAc)2] (both are chitin derived sugars), thymidine and N-acetylneuraminic acid (NANA). Only one of these mutants, vfcA, displayed any change in behaviour. It did not display a chemotactic response to serine but otherwise functioned as the wild types. vfcA is regulated by the flagellar master regulator FlrA so was thought to be a likely candidate for chemoctactic regulation. The lack of response to serine strongly indicates the involvement of VfcA in serine chemotaxis. In a capillary assay, performed in suspension, the vfcA mutant, again, showed none of the chemotactic response to serine that the wild type strains did, this further confirmed serine attraction as one of its functions. Serine attraction was restored in the mutant with heterologous expression of vfcA meaning that it it is the product not the gene that is responsible for the attraction.
To discover if other amino acids also induced vfcA mediated chemotaxis, a capillary assay was performed for all 20. Serine, cysteine, threonine and alanine all displayed strong a chemotactic response in the wid type but not in the vfcA mutant. The mutant did however have an increased response to the hydrophobic aromatic amino acids. This could be due to an increase in expression of other MCPs or that these act as a chemorepellant in the presence of VcfA.
To test if this was the primary driver in colonization of squid, wild type strains, vfcA mutants and a combination of the two were allowed to colonise squid. Both mutant and wild type colonized to comparable levels so there must be other more important factors driving colonization. These results indicate that chemotaxis towards amino acids is not of great importance in the colonization of the squid, however it could be that attraction to the hydrophobic amino acids compensates for that of serine, alanine, threonine and cysteine. There are another 42 MCPs in V. fischeri that might yield the answer to what drives colonization and the other 3 MCPs regulated by the flagellar master regulator would be a good place to start. The most beneficial aspect of this work is the development of the capillary assay which allows chemotaxis to be quantified in suspension rather than on plates, This is much more similar to the natural environment so results will hopefully be more comparable.
Brennan, C. A., DeLoney-Marino, C. R., & Mandel, M. J. (2013). Chemoreceptor VfcA mediates amino acid chemotaxis in Vibrio fischeri. Applied and environmental microbiology, 79(6), 1889–96. doi:10.1128/AEM.03794-12