Diagrama de Flujo

Figure 4.8Comparison ofa)M. xanthusandb)St. aurantiaca crtWgene clusters and neighbouring genes. Arrows indicate the direction in which the gene is transcribed. All labelled genes forSt. aurantiacaif not stated are putative.

Due to its strong similarity with M. xanthus and as a result the little information that could be obtained, the bacteriumStigmatella aurantiacahas not been considered in detail throughout this study. In terms of thecrtWoperon this is true, as a similar structure exists within St. aurantiaca, leading to identical carotenoids being

Heat Shock

protein _{pdp crtW yedY yedZ}

Putative lipoprotein Histidine kinase TonB dependent regulator TPR-domain containing protein Sensor histidine kinase Anthranilate phosphoribosyl - transferase Histidine kinase-like

ATPase pdp crtW yedY yedZ

Putative mmb Carbohydrate binding protein RND transporter caax amino terminal protease a) b) crtWoperon crtWgene cluster

produced by the bacterium (Figure 4.8). The operon is however located in a different place to the one from M. xanthus. It is not immediately preceded by a heat shock protein transcribed in the opposite direction, thus ruling out a link between this gene and the operon. This includes the possibility of a promoter region for the operon being encoded in part of the gene region. At the opposite end of the operon two different genes are encoded after it, transcribed in the opposite direction which is also observed inM. xanthus. These appear to encode a putative lipoprotein and a histidine kinase. In M. xanthusthecrtW operon is followed by a series of unlinked genes that are not found in St. aurantiaca. High conservation of the gene sequences in St. aurantiaca and M. xanthus when the surrounding genes are altered further indicates that the four genes form an operon.

4.3. Discussion

By repeating initial gene comparison studies on NCBI BLAST, results indicate M. xanthus crtW is most similar to other genes encoding a CrtW carotenoid ketolase. The two genes downstream of crtW in the crtW operon, previously annotated asordandmmb, encode a periplasmic molybdopterin and haeme-dependent oxidoreductase, YedYZ. These are intrinsically linked in a host of bacterial organisms, as they are in the operon. The remaining operon gene, pdp, also appears to encode a protein linked to the MutT/Nudix family. In this respect it is likely the enzyme plays a role within the cell as a hydrolase, involved in the formation or destruction of a cell substrate. Principally, with the exception of M. xanthus andSt. aurantiaca, the putative MutT/Nudix gene,crtWand theyedYZpair were never found transcribed together in any of the species identified during NCBI BLAST analysis.

EMBL STRING studies also indicate that none of the operon genes are usually found linked together in any other species, with the exception ofyedYZ. SimilarcrtW gene sequences are however identified usually linked to other carotenoid biosynthetic enzymes. The search provided no additional information on the MutT/Nudix protein as there were not enough matches identified in the EMBL database. In contrast the strength of theyedYandyedZlink was indicated by large linkage values for the two in a number of bacterial species. For thecrtWgene in particular links were identified in a number of organisms to genes that encoded ribosomal subunits and transport proteins, although again there is no evidence for similar linkage in M. xanthus.

Conversely, the yedY and yedZ homologues displayed no genetic linkage to crtW in any bacteria other than M. xanthus and St. aurantiaca. Overall the STRING results suggest an increased number of roles for each of the genes encoded by the crtW operon and that the gene arrangement in the crtW operon is a novel bacterial arrangement.

Further study of carotenoid producing organisms suggests that the biosynthetic enzyme encoding genes are usually found located together in a cluster. However in the case of M. xanthusthere is a major cluster and two separate genes,crtIandcrtW. This is unusual as crtI is an essential carotenoid biosynthetic gene, found clustered with the other essential biosynthetic genes in the majority of carotenoid-producing bacteria. When crtW is present in a larger gene cluster it is sometimes found associated with a hydroxylase gene, crtZ, but more usually it is either crtW or crtZ alone. Joint activity of the two can give rise to a carotenoid that possesses both a ketone and a hydroxyl group. This is highly stable and very effective at dealing with a number of reactive, cell damaging reactive species. InM. xanthus crtWis not linked to acrtZ, and nowhere in the genome is a carotenoid hydroxylase encoded. More in

depth study of the effects of ketonisation and hydroxylation on a β-carotene substrate

indicates that in most cases the former produces a carotenoid that is more effective at quenching reactive species. The primary exception to this rule is when negating the effect of reactive peroxyl species, where a solely hydroxylated carotenoid is far more effective.

The evolutionary relationship between M. xanthus and other carotenoid producing species is found to vary depending on which gene sequences are being compared. By initially comparing thecrtYsequence it was clear that the phylogenetic comparisons were often intermingled. When repeating the process withcrtWand also crtB, similar organisms grouped together once more, although M. xanthus is paired with different individuals in each case. Overall comparison of the biosynthetic gene sequences revealed very little phylogenetic correlation. Considering both the phylogenetic results and the data obtained from direct comparison of additional biosynthetic enzymes involved in M. xanthus carotenogenesis, it is implied that the genes responsible for their creation have originated from a number of sources prior to M. xanthus acquisition. The obtained results show a distinct difference between the origin of the essential genescrtE,crtBandcrtI, semi-essential genecrtY, and the non- essentialcrtgenecrtW, ofM. xanthus.

The bioinformatic work suggests that with the exception of Stigmatella aurantiaca the four operon genes are not found located together in any other organisms. The MutT/Nudix protein is not even encoded in most of the organisms that contain homologues of the other three genes. As a result it is unclear the role that the operon plays inM. xanthus, although a number of separate roles for the genes and the pathways in which they function have been suggested. No evidence as of yet indicates a benefit of possessing thecrtWoperon and the four genes being grouped.

Chapter 5