Modernización de las instituciones de P.I

Sigma factors are key regulators of bacterial transcription, able to recruit the RNA polymerase core enzyme to relevant promoter sequences. Over twenty years ago, a new sigma factor was purified from E. coli RNA polymerase and named σ^E or σ²⁴ (Erickson and Gross 1989). The subsequent discovery of homologues in other bacteria including the σ^E of S. coelicolor revealed that these sigma factors form a distinctive subfamily within the σ⁷⁰ family (Lonetto et al. 1994). These sigma factors were found to be involved in the response to cell envelope or oxidative stress as well as the regulation of iron uptake, development and virulence, so were named the extracytoplasmic function (ECF) sigma factors (Missiakas and Raina 1998). Members of the σ⁷⁰ family have four regions that are generally conserved, of which ECF sigma factors share similarity across three. Region 1 ensures that the sigma factor will only bind the promoter when it is complexed with core RNA polymerase, but is only found in some ECF sigma factors (Lonetto et al. 1994). pspX encodes a 185 amino acid protein containing the conserved domain pfam04542, part of the cl08419 superfamily. Pfam04542 corresponds to the most conserved region of the σ⁷⁰ class, region 2. This contains the primary core RNA polymerase binding determinant (region 2.1), which interacts with the clamp domain of beta prime, the largest polymerase subunit (Malhotra et al. 1996). Region 2.3 includes four aromatic residues involved in promoter melting, while region 2.4 contains the -10 sequence recognition determinants (Lonetto et al. 1994). Region 3 is not present in ECF sigma factors, with the consequence that these proteins are often shorter than others in the σ⁷⁰ family (Lonetto et al. 1994).

Region 4 contains a helix-turn-helix DNA binding motif that recognises the -35 element of promoters (Campbell et al. 2002). In accordance with this, all sequences with a significant alignment to PspX are annotated as RNA polymerase sigma factors of either the σ⁷⁰ family or more specifically the σ²⁴/σ^E extracytoplasmic function (ECF) subfamily.

The two protein sequences bearing most similarity to PspX are ECF sigma factors MibX from M. corallina (58 % identity end-to-end) and Tbis_3432 from T. bispora DSM 43833 (56 % identity end-to-end) (Table 5.1). Five further proteins from actinomycetes make up the top seven sequences with most similarity to PspX. These are ECF sigma factors Franean1_3991 from Frankia sp. EAN1pec (52 % over 163 residues), Sros_1172 from S.

182 roseum DSM 43021 (49 % over 168 residues), ZP_06162157.1 from Actinomyces sp. oral

taxon 848 str. F0332 (43 % over 176 residues), Blon_1401 from B. longum subsp. infantis ATCC 15697 (42 % over 170 residues) and Bbr_1068 from B. breve UCC2003 (40 % over 170 residues). Slightly less conserved was Elen_1454 from E. lenta DSM 2243 (42 % over 161 residues) and CAI_78578 from an uncultured candidate division OP8 bacterium (34 % over 183 residues). All nine of these PspX-like proteins are encoded in gene clusters containing minimally lanXWJYZ (Figure 5.5).

A couple of years ago, Hidden-Markov Model analysis grouped 1735 ECF proteins into 43 major phylogenetically distinct groups on the basis of sequence similarity (Staron et al.

2009). The associated web tool, ECFfinder, was used to classify PspX into one of the groups. The best match had a score of 184.1 to group ECF01, a group of RpoE-like ECF sigma factors from nine different phyla. In contrast, it had previously been noted that group ECF33 contained three sigma factors with sequence similarity to MibX (Foulston 2010). These three proteins also shared a significant percentage identity with PspX.

These were BBta_3011 from Bradyrhizobium sp. BTAi1 (37 % over 184 residues), M115118 from Mesorhizobium loti MAFF303099 (38 % over 174 residues) and Rpal_2022 from Rhodopseudomonas palustris TIE-1 (35 % across 175 residues).

5.4.2 PspW

Very few sequences in the current NCBI database produce a significant alignment with the 314 residue protein PspW. The two most similar sequences are the hypothetical protein Tbis_3433 from T. bispora DSM 43833 (43 % over 201 residues) and putative anti-sigma factor MibW from M. corallina (38 % over 201 residues) (Table 5.1). Of the total of nine hits, four more are of particular interest. These are the hypothetical proteins Sros_1171 from S. roseum DSM 43021 (31 % over 210 residues), Krac_2613 and Krac_2809 from Ktedonobacter racemifer DSM 44963 (30 % over 142 residues and 21 % over 156 residues, respectively) and CAI78580 from uncultured candidate division OP8 bacterium (26 % over 170 residues). In all six cases the PspW-like protein is encoded downstream of a gene previously identified to encode a PspX-like protein (Figure 5.5).

Subsequently, these PspW-like proteins were also used as a query in a BLASTP search against the NCBI database to reveal more PspW-like proteins. Sros_1171 from S. roseum DSM 43021, ZP_06162158 from Actinomyces sp. oral taxon 848 and Franean1_3992 from Frankia sp. EAN1pec were shown to have significant similarity to MibW. Proteins sharing a high percentage identity to ZP_06162159 from Actinomyces sp. oral taxon 848 include E. lenta DSM 2243 Elen_1456, B. breve UCC2003 Bbr_1070 and B. longum

183 subsp. infantis ATCC 15697 Blon_1403. These results have been incorporated into the

schematic in Figure 5.5.

The location of pspW within the psp cluster gives a clue as to its possible function. pspW appears translationally coupled to the ECF sigma factor pspX. It has been noted that both groups linked to PspX, ECF01 and ECF33, contain characteristic membrane-anchored anti-σ factors (Staron et al. 2009). In accordance with this, TMHMM v.2 was used to predict the number of TM helices in each of these proteins (Krogh et al. 2001). PspW, Sros_1171, Krac_2613 and CAI78580.1 were predicted to have five TM helices, whereas Tbis_3433, MibW, Krac_2809, ZP_06162158 and Franean1_3992 were all predicted to have six (Figure 5.8). All nine proteins had at least 60 amino acids at the N-terminus that did not form part of a helix. The C-terminus of all nine proteins is cytoplasmic (22 residues in PspW) and could serve to bind the cognate sigma factor encoded by the neighbouring gene, sequestering it away from the RNA polymerase sigma factor. The proteins with six TM helices also have a cytoplasmic N-terminus, whereas those with only five TM helices have an extracellular N-terminus. However this interpretation relies on all TM helices being predicted successfully. The six TM helices of MibW are predicted to occur within the region from 74-244 residues, which coincides with the region showing significant similarity to PspW, implying PspW may instead form six TM helices. A comparison of 17 programs that can identify transmembrane regions concluded that TMHMM v2.0 was the most effective (Moller et al. 2001). However many other programs are available. The preferred model from TMpred predicted six TM helices in PspW with a cytoplasmic N-terminus (Hofmann and Stoffel 1993). Five helices were the same as predicted by TMHMM, but an additional helix was also predicted between residues 106 to 133 with the consequence that the N-terminus becomes cytoplasmic. Therefore it is more likely that the longer cytoplasmic N-termini serve to sequester the cognate sigma factor, but in any case it is likely that the sigma factor binding domain will be positioned at the same location in all PspW-like anti-sigma factors.

An alignment of all nine amino acid sequences did not indicate any conserved motifs that could be involved in interacting with the ECF sigma factors. The anti-sigma factor domain (ASD) was initially identified in the N-terminus of Rhodobacter sphaerorides ChrR and E.

coli RseA (Campbell et al. 2007). The ASD consensus represents a structural fold conserved in an estimated 33 % of anti-sigma factors. The ASD is involved in contacting the cognate ECF sigma factor in an interaction which is mediated in response to specific environmental signals by a C-terminal domain (Campbell et al. 2007). However no such motif was identified in PspW-like proteins.

184

Figure 5.8 : A schematic showing the transmembrane helices predicted to occur in PspW and eight PspW-like proteins.

Namely Tbis_3433, MibW, Sros_1171, Krac_2613, Krac_2809, CAI78580.1, ZP_06162158 and Franean1_3992. Predictions were made using the full length protein sequences submitted to TMHMM (Krogh et al. 2001). Transmembrane helices are illustrated in red, cytoplasmic loops in blue and external loops in pink.

185 Although the membrane topology of PspW is not fully defined, it is likely that PspW does

function to regulate the activity of PspX. The membrane location of PspW means that on binding PspX, it could sequester the sigma factor away from its promoter binding sites thus preventing transcription initiation. In accordance with characterised systems, an extracellular signal (such as cell wall stress) would then be detected either directly by the anti-sigma factor or be mediated through an additional protein, allowing the release of the sigma factor and expression of the relevant regulon. In group ECF01, which PspX bears most similarity to, the best characterised example is σ^W from B. subtilis (Staron et al.

2009). σ^W is regulated by the anti-sigma factor RsiW which is in turn regulated through proteolytic degradation (Schobel et al. 2004; Zellmeier et al. 2006). Indeed proteolytic degradation is a common mechanism for the release of the sigma factor upon receipt of the appropriate signal (Heinrich and Wiegert 2009). Alternatively, a conformational change can regulate the binding of the anti-sigma factor to its cognate sigma factor, such as with S. coelicolor σ^R. Here regulation occurs through a conformational change in the anti-sigma factor, the zinc metalloprotein RsrA, due to a thiol-disulphide redox switch (Paget et al. 2001). In addition, PspX activity could be regulated at the level of transcription or by the protoeolytic processing of a pro-σ-factor. Examples of both of these mechanisms have been characterised in S. coelicolor. Transcription of sigE is regulated by the CseB/CseC two-component system (Hutchings et al. 2006). σ^BldN is synthesised as an inactive pro-σ factor which is subject to proteolytic processing to release active BldN (Bibb et al. 2000).

5.4.3 PspR

The 260 amino acid PspR contains the conserved domain pfam cd06170 (part of the superfamily cl10457) between residues 190 to 250. This corresponds to a helix-turn-helix motif responsible for DNA binding. Proteins belonging to this group include the LuxR-like transcriptional regulators. Within this family, there are both transcriptional activators and repressors. In Vibrio fisheri, N-acyl derivatives of homoserine lactone act as signalling molecules which bind to the N-terminal domain of LuxR, resulting in dimerisation and activation of gene expression through quorum-sensing (Stevens et al. 1994). In contrast, in Sinorhizobium meliloti it is phophorylation of the N-terminal domain of FixJ which leads to multimerisation and the subsequent transcriptional activation of nitrogen fixation genes (Da Re et al. 1999). In PspR, there is an N-terminal domain which may be modified to modulate the DNA binding property of the C-terminal LuxR-like DNA binding domain.

PspR bears most similarity to MibR, the putative DNA-binding protein from M. corallina (33 % identity over 205 residues) (Table 5.1). Other proteins with similarity to PspR come from a range of organisms. The two-component response regulator YP_075969 from

186 Symbiobacterium thermophilum IAM 14863 shares 38 % identity over 82 residues. S.

thermophilum depends on microbial commensalism so is currently uncultivable under laboratory conditions. The 3.57 Mb genome has been sequenced, revealing a GC content of 68.7 %, yet the genome bears most similarity to those in the Firmicutes phylum (Ueda et al. 2004). Aside from these two proteins, a majority of other alignments show similarity only within the putative helix-turn-helix domain of the protein. Thus PspR is a putative transcriptional regulator of the psp gene cluster.

5.5 Unknown proteins