Additional key proteins

As CarQ directly controls CarS production, it is effectively the principle protein responsible for the control of carotenoid synthesis. This is supported by the fact that it also plays a role in the promotion of the separatecrtIgene (Ruiz-Vazquez

et al., 1993). crtIencodes the biosynthetic enzyme phytoene dehydrogenase,which is

responsible for two steps during primary carotenoid production (Figures 1.7a and 1.7b). In vitrostudies of transcription activation have found that CarQ is incapable of

crtI transcription alone, and the entire process also requires the host cells to have

undergone a period of carbon starvation before it can occur (Browning et al., 2003).

It is hypothesised that additional regulator and catabolites and possibly enhancer sequences, may be involved incrtIcontrol (personal communication, F. J. Murilllo).

An additional carotenoid protein, CarD, is also required for the successful transcription of both crtI and carQRS (Nicolas et al., 1996). Although the mode of

action is still currently not fully understood, preliminary studies indicate that it functions as a transcription factor, essential in the activation of carotenogenesis. Besides its role in the reaction to blue light, it also plays a role in the cellular response to nutrient starvation, which may in part explain its involvement in crtI transcription

(Galbis-Martínez et al., 2004). It is the only known protein that is involved in both

fruiting body formation and carotenogenesis in M. xanthus (Nicolaset al., 1994). A

similar homologue has also been detected in the closely related Stigmatella

aurantiaca(Cayuelaet al., 2003).

CarG is another carotenogenesis transcription factor, which is zinc- associated, suggesting it plays some sort of structural role. The protein is unable to bind directly to DNA, but forms a reaction complex with CarD (Peñalver-Mellado et

al., 2006) Data indicate that when either of the two proteins is present in a cellular

reaction then so is the other, and that binding between the two involves the N- terminus of CarD. The 1:1 ratio bind ratio observed for the two proteins in the cell is similar to that recorded for CarA and CarS, and it has been shown that CarD binding with CarG is essential for either to function effectively as a transcription factor.

Aside from the principal proteins controlling carotenogenesis, a number of additional elements have also been recorded to have a direct effect on the efficiency of the process. The best example of this is observed when increased levels of copper are present in the bacterial growth media, resulting in the stimulation of carotenoid synthesis (Moraleda-Munoz et al., 2005). Copper, particularly in the Cu2+ form, is

known to generate oxidising agents, hence the cellular response (Valko et al., 2005).

Studies indicate that each of the known genes involved directly in carotenogenesis reacts to the presence of copper ions, with the sole exception of carF. This suggests

that CarF is designed to specifically react to blue light and no other external stimuli. In turn this means that the copper ions, either directly or through an entirely different protein cascade, disrupt the CarR-CarQ complex in an alternative mechanism to carotenogenesis, still resulting in carotenoid formation.

1.7. CrtW

The final stage in the production of myxobacton, the more prevalent of the carotenoid esters in M. xanthus, is a ketonisation step. The enzyme believed to be

responsible for this is a β-carotene ketolase. Similar enzymes that function at the

equivalent stage of carotenogenesis have already been identified in a range of other bacteria, including Bradyrhizobium species (Hannibal et al., 2000), Synechocystis

species (Fernández-González et al., 1997), Synechococcus species (Albrecht et al.,

2001) and Brevundimonas species (Tao et al., 2006). They are usually annotated as

either CrtO or CrtW, with the primary differences between the two ketolases being their lack of sequence similarity with each other and the former being generally twice the size of the latter (Takaichi and Mochimaru, 2007). Despite the final step in carotenoid production appearing to be a ketonisation, no candidate gene or enzyme for this function had previously been identified inM. xanthus.

Following preliminary analysis of the M. xanthus DK1622 genome sequence

(D. Whitworth, unpublished), a candidate ketolase-encoding gene was identified. This was achieved through scanning the entire genome for potential open reading frames and identifying any homologues through online genome database searches. The product of one such gene, situated approximately 1.45kb from the M. xanthus

origin of replication, displayed significant sequence similarity with the product of a crtW gene previously identified in Gloeobacter violaceus; a cyanobacterium and

carotenoid producer (Steiger et al., 2005). The enzyme encoded by the gene was

responsible for part of the beta-carotene production pathway, functioning as a ketolase and was designated CrtW.

The preliminary sequence analysis of M. xanthus crtW (as the gene shall be

referred to henceforth) and its associated flanking regions also identified three additional potential genes situated nearby, with the closest two less than 100bp either side of the sequence (Figure 1.10). Following further sequence analysis, the products of the pair of genes immediately downstream of crtW were identified as a putative

oxidoreductase (product of ord) and an integral, inner membrane protein (product of

mmb) respectively. Upstream of crtW a possible putative dipeptidylphosphatase

encoding gene (pdp) was identified. In addition to the three genes, a gene encoding a

potential heat-shock protein appeared to be located immediately upstream of the pdp

gene, but transcribed in the opposite orientation to the pdp crtW ord mmb cluster.

Sequence analysis suggests this encodes HtpG, a protein believed to be involved in the biosynthesis of tetrapyrroles in a range of cyanobacteria (Watanabe et al., 2006).

Given the close proximity of the pdp, ord and mmb sequences to crtW, and the fact

that all four of the genes are transcribed in the same orientation, it appears possible that they were arranged in an operon.

7484620 CGTCCACGCCAGCGGCGCTTCCCAGTCGTGCGTCAGGTGCTTGTAGAACTCCTGGTACTGCTCGTCCGTGATTTC GGACTTGGAGCGCTGCCACAGGGCGCTGGCCTTGTTGACGACCTCCAACGACGTCTCCGTCTTCGCCTCGTCGCCGGTCCCCGTCG TCTTGCTCACCTGGAGCTTGATGGGGTGGCCCACGTAGTCGGAGTACTGCGTGATGAGCGACCGCAGCCGCCACTCGCCCAGGAAC TCCTTCTGGTCCTCCTTCAGGTGCAGGGTGATGGAGGTGCCGCGCGCGGCGCGCTCGGCGGGCTCCACCGTGAAGGAGCCCTTGGC TTCCGACGTCCACCGCCAGGCGGACTGGCCCTGGCCGGCGGCGCGGCTGACCACCTCCACGCGGTCCGCGACCAGATAGGCGCTGT AGAAGCCCACGCCGAACTGGCCGATGAGCTGCATGTCCTTCTGCTGGCCCTTCTGCGCCAGCGCCTCGATGAACTCGCGCGAGCCG GAGTGGGCGATGGTGCCCAGGTTCTTCACCAGCTCGTCATGCGACATGCCGATGCCGGTGTCCTCGATGGTGAGGGTGCCCTTCGC CTCGTCCGGGATGAGGCGCAGCTCCAGGGCCGGCTCGTCCGCCAGCAACTCCGGCTCCGTAATCGCGCGGAACCGGAGCTTGTCGA GCGCGTCGGACGCGTTGGACACCAGCTCGCGGAGAAAAATCTCCTTGTGGCTGTAGAGCGAATTGATGACCAGGCTGAGGAGCTGA TTGATTTCCGCCTGGAATGCGTGGGTCTCCCGCTGGGGGGCGTTTTCGACGGTCATGGTGACAGGGGCCTCCGGGGCGCCCGGTGG GGCGCCAGCGTCGCTTTCCCTTAACCATGGAAAACCGCTTGTCGAGTCCGGACTGTCCGCCCGCCCGCTCAAGGGCGGGTGTGCTG GTGGTCCATTCCGGGTGCGCGCGAATGAGGGGGAAATGTGGGACGTTGGGTTTTCAGATGTGAGGACCCCTGGGAGGGTGGGCCCG GGTTGGAGTAGGGAAGGGGACATGAACCGTACCCTCCTGTCCCTGCTCGGCGCGGCGATGCTGTCGGGCGCCGCTACCGCCGCGGA GAAGACGCCCCCTGCCCGTTTCCCGGATGCCGCGGAGCTCCAGCGCCTGACGGCGCGCTTCGCGCCGGTGGAGCTCCGGGTGGACC TGAAGGCGCTGCCGGAGTCCGAGCGCCGCGCCCTGGCCCGCATTGTCCAGGCCTCGAAGCTGATGGACACGCTCTTCCTGCGTCAG CGGTGGGCGGGCAACGAGCCGCTGCTGCTGGACCTGGTCCAGGACACGACGCCGCTGGGCCGCGCGCGGCTGCAGGCGTTCCTGTT GGACAAGGGGCCCTGGAACAGCCTCGACGAGGCGCGGCCCTTCATCCCGGGCGTGCCGGCCAAGCCCGCGTCCGCGAACTTCTATC CGGCTGGCGCCACCCAGGCGGAGGTGGAGGCGTGGGTGAAGTCGCTGCCCGAGGCGCAGCAGAAGGAGGCCACCGGCTTCTACACC ACCATCCGCCGCGGCACGGATGGCCGCTTCATCACGGTGCCCTACAGCGTGGAGTACCAGGGCGAGCTGGCCCTGGCCGCCGCGTT GCTGCGTGAGGCCGCTGCGCTCACCCAGCAGCCCACGCTGAAGGCGTTCCTCACCTCGCGCGCGGACGCGTTCCTGTCCAACGACT ACTACGCCAGCGAGGTGGCGTGGATGGAGCTGGACGCCAGCATCGAGCCCACCATCGGGCCCTACGAGGTCTACGAGGACGAGTGG TTCAACTACAAGGCCGCCTTCGAGGCCTTCGTGGGCCTGCGCGACGACGCGGAGACGCAGAAGCTGGCGAAGTTCAGCGGGCAGCT CCAGGGGCTGGAGAACAACCTCCCCATCGACCCGAAGCTGCGCAACCCGAAGCTGGGCGCGCTGGCGCCCATCCGCGTCATCAACA GCCTGTTCTCCTCCGGTGACGGCAACCGGGGCGTGCAGACGGCCGCCTTCAACCTGCCCAACGACGAGCGGGTGTCGGAGAAGATG GGCTCCAAGCGCGTGATGCTGAAGAACGTGCAGGAGGCCAAGTTCGAGCGCGTGCTGCTGCCCATCGCCAAGGTGGCCCTCACCCC GGCGGACCAGAAGGACGTCTCCTTCGATGCCTTCTTCACGCACATCTTGATGCATGAGCTGATGCACGGCCTGGGGCCCAGCAACA TCACCGTGGGTGGCAAGGCCACCACCGTGCGCAAGGAGCTCCAGTCGGCCTCCAGCGCCATCGAAGAGGCGAAGGCGGACATCTCC GGCCTGTGGGCGCTCCAGCGCCTGGTGGACACCGGCGTCATCGACAAGTCGCTGGAGCGCACCATGTACACGACGTTCCTGGCCTC CGCCTTCCGCTCCATCCGCTTCGGCGTGGACGAGGCGCACGGCAAGGGCATCGCCGTGCAGCTCAACTACTTCCTGGACACCGGCG CGGTGAAGGTGAACGCGGACGGCACTTTCTCCGTGGTGCCGGCGAAGATGAAGAAGGCCGTCATCTCGCTGACGAAGCAGCTCATG GAGATTCAGGGCCGCGGCGACCGGAAGGCCGCCGAGGCGCTGCTGGCGAAGCTCGGCGTGGTGCGCCCGCCCGTGCAGCGCGTGCT GGAGCGCCTCAAGGACGTGCCGGTGGACATCGAGCCGCGCTACGTCACCGCGGAGGAGCTGGTACGCGACGTGAAGAAGTAGCCAC CGGCCCATTGCCCCGCCCCGCCGCCTGGGTATTTCCCATGCGCGGCGGGCCGGCGATGGCCAGAAAGCAGGTGAGATGGAGACTTC CGCCCGCCAACTCCGTCCAGCGCCGCCCGGTCCCTGGGGCGTCGTCATCGCGCTCATCATCATGGGCGCGTGGGGTGGGCACCTTG CCTGGGCGCTGACACGAGCGGAGCTGCCGTGGGTGGAGCCGCTCACCTGGCTGCACGTCGCTCTCCAGGCCTGGCTGTGCACGGGC CTCTTCATCACCGGCCACGACGCCATGCACGGCACGGTGTCCGGCCGGCGCTGGGTGAACGAGGCCGTGGGCACGGTCGCCTGCTT CCTCTTCGCGGGGCTGTCCTACCGTCGGCTGGTGGTGAACCACCGTGCCCACCATGCCCGACCCACGAGCGACGCGGACCCGGACT TTTCCACCCACAGCCAGTCCTTCTGGCCGTGGCTGGGTACCTTCATGGCCCGCTACACCACGCTGCCCCAGCTTGGGGTGATGGCG GCCAAGTTCAACGTGTTGCTCTTCCTGGGCGTCTCCCAGCCACACATCCTCGGCTATTGGGTGCTGCCCTCGGTGTTGGGCACGTT GCAGCTCTTCTACTTCGGCACCTACCTGCCGCACCGGCGCCCGGAGACGCCGGACATGGCCCCTCACCACGCGCGCACGTTGCCGC GCAATCACCTGTGGGCCCTGCTGTCGTGCTTCTTCTTCGGCTACCACTGGGAGCACCACGAGTCCCCGGGCACACCCTGGTGGCGG CTGTGGCGCCTGAAGGACGCCCGGGCCCGTGAGGCCGCGCTGACGCAGAGCACCGGGACGCTCCCGGGACAGGAAGGCACCGCCCG GTAACGCGCGGGCGGCCCGGGCCGTTATACAGGGGCCATGCGCGACAAGCCGCCCGCTGAGCCGCCCAGCTCCGAAGTCACCCCCG AGAAGACGTACCTGCGCCGGCGCGAGCTGCTGAAGAACGCGGGCCTCTTCGCGGGCACGGCCGTCGCCGTCGCCGGAGGGCTGCAC CTGCTGGGCCGCAAGCAGACGCGCCCCATGGAGCGCTTCGTCCCGGACGCGGGCCTGGTGGAGCAACCGGTGGCGCAGGCGATGGG CCCCTTCGACACGGACGAGCCGCGCACGCCCTACGAGGACGTCACCACCTACAACAACTTCTACGAGTTCGGCTTCGACAAGAACG ACCCGGCCCGCTTCGCGCACACGCTGAAGCCGAAGCCGTGGAGCGTCGTCATCGACGGCGAGGTGCATAAACCGCGGACGGTGGAC GTGGAGCAGCTCACGTCCTGGTTCTCCCTGGAGGAGCGCGTCTACCGCATGCGCTGCGTGGAGGCCTGGTCCATGGTGATTCCGTG GCTGGGCTTCCCGCTGGCGGCGCTGCTCCAGCGCGTGGAGCCCACCAGCCATGCGAAGTACGTCGCCTTCACCACGCTGCTGGACC CGGAGCAGATGCCGGGCCAGCGCCGCGCCCTGTTGGATTGGCCGTACACGGAAGGACTGCGCCTGGACGAGGCGATGAACCCGCTC ACGCTGCTGGCCACGGGGCTCTACGGCCGGCAACTGCCCAACCAGAACGGCGCGCCGCTGCGGCTCGTGGCTCCTTGGAAGTATGG ATTCAAGGGCATCAAGTCCATTGTCCGCATCAGCCTCACGCGGGAGGAGCCCATGACGACGTGGCGCCTGTCCGCGCCGCGCGAGT ATGGCTTCTACGCCAACGTGAATCCTTCCGTGCCCCATCCGCGCTGGAGCCAGGCCAGCGAGCGCCGCATCGGCGACTTCGAGCGC CGCCCCACGCTGCCCTTCAATGGCTACGCGGAGCAGGTGGCCCACCTCTACACCGGCATGGACCTGCGCCGGTTCTACTGAGTCCC CGCGCCATGGCCTCGTCTCCGTATCCCTGGCTCAACCCCGCGCTCGTCGTGGGTGGCCTGTCGCCGCTGCTGATGCTCGCCGTCCA GGGGCCCCGGGGCGAGCTGGGGCCCAACGCGATTGAAGCCGCGCTCCACCAGACGGGGCTGCTGACGCTGGTGCTGCTGGTGGCCT CGCTGACGTGCACGCCGCTGCGGCTGGTGGCGGGGTGGACGTGGCCCGCGCGCGTGCGCCGCACCCTGGGCCTCTTGGCCTTCACC TACGCGGTGGCGCACTTCCTCGTGTACGCGGTGCTGGACCAGGGGCTGGCGTGGGGCGCGCTGTGGGCGGACGTCACCGAGCGCCC CTTCATCACCGTGGGCTTCGCCGCGCTGGTGCTGCTGGTGCCCCTGGCCGTGACGTCGACGAACCGGTGGGTGCGGCGGCTGGGCT TTCCACGCTGGCAGCGCCTGCACCGGCTGGCCTATGGGGCCGCGGCGCTGGGCGTGGTGCACTTCGTGTGGCGCGTGAAGAAGGAC GTCACCGAGCCGCTCATCTACGGCGCGGTGCTGGCGCTCCTGATGGCCATTCGCGTGGGTGAAGCCATGCGAAAACGCCGGGCCCG CGCCGCTGCGGCGGCCCGGAACCCGGCGTGAGTGAAGCGGTGCCCAGACGGGCGCGGCGCGCTACTTGCGCGGCAGCGGGAGGATG GTGTCCACCAGCGTCATGAGCTGCGCGCAGCTCACCGGCTTGCGGACGAAGGCGCTGATGCCGGCCTTCTGACCCAGCGCGCGCAC CTCCGCCACGTTCGGGTCGCCCGT 7490019

Green: Heat shock protein Red: pdp

Orange: crtW Blue: Oxidoreductase (ord)

Rose: Transmembrane protein (mmb) Bold bases: Start/stop codons

Bold and underlined bases: Possible CarQ dependent promoter P1

Bold and underlined bases: Possible CarQ dependent promoter P2

Figure 1.10The DNA sequence of the putativecrtWoperon, with gene regions labelled. The numbers represent sequence location in theM. xanthusgenome.