The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5′ untranslated mRNA regions (5′-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5′-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5′-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5′-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.
Citation: Accetto T, Avguštin G (2011) Inability of Prevotella bryantii to Form a Functional Shine-Dalgarno Interaction Reflects Unique Evolution of Ribosome Binding Sites in Bacteroidetes. PLoS ONE 6(8): e22914. https://doi.org/10.1371/journal.pone.0022914
Editor: Grzegorz Kudla, University of Edinburgh, United Kingdom
Received: February 22, 2011; Accepted: July 1, 2011; Published: August 12, 2011
Copyright: © 2011 Accetto, Avguštin. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Slovenian Research Agency, research project P4-0097 (http://www.arrs.gov.si/en/dobrodoslica.asp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Shine-Dalgarno (SD) sequence of the prokaryotic mRNA is commonly regarded as a key element involved in the selection of the authentic start codons versus internal AUG codons. It is usually 4–5 bp long, positioned 5–8 bp upstream from the start codon, and basepairs with the complementary sequence (anti SD) at the 3′ end of 16S rRNA of the 30S ribosomal subunit. This interaction also makes possible the translation initiation in the presence of mRNA secondary structures weaker than −6 kcal/mol, while translation initiation rapidly becomes inefficient in the presence of more stable secondary structures in Escherichia coli. –. The SD sequence is found in majority of translation initiation regions of Escherichia coli . It is thought that 4–5 bp Shine-Dalgarno sequence-16S rRNA interaction is usually sufficient since SD sequence lengthening only rarely results in increased translation efficiency and longer, 8 or 10 bp interaction actually inhibits translation , , . It was shown, on the other hand, that E. coli can efficiently initiate translation also in A/U rich translation initiation sites lacking SD sequence altogether  and that translation initiation of leaderless mRNA proceeds without mRNA-16S rRNA interaction . It has recently been observed, as the number of sequenced bacterial genomes increased, that a significant share of genes is not preceded by SD sequence . Further, the share of genes preceded by SD sequence was found to be phylum specific. It was suggested that phyla exhibiting low fractions of genes preceded by SD sequence in their member's genomes rely primarily on the ability of ribosomal protein S1 to mediate translation initiation . The ribosomal protein S1 binds to the A/U rich stretch of mRNA upstream of the start codon and is not universally conserved in bacteria: in Firmicutes which possess the largest fraction of genes preceded by SD sequence, the protein S1 is predicted to be non-functional in translation initiation .
Ribosome binding sites of an organism may be conveniently represented by a sequence logo. Sequence logo primarily describes the information content of individual sites in the sequence alignment. The information content is a measure of sequence conservation at the specific site and is represented by the height of the logo at that position. Relative frequency of bases, for a nucleic acid logo, is described by the height of individual stacked symbols specific for given bases at that site. Sequence logo thus combines different information in one picture and is usually used to display protein binding sites of nucleic acids, and protein motifs .
Prevotella bryantii is a strictly anaerobic gram negative bacterium involved in the degradation of plant cell wall polysaccharides in the rumen of cattle and sheep . It belongs to the large bacterial phylum Bacteroidetes whose representatives inhabit diverse habitats such as fresh and salt water, sediments and animal gastrointestinal tract . The Bacteroidetes are known for their somewhat unique genetic make up. The consensus promoter sequences and their spacing are for example distinct from those known in E. coli , , which is thought to be the consequence of the unusual primary sigma factor . The development of general gene manipulation techniques in Bacteroidetes was therefore rather slow, with the exception of the genus Bacteroides  to a certain extent. Recently we developed a gene expression system for Prevotella bryantii TC1-1 strain based on a shuttle plasmid pRH3. The expression was regulated by transcriptional fusion of the expressed gene with tetQ. Expression was not seen in the absence of tetracycline when nucB, a gene coding for a non-specific nuclease, and 108 bp of its upstream sequence were cloned into pRH3 . The abovementioned studies on translation initiation diversity in bacteria by Chang et al.  and Nakagawa et al.  touched the Bacteroidetes only briefly by analyzing genomes of four species in total. These studies found that the genomes from Bacteroidetes genera Bacteroides, Porphyromonas and Cytophaga contain a low fraction of genes preceded by SD sequence. The translation initiation in Bacteroidetes was predicted to rely on ribosomal protein S1 which should be functional according to sequence analysis . In order to comprehensively analyze the start codon upstream regions in Bacteroidetes we examined almost forty Bacteroidetes genomes which span the wide diversity of this phylum. The Prevotella bryantii TC1-1 expression system was used to examine the effects of sequence manipulation involving the start codon upstream region on NucB protein level. Bioinformatic analysis confirmed that the low proportion of genes preceded by SD sequence is truly a phylum wide trait in Bacteroidetes and also presented the way the translation initiation region evolved in Bacteroidetes presumably to serve in ribosomal protein S1 mediated initiation. Experimental evidence suggests that the ability to form a functional SD-anti SD interaction during translation initiation was lost in Bacteroidetes evolution explaining lack of SD sequence in the majority of genes.
Sequence logos detect SD sequence in the genomes of many prokaryote phyla
The genome wide presence of SD sequence leads to the enrichment of adenine and guanine bases in sequence logo spanning the −5 to −10 bp region relative to the start codon of a gene , . Such enrichment can be clearly found in sequence logos of major bacterial phyla: Proteobacteria, Firmicutes, Actinobacteria, Thermotogae, Chloroflexi, and Aquificae (Fig. 1 A and S2, S6, S9, S10, S12, S14). Notable exception in Proteobacteria are the low GC % parasites or symbionts from the family of Rickettsiales and the Tenericutes from the genus Mycoplasma, which possess only slight enrichment in guanine in SD sequence region (Fig. S10 and S15). The sequence logos of species containing the SD sequence and belonging to the same phylum are not uniform, however. The heterogeneity sometimes reflects the differences in GC content of the species in the absence of drastic logo changes in SD sequence area e.g. Moorella thermoacetica with 55,8% GC and Clostridium acetobutylicum with 30,9% GC genomic content (Fig. S9), but in other cases the overall shape of a logo in SD sequence area is changed e.g. between Bifidobacterium longum and Arthrobacter aurescens (Fig. S14). The SD sequence is less obvious but still detectable in sequence logos of certain phyla e.g. Spirochetes, Acidobacteria, Chlamydiae, Deinococcus-Thermus, Fibrobacteres and some Cyanobacteria (Fig. S1, S3, S7, S8, S11, S13).
A: Sequence logos showing the presence of SD sequence in some well known bacteria. Sequence logos of Bacteroidetes (B) and Chlorobi (C) demonstrate the lack of SD sequence. The position 0 represents the nucleotide position of start codon.
The genomes from the phyla Bacteroidetes and Chlorobi lack detectable SD sequence
When the regions preceding the start codons in almost forty available genomes from the phylum Bacteroidetes, spanning the diversity of this phylum, are examined, no similarity to above mentioned logos of other major phyla is apparent. Instead, the major feature of Bacteroidetes logo is an adenine and thymine enrichment centered at 12–13 bases ahead of the start codon (Fig. 1 B, S5). Chlorobi, however, lack the information almost totally i.e. they have random base composition in regions preceding the start codons. The only recognizable features shared by most Chlorobi species is a slight adenine and thymine enrichment in the region spanning 23–11 bases ahead of the start codon and an enrichment in cytosine and thymine directly before the start codon (Fig. 1 C, S4). The exceptions in Bacteroidetes are Rhodothermus marinus DSM 4252 and Salinibacter ruber DSM 13855 whose logos are aberrant (Fig. S5), the logo of the former more resembling those of Chlorobi.
Sequence logos of regions preceding the start codons in Bacteroidetes are conserved at the genus-family level
The logos of all of the eight sequenced species belonging to genus Bacteroides examined are characterized in addition to abovementioned AT enrichment centered at −13 bp relative to start codon (Fig. 1 B) also by (i) a similar yet smaller enrichment spanning the −8 to −6 region, (ii) A enrichment at −3 position and (iii) T enrichment at -1 (Fig. 1 B, S5). This type of logo is conserved also in all four strains of genus Prevotella, all three strains of genus Parabacteroides, strain of Alistipes, and all four strains of genus Porphyromonas examined, the latter containing very low information content and resembling Chlorobi in this respect. In three strains from the genus Porphyromonas a shift of the AT enrichment center from −13 to −14 (Fig. S5) can be seen. The essential features of the logo excluding its height are thus conserved also at the level of order Bacteroidales encompassing Prevotellaceae, Bacteroidaceae, Porphyromonadaceae and Rikenellaceae. The shape of a logo is, however, not completely conserved e.g. the enrichment centered at −13 bp is not so pronounced in Prevotella as in Bacteroides or is moved as mentioned above. The variation in logo height in genera Bacteroides, Prevotella, Porphyromonas and Parabacteroides can be observed too. The species with lower % GC in the 30 bp preceding the start codons predictably produce higher logos and vice versa (Fig. S5). The Flavobacteriaceae logos differ from those in Bacteroidales in the placement of the main AT enrichment which is centered at −12, except for the Capnocytophaga ochracea DSM 7271, and by the shape of the −8 enrichment (Fig. S5). Cytophagaceae are distinguished by a flat logo, with the enrichment at -13 sometimes barely detectable.
Translation initiation regions of the Bacteroidetes and Chlorobi genomes have low GC ratio in comparison to coding regions
Generally, the species from the Bacteroidetes phylum have genome GC content below 50%. When the 30 bp regions preceding the start codons are examined the GC content decreases by 12,6±2,3% relative to genome GC content (Table S1). This decrease can be observed also in other phyla but is typically around 5% relative to genome GC content with the exception of the Chlorobi with 9,9±1,3% GC decrease (Table S1). The % GC decrease in regions preceding the start codons is conserved also in those rare species from the phylum Bacteroidetes which are characterized by somewhat higher GC genome content. In Robiginitalea biformata for example, which has a 55% GC genome content and belongs to the family Flavobacteriaceae with the predominantly 32–39% GC genome content, a 13% GC decrease can be seen. It is similar in Dyadobacter fermentans and Spirosoma linguale, members of the family Cytophagaceae (Table S1). Again, Rhodothermus marinus and Salinibacter ruber are the exceptions among Bacteroidetes with merely 8,5 and 5,4% GC drop in regions preceding the start codons, respectively.
16S rRNA genes of all the analyzed Bacteroidetes and Chlorobi genomes contain anti SD sequence
All of the 16S rRNA coding Bacteroidetes genes contain the anti SD sequence 5′ACCUCCUU 3′ at the expected position at the 3′ end of the gene (Fig. S16) with the exception of Rhodothermus marinus and Salinibacter ruber, where the last U in the anti SD sequence is replaced by A. It is also apparent from the alignment that Chlorobi and Bacteroidetes 16S rRNA genes, excluding those of Rhodothermus marinus and Salinibacter ruber, contain a deletion of approximately 9 nucleotides in comparison to representative 16S rRNA sequences of other phyla. The deletion is located 80 bp upstream of anti SD sequence in the helix 44 of the 16S rRNA .
The SD sequence does not significantly influence NucB protein level in Prevotella bryantii TC1-1 strain
Since bioinformatic analysis demonstrated a general lack of SD sequence in Bacteroidetes, a question arises whether the SD sequence in the 5′-UTR still has any effect on the translation initiation in these bacteria. To resolve this we used a recently developed gene expression system for Prevotella bryantii TC1-1 strain  which is one of only four systems for gene delivery and expression in Bacteroidetes to our knowledge.
The effect of SD sequence addition or omission on NucB level was tested using pRH3 constructs containing the cognate nucB start codon upstream sequence to which the SD sequence was added, or the upstream sequence of PINA_1201, a gene of Prevotella intermedia 17, which originally contains the SD sequence, but from which the SD sequence was omitted partially or fully (Fig. 2 A and B). The results are presented in tables 1 and 2. Although there may be minor reductions in the NucB protein levels, both when SD sequence was added or omitted, the NucB levels stayed within the experimental error in strains harboring plasmid constructs with omitted or added SD sequence.
The sequence starts with the PstI site of the pRH3 or pUC19 vector (shown in italics). The stop codon at the start of the upstream sequence is underlined and the start codon of nucB or hMGFP is in boldface. A and B: nucB constructs with added (A) or removed (B) SD sequences. C: start codon upstream sequences containing SD sequences that are involved in formation of mRNA secondary structures with different stability. The sequences starting with mg were used in E. coli to translate the MGFP. D: Start codon upstream sequences of plasmid pRH3 borne nucB constructs containing SD sequence length of 10, 8, and 6 nucleotides. E: nucB upstream sequence used to asses the effect of secondary structure 20 bp upstream of the start codon. The secondary structure element was inserted into construct 0 from C after the designated adenosine.
mRNA Secondary structure in the SD sequence region interferes with translation to a different degree in Prevotella bryantii TC1-1 and Escherichia coli
On the basis of PINA_1201 upstream sequence, further upstream sequences in which SD sequence was part of a secondary structure of different stability were designed (Fig. 2 C). The upstream sequences and pRH3 plasmid constructs were named according to the stability of secondary structure as computed by mFold , thus the upstream sequence forming the secondary structure of stability ΔG = −11,1 kcal/mol was named 11,1 and so forth (Fig. S17). The NucB protein levels in P. bryantii TC1-1 strains harboring these constructs as determined by western blot can be seen in table 3. The essentially same upstream sequences were used to initiate translation of hMGFP mRNA in Escherichia coli TOP10 where upstream sequences and plasmid constructs were named as above and adding the mg prefix. The relative amount of MGFP was determined by western blot and fluorescence. In contrast to P. bryantii TC1-1, the upstream variant, which forms a secondary structure containing SD sequence with the stability of -5,2 kcal/mol at 37°C, produced roughly the same amount of protein and fluorescence as the variant forming no secondary structure (table 4). We further designed upstream sequence variants based on 11,1 and 5,2 sequences in which the SD element and its complementary sequence were swapped and named them 11,1inv and 5,4inv (Fig. 2 C, S17). We anticipated that by moving SD sequence further upstream, any extant SD-anti SD interaction would be impaired even more. The amounts of NucB produced from these constructs in P. bryantii TC1-1 were comparable to those obtained with constructs 11,1 and 5,2 (table 3) thus strengthening the validity of the former experiments. Minor changes within the experimental error are possible yet while the analysis of construct 11,1inv suggests a rise in NucB protein levels it is the opposite in the case of construct 5,4inv.
Strong secondary structure 20 bp upstream from start codon does not inhibit translation in P. bryantii TC1-1
A hairpin loop was inserted at -21 bp relative to start codon in the upstream sequence 0 (Fig 2 E). The stability of the secondary structure in resulting upstream sequence -20 was computed by mFold to be −9 kcal/mol (Fig. S17). The amount of NucB in P. bryantii TC1-1 cultures harboring plasmid construct -20 relative to the plasmid construct 0 was 1,34±0,06. The nucB mRNA level was not different in construct −20 relative to the amount in wild type nucB: 1,12±0,11.
Long SD sequence does not affect NucB protein amount in Prevotella bryantii TC1-1
The pRH3 based plasmid constructs SD6, SD8 and SD10 containing nucB and the identical upstream regions as used in study by Komarova et al.  in Escherichia coli (Fig.2 D) were produced and the NucB protein level was quantified. No correlation between SD sequence length and NucB protein levels was observed (table 5). Typical blot from which the quantifications were made is presented in figure S18.
Comparison of the NucB protein levels in constructs containing wild-type nucB, PINA_1201 or SD10 upstream sequences in Prevotella bryantii TC1-1
NucB level was highest in cultures containing PINA_1201 plasmid construct. The NucB protein level in cultures containing nucB and SD10 was lower: 0,4±0,17 and 0,13±0,09 respectively, relative to PINA_1201. The mFold mRNA secondary structure prediction in the upstream sequence region revealed lack of secondary structure in PINA_1201 derived mRNA whereas the mRNA from nucB and SD series of constructs form a hairpin loop starting at positions preceding the start codon -18 and -14, -16, -18 bp, respectively, (Fig. S19).
The folding energies of start codon and intra gene methionine codon upstream regions in Prevotella bryantii B14 genome
In P. bryantii B14, the distribution of folding energies of start codon upstream regions differs markedly from those of successive methionine codon upstream regions, the considerable share of the latter being more prone to fold a stable mRNA secondary structure (Fig 3). The same result was obtained using other Prevotella genomes. The difference is much smaller in E. coli K12 and Bacillus subtilis subsp. subtilis strain 168 (Table S2).
Any bioinformatic analysis, including the sequence logo of the ribosome binding site region, critically depends on accurately annotated coding sequence start sites. Since the most of the available genome data from various members of the phylum Bacteroidetes is currently incomplete and annotation was done generally without human intervention, we focused initially on the complete published genomes of the bacteria from the genus Bacteroides only –. In these genomes, the start site annotation was done or verified manually except for the genomes of B. vulgatus and B. distasonis, now often referred to as Parabacteroides distasonis , where an additional step using a self training program MED-Start  was introduced to refine the start site placement. It was expected that manual annotation favored start sites preceded by canonical SD sequence which was also stressed by Kuwahara et al.  in B. fragilis YCH46 annotation. This did not affect the sequence logos much since they do not reveal canonical SD sequence and do not differ from the B. vulgatus and B. distasonis logos. Subsequently, we additionally examined almost forty mostly unfinished Bacteroidetes genomes and discovered logos similar to ones obtained from genus Bacteroides, as described. The only two species lacking the conserved Bacteroidetes logo but still not exhibiting the SD sequence, Rhodothermus marinus and Salinibacter ruber, belong to the Bacteroidetes family Rhodotermaceae which is only distantly related to other Bacteroidetes . Given its AT richness, the Bacteroidetes logo superficially resembles logos of parasites from the proteobacterial order Rickettsiales or from Mycoplasma. Yet there is no −13 AT enrichment in the latter two groups. Further, strains from species with low % GC e.g. Clostridium acetobutylicum ATCC 824 and Streptococcus pneumoniae D39 with 28,7 and 33,4% GC in the regions preceding the start codon, respectively, resembling the Bacteroidetes in this respect, have logos exhibiting typical SD sequences (Fig. S9).
The Bacteroidetes logo with the lack of SD sequence and -13 AT enrichment thus represents a true phylum wide characteristic which may reflect the basic features of translation initiation as does the SD sequence in other phyla. Also, the lack of SD sequence in Bacteroidetes sequence logos is in agreement with findings of Nagakawa et al.  who used the calculation of free energy change resulting from basepairing between anti SD sequence and 5′ untranslated regions of mRNA to identify genes preceded by SD sequence. The logo approach not only detects the SD sequence, but also informs of the translation initiation site structure at the same time. The other prominent characteristic of the Bacteroidetes regions preceding the start codons beside the sequence logo is that they are GC poor relative to the whole genome, which is a unique trait shared only by Chlorobi in analyzed bacterial phyla (Table S1). Interestingly, as described in results, the 12,6±2,3% GC drop is conserved universally in Bacteroidetes species whether their % GC genome content is high or not, e.g. Alistipes putredinis with 53% GC and Flavobacterium psychrophilum with 32% GC (Table S1). This suggests that the % GC drop itself, not the absolute % GC, is important. This fits the extended Unique accessibility hypothesis of translation initiation  well. The hypothesis claims that the decisive factor in authentic start codon selection is not the SD sequence but rather the masking of gene internal methionine codons in mRNA by secondary structure. In Bacteroidetes therefore, the local drop in % GC in regions preceding the start codons may result in a stretch of mRNA lacking or forming less stable secondary structures than the rest of the mRNA thereby exposing the authentic start codon regions. The start codon is then chosen according to Nakamoto  by multiple interactions with mRNA in the ribosome binding site including SD sequence if extant. Bacteroidetes, exhibiting the −13 AT enrichment, which is a possible target for mRNA binding ribosomal protein S1 , lack the SD sequence, and it appears that they don't make use of the SD sequence in this step. This is in agreement with our experiments in P. bryantii TC1-1 where the addition or omission of SD sequence preceding the start codon did not change the NucB yield appreciably. Lengthening the SD sequence does not result in lower reporter protein level as in E. coli ,  where it was suggested that strong SD duplex deriving from mRNA and 16S rRNA stalls the ribosome and thereby slows the translation. This poses the question whether P. bryantii is able to form the above mentioned duplex at all. We addressed the issue by constructing series of start codon upstream sequences which trap the SD sequence in secondary structures of different stability. It was found that the secondary structure with the folding energy of -5,2 kcal/mol reduced the NucB protein level in P. bryantii TC1-1 to approximately one fifth, whereas in Escherichia coli the protein level produced from hMGFP preceded by the identical upstream sequence was not affected. The latter is in the agreement with de Smit and van Duin  who found that secondary structures less stable than -6 kcal/mol usually don't affect translation initiation in E. coli. Also, when the nucB, PINA_1201 and SD series of upstream sequences driving the translation are compared, the upstream sequence PINA_1201, lacking secondary structure as predicted by mFold, is the most efficient while the SD6 construct which contains a hairpin loop 14 bp upstream of start codon is the least efficient. Taken together, these experiments suggest that the translation in P. bryantii and possibly other Bacteroidetes is easily inhibited by secondary structure since there is no SD interaction to compensate for it as in E. coli . This could therefore explain both the lack of SD sequence in Bacteroidetes sequence logos and the % GC drop in start codon regions. We also verified that the latter leads to a diminished propensity of these regions in mRNA to fold into stable secondary structures relative to intra gene methionine codon upstream regions (Fig 3, table S2) thereby favoring the translation initiation at the authentic start codon.
The % GC drop in genome regions preceding the start codons and the sequence logos of these regions, which lack the SD sequence, clearly separate Bacteroidetes and Chlorobi from other phyla which is in agreement with a notion that these two groups evolved from a common ancestor not shared by other bacteria . In results we showed that merely by visual inspection of sequence logos one could discriminate genera and families in Bacteroidetes. It is similar in Spirochaetes where the sequence logo clearly separates Leptospiraceae from Spirochaetaceae and Brachyspiraceae (Fig. S11) and more such cases can be found. If each position in the logo was represented numerically, by its information content and by frequencies of individual bases at each position, one could construct a signature sequence logo along with deviations for each position at the genus or family level which could be used as an additional taxonomic marker. At the phylum level, however, the logo may not be very conserved as exemplified by % AT rich parasites and symbionts in Proteobacteria and Firmicutes. Excluding such, the GC genome content at the phylum level still varies enough to introduce bias in a sequence logo e.g. Moorella thermoacetica and Listeria monocytogenes with 55,8 and 38% GC, respectively. This two organisms share a similar shape of SD sequence region, yet the L. monocytogenes logo is enriched by AT (Fig. S9) and thus drastically changed as a whole. A correction for % GC deviation at the level of phylum seems needed yet it is not justified since it is evident that the % GC is only one of the factors influencing the start codon upstream region make up. Consequently the shape and placement of SD sequence region and any additional phylum-wide peculiarity of a logo, not the whole logo including its height, is a better candidate marker distinguishing at least some additional bacterial phyla.
Materials and Methods
Strains, plasmid, primers and growth conditions
The P. bryantii TC1-1  was cultured anaerobically at 37°C in rumen fluid containing M2 medium  or modified DSMZ medium 330 . When appropriate, tetracycline was added to the medium prior to inoculation at the final concentration of 3.75 µg ml−1. The shuttle vector pRH3  originates from the laboratory of Harry J. Flint (Rowett Research Institute, Aberdeen, Scotland). The phMGFP vector coding for Monster green fluorescent protein was obtained from Promega (USA). The Escherichia coli TOP10 and pUC19 vector were from Invitrogen (USA). The oligonucleotide primers were synthesized at Eurofins MWG OPERON (Germany) and Microsynth GmbH (Switzerland) and are shown in table S3.
Nucleotide sequences of bacterial genomes were obtained from the EBI Genomes server (http://www.ebi.ac.uk/genomes/). When the unfinished, but annotated whole genome shotgun data for Bacteroidetes genomes was examined, the contigs were joined using the union of the EMBOSS package . The 30 bp of upstream sequence of all coding sequences in a given genome were retrieved with Artemis v9 . Sequence logos were then created by WebLogo  and the information axis was limited to 1 bit for clarity. The logo position 30 corresponds to nucleotide site directly preceding the start codon. RNA secondary structure was predicted by mfold 3,2 . The nucleotide sequences of 16S rRNA coding genes from the Bacteroidetes, Chlorobi and other phyla representative genomes were extracted using Artemis and aligned by ClustalX 2.0.12 . The folding energies of the start codon and intra gene methionine codon 30 bp long upstream regions in genome were calculated using UNAFold's hybrid-ss-min . The intra gene mathionine upstream sequences were obtained using Artemis v9.
PCR, cloning and expression of the nucB and hMGFP genes
These were essentially performed as described earlier . Briefly, the nucB gene variants containing different upstream sequences were produced by PCR, cloned into pRH3, checked for orientation, protected toward P. bryantii TC1-1 type II restriction and electroporated into P. bryantii TC1-1. The plasmid DNA of the prepared constructs was isolated, sequenced and inspected. The recombinant strains expressed nucB in modified DSMZ 330 medium containing tetracycline for 24 hours upon subculturing from overnight culture. All nucB constructs contained a stop codon immediately after the PstI site in order to terminate any possible translation coming from the truncated rteA gene which was cloned into pRH3 during its construction along with tetQ. Constructs with added or omitted SD sequence had 41 bp of upstream sequence. The constructs containing variable SD sequence length, namely SD10, SD8 and SD6, had a 41, 39 and 37 long upstream sequence, respectively. The constructs containing SD sequence involved in secondary structures had 50 bp of upstream sequence.
hMGFP variants were also produced by PCR. These hMGFP variants contained the same upstream regions as nucB variants containing SD sequence involved in secondary structure, except for a cytosine which was inserted immediately downstream of PstI site in order to bring the stop codon following it in frame to end the translation of the lacZ alpha fragment of pUC19 (Fig 2 C). The hMGFP variants were digested using PstI, ligated with pUC19 and transformed into E. coli TOP10. The resulting recombinant strains were checked for orientation using PCR and verified by sequencing. The strains were grown overnight in LB containing 100 µg/ml of ampicilin, diluted 100x into fresh medium containing ampicillin and grown to OD600 = 0,5. For western blot, cells from 0,1 ml of culture were analyzed using anti-his6 antibodies since His6 was added to MGFP at the C-terminal end. For fluorescence measurement, 1 ml of culture was centrifuged and the cells were resuspended in 0,1 ml of 50 mM Tris-HCl pH = 7,5 0,15 mM NaCl. 50 µl of cell suspension was then transferred to 384 well transparent bottom microplate (Brand, Germany) and the MGFP was excited using blue light converter placed over the transiluminator of Chemigenius2 bio imaging system (Syngene, UK).The fluorescence was quantified using GeneTools from Syngene from at least three independent experiments. Prior to that the cultures expressing hMGFP were checked using epifluorescence microscopy to assure all cells fluoresce i.e. the plasmid constructs were not lost during cultivation.
nucB and hMGFP mRNA quantification
The amount of nucB mRNA in P. bryantii TC1-1 harboring pRH3 constructs and hMGFP m RNA in E. coli was measured as described earlier  during the exponential growth at OD600 = 0,5. Briefly: the total RNA was reverse transcribed using specific primers and cDNA obtained was quantified by real-time PCR using standard curve method and Sybr green as the reporter dye. The amount of the 16S rRNA amplicon was used for nucB normalization. Real-time PCR quantification was performed for each sample in triplicates in at least three real-time PCR runs. For hMGFP mRNA quantitation new primers were designed: mgrtF and mgrtR amplyfing the hMGFP and ecort16SF and ecort16SR for endogenous control which was used for normalization (Table S3).
NucB and MGFP quantification using western blot
The P. bryantii culture supernatans containing NucB were concentrated using Amicon ultra-4 10000 MWCO centrifugal filter devices from Millipore and proteins separated by standard SDS-PAGE gel. When analysing MGFP content, the cells from 0,1 ml of culture were centrifuged and resuspenced in 50 µl of water. The western blot and immunodetection was performed as described earlier  except for the change of the HRP substrate, which was SuperSignal from Novagen (Merck, Germany). The chemiluminiscence was recorded using the Chemigenius2 bio imaging system (Syngene, UK) and relative quantification was done using GeneTools from Syngene from at least three independent experiments.
Sequence logos of start codon upstream regions of Acidobacteria .
Sequence logos of start codon upstream regions of Aquificae .
Sequence logos of start codon upstream regions of Chlamydiae .
Sequence logos of start codon upstream regions of Chlorobi .
Sequence logos of start codon upstream regions of Bacteroidetes .
Sequence logos of start codon upstream regions of Chloroflexi .
Sequence logos of start codon upstream regions of Cyanobacteria .
Sequence logos of start codon upstream regions of Fibrobacteres .
Sequence logos of start codon upstream regions of Firmicutes .
Sequence logos of start codon upstream regions of Proteobacteria .
Sequence logos of start codon upstream regions of Spirochaetae .
Sequence logos of start codon upstream regions of Thermotogae .
Sequence logos of start codon upstream regions of Deinococcus-Thermus .
Sequence logos of start codon upstream regions of Actinobacteria .
Sequence logos of start codon upstream regions of Tenericutes .
Multiple sequence alignment of 16S rRNA genes from Bacteroidetes and representatives of other bacterial phyla.
mRNA secondary structure prediction of 11,1, 5,2, 2,1, 0, 11,1inv, 5,4inv and -20 start codon upstream regions.
A typical western blot used for quantification.
mRNA secondary structure prediction of PINA_1201, nucB , SD6, SD8 and SD10 start codon upstream regions.
GC content in the start codon upstream regions in major bacterial phyla.
Folding energy of the annotated start codon and successive methionine codons 30 bp upstream regions in genus Prevotella , Escherichia coli K12 MG1655 and Bacillus subtilis subsp. subtilis 168.
Primers used in the study.
Conceived and designed the experiments: TA. Performed the experiments: TA. Analyzed the data: TA GA. Contributed reagents/materials/analysis tools: GA. Wrote the paper: TA GA.
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