To identify candidate genes for the E. coli biotin transporter, homology searches with genes encoding biotin transport systems from other organisms like SMVT from mammals, VHT1 from S. cerevisae, vht1+ from Sz. pombe in the E. coli genome were performed. Searches did not lead to high-scoring homologues.
For modular transport systems that were identified among many species in gram-positive bacteria and archea no homologues in the subdivisions of β-, γ-, δ- and ǫ-proteobacteria (including E. coli) could be found [157]. Homology search with B.
subtilis bioY inE. coli did not lead to genes with high homology. In conclusion this results suggested, E. coli must contain a new transport system for biotin.
3.1.1 Candidate genes
Eisenberg et al. were able to generate four classes ofE. coli mutants resistant to α-dehydrobiotin, a toxic structure analogon of biotin via random mutagenesis with nitrosoguanidine [55]. One group showed derepressed levels of the biotin biosynthetic enzymes, the second one showed lesions in the bio-operon and a third one general permeability defects and weak growth. The fourth one was affected in biotin uptake and desiganted bioP. Mapping by P1-phage transduction experiments revealed the bioP-mutation lying at 75 min on the E. coli chromosome between the ilv-operon and the metE-gene, but much closer to metE [55]. So a search for genes encoding proteins with at least 10 predicted transmembrane-domains as candiadate-genes for the biotin-transport protein in theilv-metE region was carried out. Transmembrane domains were predicted by TOPCONS. As shown in fig. 3.1 and tab. 3.1 three candidate genes were identified.
To analyze if these candidate genes have any influence on biotin transport, uptake experiments with [14]C-labeled biotin and knockout-mutants for the three mentioned genes and a corresponding wt-strain were performed. In this experiment yifK∆and
56
Figure 3.1: Region between ilvG and metE on the E. coli chromosome.
Open reading frames between ilvG and metE. Candiate genes for the biotin transport protein with 10 or 12 TMDs are marked with a red box.
gene name TMDs annotation on ECOCYC
yifK 10 uncharacterized member of the APC superfamily of amino acid transporters
rarD 10 predicted chloramphenicol resistance permease yigM 10 putative uncharacterized transport protein
Table 3.1: Candidate ORFs for the E. coli biotin transporter. Candidate genes from theilv-metE region with their predicted TMDs and annotated predicted gene functions from the ECOCYC-database (www.ecocyc.org) rarD∆showed similar uptake to the wildtype, whereas biotin uptake inyigM∆, that lies closest to metE, was abolished (see fig. 3.2).
0 0,5 1 1,5 2 2,5 3
0 0,5 1 1,5 2 2,5 3
time [min]
pmolbiotin/OD600
wt yifKD
rarDD
yigMD
Figure 3.2: Uptake experiments with E. coli wt cells and single knockout mutants of the candidate genes. Biotin uptake inE. coli wt, yifK∆, rarD∆ and yigM∆ mutants cells has been measured. The experiments were performed in 50 mM KPi-buffer pH 6.0 with 10 mM MgCl2and with 100 nM [14]C-biotin. Tests were started by adding biotin and aliquots taken every 30 seconds for 3 min. The mean values of three independent experiments are shown. Bars indicate standard deviations. Mutants were purchased from Coli Genetic Stock Center (CGSC).
3.1.2 In silico analysis of YigM
The YigM-protein consists of 299 amino acids with a molecular weight of 33727 Da. The 12 negatively and 20 positively charged amino acids result in an isoelectric point of 9.64. 211 of the 299, representing 71 % of all amino acids are part of the 10 predicted transmembrane domains. YigM C- and N-terminus are predicted to be located in the cytoplasm (see fig. 3.3) and the protein contains only very small hydrophilic streches. No similarity of YigM to other known biotin transport-proteins were found. There are no motifs that could give hints to the function of the protein. One domain that can be found is a domain of unknown function (DUF6) and the protein can be classified into the subfamiliy of carboxylate/amino acid/amine transporters (CAAT family TC 2.A.78) [172]. CAATs are one of ten subfamilies belonging to the family of secondary amino acid transporters found exclusively in bacteria [172]. Members of the CAAT family represent integral membrane proteins with sizes from 287 to 310 amino acids and 10 putative TMDs. CAAT proteins can be found in phylogenetically divergent bacteria and archaea, e.g. in E. coli, B. subtilis and Aspergillus fulgidus multiple paralogues are present. Representatives of the CAAT family show low degrees of sequence similarity with members of the ubiquitous L-rhamnose transporter (RhaT) family (TC 2.A.9) and with the eukaryotic triose phosphate transporter family (TC 2.A.50) [172].
The CAAT family probably arose from an internal gene-duplication event as the first halves of these proteins are homologous to the second halves. None of these prokaryotic proteins of CAATs is functionally characterized. Nevertheless functions have been ascribed to some members like the MttP protein of the Methanosarcina barkeri that is supposed to transport methylamine [60] and YtfF of Chlamydia tra-chomatis that may transport basic amino acids [182]. Additionally, MadN that is part of the malonate utilization operon ofMalonomonas rubraand was speculated to be an acetate-efflux pump [13], as well as PecM ofErwinia chrysanthemi controlling pectinase, cellulase and blue-pigment production are members of CAATs [155].
Homologous genes ofyigM can be found in differentShigella,Citrobacter,Salmonella, Klebsiella and Yersinia-strains (Fig. 3.4). These are mostly annotated as (inner) membrane or putative transport proteins, but the function of none of them has been experimentally shown.
The yigM-open reading frame can be found as a single gene on the chromosome and is not part of an operon (see fig. 3.1).
Figure 3.3: Predicted transmembrane-domains of yigM. Predictions were made by TOPOCONS (http://topcons.net/), that gives consensus pre-dictions of five single topology prediction programs. Boxes indicate transmembrane regions, red lines predicted cytoplasmic and blue lines predicted periplasmic parts of the protein.
1 1I III 1V
V V1 VI1
VIII IX X
Figure 3.4: Alignment of YigM-homologous proteins from different bacte-ria. Predicted transmembrane domains for E. coli YigM are indicated by boxes with roman numbers. Predictions were made by TOPOCONS (http://topcons.net/).
3.1.3 Immunological detection
As YigM seemed to be the most promising candidate it was cloned into a pBS vec-tor under control of the constitutively active PR-promoter of the phageλ. Versions of YigM with a C-terminal or N-terminal HA-epitope-tag have also been cloned in this vector for detection of the protein on a western-blot (see fig. 3.5). These plasmids were transformed in wt- and yigM∆-strains and uptake experiments performed in intact cells (see fig. 3.6).