BACKGROUND
proteins with less than  <dig> amino acids in prokaryotes or  <dig> amino acids in eukaryotes are defined as short proteins .
during standard genome annotation these short protein-coding genes are frequently neglected and proteomics-based analyses fail to detect this class of peptides routinely.
as a result, μ-protein-coding genes are a systematically underestimated class of gene products.
in strong contrast is the finding that small orfs constitute the most frequent essential genomic component in bacteria, even more than conventional orfs  <cit> .
indeed, the functional characterization of selected examples of μ-proteins has revealed their critical involvement in processes such as quorum sensing or interspecies communication  <cit> , regulatory functions  and in the formation of multi-subunit protein complexes.
an increasing number of μ-proteins is being discovered also in eukaryotes , and archaea  <cit> , indicating their ubiquity in all three domains of life.
nevertheless, the likely diverse functions of short proteins are largely unknown, even for simple unicellular bacteria.
photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 μ-proteins of less than  <dig> amino acids, that play a role in photosystem ii , psbi, psbl, psbj, psby, psbx, psb <dig> , psbn, psbf, psbk  <cit> ), in photosystem i , photosynthetic electron transport , or have accessory functions   <cit> ).
the shortest annotated protein conserved in cyanobacteria is with  <dig> amino acids the cytochrome b
6
f complex subunit viii, encoded by petn  <cit> .
several cyanobacterial model species have been studied by transcriptomics  and proteomics  approaches but there are no reports specifically targeting μ-proteins.
based on extensive comparative transcriptome and genome information we used the model cyanobacterium synechocystis sp.
pcc  <dig>  and the closely related strain synechocystis sp.
pcc  <dig>   for the prediction of possible μ-orfs.
we found  <dig> transcriptional units  containing candidate small orfs ≤ <dig> codons in synechocystis  <dig>  including all known mrnas encoding small proteins of the photosynthetic apparatus.
we chose  <dig> examples from synechocystis  <dig> for experimental analysis.
these were norf <dig> and norf <dig> , nsir <dig> and hlir <dig> , the latter two transcripts originally considered non-coding  <cit>  as well as the short gene ssr <dig>  which was predicted as protein-coding in the current version of the genome sequence .
all five proteins could be detected after flag tagging in vivo.
their modes of regulation, conservation and physico-chemical properties make these five μ-proteins interesting candidates for functional studies.
methods
strains and growth conditions
synechocystis  <dig>  substrain “pcc-m”  <cit> , served as wt and was grown in cu2+-free, tes-buffered  liquid bg <dig> medium  <cit>  with gentle agitation or on agar-solidified  bg <dig> supplemented with  <dig> %  sodium thiosulfate at 30 °c under continuous illumination with white light of ~40 μmol photons m− <dig> s− <dig>  to induce expression of flag - tagged μ-proteins from the cu2+-responsive pete promoter  <cit>  2 μm cuso <dig> was added to exponentially growing cells.
different environmental conditions were applied for induction of gene expression under control of native promoters:  high light, 300 μmol photons m− <dig> s−1;  dark, flasks wrapped with aluminium foil;  nitrogen deficiency, cells were pelleted by centrifugation, washed once and resuspended in no3
−-free bg <dig>  samples for protein extraction were taken just before and 6 h  or 24 h  after induction of gene expression.
ssr <dig> was expected to be most expressed in exponential growth phase, hence samples were taken from exponentially growing cells at two consecutive days.
synechocystis  <dig> strain pur-ppetj-3xflag-sfgfp  <cit>  was used as positive control for the detection of flag-tagged proteins by western blots.
e. coli strains top10f’ and j53/rp <dig> were used for generating synechocystis  <dig> mutant strains by conjugation.
in liquid bg <dig> medium 5 μg ml− <dig> gentamicin or 50 μg ml− <dig> kanamycin and 5 μg ml− <dig> gentamicin were used to maintain recombinant strains .
for examination of gene expression by northern blot analysis, exponentially growing wt cells were transferred to the different environmental conditions described above.
cultivation under high light was followed by a shift back to standard light conditions ).
cultures grown in the dark as well as nitrogen deprived cultures were additionally aerated with ambient air through a glass tube and a sterile filter for constant and fast growth.
computational methods
small orfs and their orthologs were identified and annotated in synechocystis  <dig> and  <dig> in three steps.blastn searches returning hits with e values ≤1e− <dig> were performed against the ncbi nt database  <cit>  for all intergenic regions covered by tus  <cit> .
from the blast results, multiple alignments were created with clustalw  <cit>  and analyzed for their coding potential with rnacode  <cit> .
the significant  small orf candidates were manually curated.
to annotate candidate small orfs, blastp queries with e values ≤1e− <dig> were done against the ncbi nr database  <cit> .
orthologs of existing and newly detected small orfs were identified in synechocystis  <dig> and  <dig> via a reciprocal best hit approach using blastp with a minimum e value ≤1e− <dig> and allowing a difference in length of ≤20% and a maximum length of  <dig> amino acids in both strains.
genes of small orfs that were covered by a predicted tu were considered to be expressed.
transmembrane helices were predicted with tmhmm server v.  <dig>   <cit> .
generation of mutant strains
gene constructs for ectopic expression of flag - tagged norf <dig> under control of the pete promoter or the native promoter were generated via gene synthesis .
the constructs consisted of the upstream sequence of pete  or the upstream sequence of norf <dig> , the norf <dig> coding sequence omitting the stop codon , a 3xflag coding tag  followed by a stop codon , the norf <dig> 3′utr  and the bacteriophage lambda oop terminator.
the obtained ppete::norf1::3xflag::toop and pnorf1::norf1::3xflag::toop constructs were digested with xhoi and hindiii and introduced into self-replicating vector pvz <dig>  <cit> .
the resulting plasmids were transferred into synechocystis  <dig> wt via triparental mating with e. coli j53/rp <dig> and top10f’  <cit> .
these two recombinant strains were selected on bg <dig> agar containing 10 μg ml− <dig> gentamicin.
to establish ectopic expression of flag - tagged nsir <dig>  hlir <dig>  ssr <dig> and norf <dig>  the respective genomic sequences  were amplified using the primer pairs nsir6_fw/nsir6_rev, ppete::hlir1_fw/3xflag_hlir1_rev, ppete::ssr1169_fw/3xflag_ssr1169_rev and ppete::norf4_fw/3xflag_norf4_rev.
all oligonucleotides used in this study are listed in table  <dig>  the pete promoter was amplified separately for each construct to generate overlaps with the particular μ-orfs using the primer puc19-xbai_ppete_fw in different combinations with nsir6::ppete_rev, hlir1::ppete_rev, ssr1169::ppete_rev or norf4::ppete_rev.
the 3′ segments consisting of the sequence encoding the 3xflag tag , the 3′utr of the norf <dig> mrna and the oop terminator were amplified from the plasmid obtained via gene synthesis described above using the primer 3xflag_psti-puc19_rev in combination with nsir6_3xflag_fw, hlir1_3xflag_fw, ssr1169_3xflag_fw or norf4_3xflag_fw, respectively.
fragments belonging together were combined by gibson assembly® master mix  according to the manufacturer’s instructions utilizing xbai and psti digested puc <dig> as vector backbone.
for expression of the small proteins under control of their native promoters the obtained plasmids served as templates for amplifying corresponding coding sequences associated with the 3′ segment described above using the primer 3xflag_psti-puc19_rev in combination with cdsnsir6::pnsir6_fw, cdshlir1::phlir1_fw, cdsnorf4::pnorf <dig> or cdsssr1169::pssr1169_fw.
upstream sequences of nsir <dig>  hlir <dig>  norf <dig> and ssr <dig> considered as promoter sequences  were amplified from synechocystis  <dig> genomic dna with the primer pairs puc19::pnsir6_fw/pnsir6::cdsnsir6_rev, puc19::phlir1_fw/phlir1::cdshlir1_rev, puc19::pnorf4_fw/pnorf4::cdsnorf4_rev or puc19::pssr1169_fw/pssr1169::cdsssr1169_rev.
related fragments were combined by gibson assembly® master mix as described above.
all resulting cassettes were released by restriction, introduced into pvz <dig>  <cit>  and transferred into synechocystis  <dig> wt via triparental mating.
additionally, the empty vector pvz <dig> was introduced into the wild type to create a control strain.
the recombinant strains were selected on bg <dig> agar containing 10 μg ml− <dig> gentamicin and 50 μg ml− <dig> kanamycin.table  <dig> list of oligonucleotides
orf <dig> generating overlaps with ppete and 3xflag::3′utr n
orf1::toop
<dig>  ssr <dig> or n
orf4
rna extraction and analysis
synechocystis  <dig> cells were harvested by vacuum filtration on hydrophilic polyethersulfone filters , immediately immersed in 1 ml pgtx  <cit>  and frozen in liquid nitrogen.
rna extraction was performed by 15 min incubation at 65 °c followed by chloroform washing and isopropanol precipitation as previously described  <cit> .
northern hybridization with 32p-labelled, single-stranded transcript probes was carried out as described  <cit> .
oligonucleotide sequences for pcr amplification of probe templates used for in vitro transcription are listed in table  <dig>
protein purification and immunodetection
cells for protein extraction were collected by centrifugation , resuspended in pbs buffer  in the presence of protease inhibitor cocktail  and immediately frozen in liquid nitrogen.
cells were mechanically disrupted by using glass beads  and a precellys®  <dig> homogenizer  at 6000 rpm and 4 °c applying six cycles of 3 × 10 s homogenization.
glass beads were removed by centrifugation .
to solubilize membrane proteins, samples were heated for 30 min at 50 °c with 2% sds  followed by determination of the protein concentration using direct detect spectrometer .
proteins were separated by sds-page on 15%  polyacrylamide gels and stained with gelcode® blue stain reagent .
pageruler™ prestained protein ladder  was used as molecular weight marker.
for immunoblot analysis, separated proteins were transferred to nitrocellulose membranes .
membranes were blocked over night at 4 °c with 5% low fat milk powder in tbs-t and subsequently probed with monoclonal anti-flag® m2-peroxidase  antibody raised in mouse  in tbs-t for 1 h at room temperature in the dark.
all washing steps were performed with gentle agitation in tbs-t  tween-20) at room temperature.
signals were detected with ecl™ start western blotting detection reagent  on a chemiluminescence imager system  and subsequently visualized using fusion-cap  and quantity one software .
reporter gene assays
to measure promoter activity as a function of bioluminescence the putative norf <dig> promoter sequence and its 5′utr  was fused to promoterless luxab reporter genes by pcr, followed by cloning into the promoter probe vector pila as described  <cit> .
the resulting pila derivative was used for transformation of a synechocystis  <dig> strain expressing the luxcde genes encoding enzymes for the synthesis of decanal, the luciferase substrate, under control of the strong promoter of the ncrna yfr2a  <cit> .
cells were grown in the presence of 10 mm glucose to provide energy for the luciferase reaction also in darkness.
bioluminescence was measured in vivo at different time points after inducing dark conditions as described  <cit> .
RESULTS
comparative transcriptomics for the identification of μ-proteins in synechocystis
the extensive comparative transcriptome and genome information for the model cyanobacterium synechocystis  <dig>  <cit>  and the closely related strain synechocystis  <dig>  <cit>  was utilized for the prediction of possible μ-orfs.
in our previous studies  <cit>  transcriptional units  had been defined, combining information on the transcriptional start sites, the lengths of transcribed utrs, operons, coding and non-coding regions.
here we judged all possible non-coding transcripts by the program rnacode  <cit>  for their protein-coding potential.
rnacode detects protein-coding regions in any given sequence on the basis of multiple sequence alignments and the evolutionary signatures that are associated with a coding sequence  <cit> .
after combination with the pre-existing annotation, this analysis led to the prediction of  <dig> potential small proteins with a maximum of  <dig> amino acids in synechocystis  <dig> and possibly  <dig> in synechocystis  <dig> .fig.
<dig> scheme of computational prediction of small orfs in cyanobacteria.
small orfs were detected based on the transcriptome information for expressed intergenic regions  and their coding potential evaluated with rnacode  <cit> .
this information was merged with the pre-existing annotation  <cit> .
orthologs between the two small orf populations were detected when they were identified as reciprocal best hits  by blastp with e ≤ 1e−2
the resulting sets of candidate μ-proteins were compared against the predicted proteome of the respective other synechocystis strain, against e. coli and the higher plant arabidopsis thaliana as reference organisms for proteins possibly conserved among bacteria or among photosynthetic organisms.
this procedure led to the identification of 146 μ-proteins shared between the two synechocystis strains, as well as  <dig> and 29 μ-proteins which are shared between synechocystis  <dig> and a. thaliana or e. coli, respectively.
interestingly, we found the  <dig> proteins shared with higher plants to be identical in both synechocystis strains.
in contrast to observations in other bacteria, a relatively high number of the predicted proteins in the smallest fraction  had assigned functions  and a matching protein in the higher plant arabidopsis thaliana or in e. coli .table  <dig> predicted and previously annotated proteins ≤ <dig> amino acids in synechocystis 6803
petn
6
psbt, ycf8
psam
1148230
1148325
-
31
norf4
norf4
norf4
y
n
n
petl
6
psbm
petm
6
1606868
1606978
+
37
hlir1
hlir1
hlir1
y
n
n
rpl36
petg
6
psbi
psbl
psbj
psby
psbx
ycf12
psaj
psai
ndhp
psbn
psbf
psbk
rpl34
ndhq
hlic, scpb
298826
298972
-
48
norf1
norf1
norf1
n
n
the start and end positions according to the chromosomal or plasmid sequences in genbank files , the location  on the forward  or reverse strand  and respective length  are given, followed by the locus tag id, gene name and product if assigned.
location on chromosome or one of the plasmids is prefixed by “chr” or the name of the plasmid.
the existence of homologs in synechocystis  <dig>  a. thaliana and e. coli is indicated by “y” for yes or “n” for no.
homologs tagged and detected in this study are highlighted in boldface letters.
names of genes tested in this work are in boldface
in vivo tagging and detection of cyanobacterial μ-proteins
we chose  <dig> examples for closer analysis: norf <dig>  nsir <dig>  hlir <dig>  ssr <dig> and norf <dig>  norf <dig> and norf <dig> were previously defined based on transcriptomic evidence  <cit> .
the protein ssr <dig> was previously modelled as part of the existing annotation, but there is no information on possible functions nor that their very existence was shown thus far.
nsir <dig> and hlir <dig> are not annotated in the genome but were found by transcriptomics  <cit> .
although these rnas harbor potential open reading frames they were initially indicated as non-coding.
after flag - tagging and inducing their expression in synechocystis  <dig>  all five proteins were detected by western blotting .
hlir <dig> and ssr <dig> showed a tendency for aggregation, even under the used denaturing conditions, possibly related to their hydrophobicity and the predicted presence of transmembrane regions .fig.
<dig> western blot detection of small proteins.
recombinant synechocystis  <dig> cells carrying the genes of interest  under control of the pete promoter on pvz <dig> vector were collected before  or 24 h after induction of gene expression  for the extraction of total proteins.
flag-tagged superfolder gfp  under the control of the petj promoter  <cit>  served as positive control, a wt strain carrying an empty pvz <dig> vector was used as negative control .
theoretical protein masses are listed in table  <dig>  two gels were run in parallel.
a proteins  were separated on a 15%  sds polyacrylamide gel and subjected to colloidal coomassie g- <dig> staining as a loading control.
b immunoblot with the same loading order probed with specific anti-flag® m2-peroxidase  antibody
the nsir <dig> transcript is highly induced upon nitrogen deprivation
nsir <dig> was not previously known as a protein-coding gene.
its mrna originates from a tss at position 729645f in the chromosome of synechocystis  <dig> .
previously, we introduced the uef  to identify genes whose expression was enhanced at a single from ten tested environmental conditions  <cit> .
this factor gives the ratio of the transcriptome read counts for the condition with the highest and the one with the second highest expression for a single tu.
thus, tus with a high uef respond strongly to a particular stimulus.
for nsir <dig>  the uef was  <dig> , ranking on position  <dig> of the most-strongly induced genes, both in synechocystis  <dig> as well as in strain  <dig>  <cit> , when the cells were deprived of sources of combined nitrogen .
this induction was confirmed by independently performed northern blots, indicating a rapid induction of expression, reaching a peak at 6 h with an about 10-fold higher transcript accumulation, followed by a declining abundance which remained higher than at the beginning of the experiment .
the nitrogen-stress-dependent induction is likely mediated via a conserved ntca binding site 5′-gtaacatttgtgac-3′, centered 42 nt upstream the transcription initiation site in both strains .
ntca-binding sites frequently overlap the − <dig> promoter region and are centered close to position − <dig>  with respect to the tss when they mediate activation  <cit> .
homologs of nsir <dig> are widely conserved throughout the cyanobacterial phylum and in the paulinella chromatophora chromatophore genome, consistent with its occurrence in the genomes of α-cyanobacteria, but not in any other bacteria or plants.
the alignment of these homologs shows two pairs of conserved cysteine residues which might be involved in redox control, protein-protein interactions or structure formation .
two pairs of cysteine residues occur also in another short protein, the  <dig> amino acid cp <dig> protein, which mediates the formation of a complex between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase in response to changes in light intensity, characterizing it as a thioredoxin-mediated metabolic switch  <cit> .
in cp <dig>  the cysteine pairs confer the redox input via post-translational thiol-disulfide bridge conversion.
the arrangement ‘cpvc’ of the first cysteine pair  matches the c-2-c motif, which frequently is involved in metal-binding  <cit> .
hence, the putative cysteine pairs in nsir <dig> may confer redox control or metal binding.fig.
<dig> heatmap indicating the expression of the genes encoding the five investigated small proteins in synechocystis strains pcc  <dig> and pcc  <dig> under  <dig> different growth conditions: exponential  and stationary growth phase ; cold  and heat  stress for 30 min each; depletion of inorganic carbon , cells were washed  <dig> times with carbon-free bg <dig> and cultivated further for 20 h; dark, no light for 12 h; fe2+ limitation , addition of iron-specific chelator desferrioxamine b  and further cultivation for 24 h; high light , 470 μmol photons m−2s− <dig> for 30 min; nitrogen depletion , cells were washed  <dig> times with nitrogen-free bg <dig> and cultivated further for 12 h; phosphate depletion , cells were washed  <dig> times with phosphate-free bg <dig> and further incubated for 12 h. data derived from previous genome-wide expression analysis by differential rna-seq  <cit> .
values indicate sequencing read counts for the primary 5′ end  of the corresponding transcriptional unit .
the tss positions are given for the synechocystis genomes available under accession numbers ba <dig> and cp <dig>  the colour varies from red  to yellow  to green
fig.
<dig> the nsir <dig> peptide.
a transcriptomic datasets indicated high read coverage in a region without annotation in synechocystis  <dig>  <cit> , which contains the here defined nsir <dig> gene.
the homolog in synechocystis  <dig> is d082_ <dig>  <cit> .
shown is the read coverage  resulting from previous transcriptome analysis, including the respective transcriptional units  defined in that work  <cit>  and a putative ntca binding site, centered 42 nt upstream the transcription initiation site in both strains.
relevant transcription initiation sites appear as steep increase in read coverage and are labelled by a black arrow.
the length of the 5’-utrs is 26 nt in both strains.
other non-coding tus are colored orange.
there is transcription in antisense orientation in both strains but with much lower coverage.
b northern blot showing the nitrogen stress-induced transcript accumulation of the nsir <dig> mrna in synechocystis  <dig> over 72 h. time point  <dig> refers to the nitrogen-replete condition.
c time course of nsir <dig> mrna accumulation after normalization to 5s rrna.
the data are presented as relative to the signal at 6 h after diminishing n .
d sequence comparison of nsir <dig> homologs from the two synechocystis strains, cyanothece atcc  <dig>  crocosphaera watsonii wh  <dig> and the two marine synechococcus strains wh  <dig> and wh  <dig> which harbor an identical protein.
four conserved cysteine residues are highlighted by arrows.
these are conserved in all  <dig> homologs detected throughout the cyanobacterial phylum
norf <dig> is highly induced upon dark incubation
norf <dig> is specific for cyanobacteria but widely conserved throughout this phylum.
it is present in  <dig>  of  <dig> cyanobacterial genomes available in the jgi database  <cit>  .
homologs are lacking in early-branching cyanobacteria such as gloeobacteria and thermophilic synechococcus ja-2-3b’a and ja-3-3ab and also in marine picocyanobacteria.
an alignment of representative homologs is shown in fig.
5a.fig.
<dig> sequence comparison and regulated expression of the norf <dig> gene.
a sequence comparison of norf <dig> homologs from the two synechocystis strains, cyanothece atcc  <dig>   <dig> different strains of crocosphaera encoding an identical protein and anabaena  sp.
pcc  <dig>  b expression of norf <dig> in synechocystis  <dig> is strongly upregulated after transfer to darkness.
c bioluminescence of a synechocystis  <dig> reporter strain harboring a transcriptional fusion of pnorf <dig>  and luxab genes in response to transfer to darkness.
representative bioluminescence dataset indicating means ± sd of measurements for two biological replicates .
a strain carrying a promoterless luxab was used as a negative control
strong accumulation of the norf <dig> mrna was observed in response to darkness .
the uef for this condition was  <dig>  in synechocystis  <dig>  but the gene was expressed also under the other tested conditions   <cit> .
to examine whether the dark-related expression of norf <dig> is under transcriptional control, we conducted reporter gene assays.
the upstream sequence of synechocystis  <dig> norf <dig> was fused to luxab reporter genes encoding luciferase, and expression was monitored as bioluminescence in vivo.
indeed, the promoter activity showed a positive response after transfer into darkness as seen for the mrna accumulation .
we conclude that the observed induction of norf <dig> in response to shifts from light exposure to darkness is under transcriptional control.
the high expression of the norf <dig> gene in darkness sets it apart from the vast majority of genes.
among the previously tested  <dig> different growth conditions, in synechocystis  <dig> only  <dig> out of  <dig> tus and in synechocystis  <dig> only  <dig> out of  <dig> tus defined in total had their maximum expression after dark incubation  <cit> .
the norf <dig> μ-protein is highly conserved and its mrna overlaps the gap <dig> gene
norf <dig> is encoded within a tu much longer than is needed to encode the  <dig> amino acids: tu <dig> in synechocystis  <dig> is 704 nt and tu <dig> in synechocystis  <dig> is 534 nt .
these tus partially overlap the gap <dig> gene encoding glyceraldehyde 3-phosphate dehydrogenase  <dig> on the complementary dna strand.
as a result, these tus overlap the gap <dig> mrna by  <dig> and 373 nt, respectively.
transcriptomic evidence suggested that both the gap <dig> and the norf <dig> mrnas were co-regulated with each other, with a mild up-regulation upon the removal of nitrogen .
thus, the norf <dig> transcript does not function as an antisense rna with a co-degradation function, which was observed previously for other pairs of overlapping transcripts in synechocystis  <dig>  <cit> .
however, co-regulation between an asrna and its cognate mrna was previously observed for the psba asrna protecting its 5′ leader from rnase e-mediated degradation  <cit> .
the expression of norf <dig> was stimulated upon removal of nitrogen, but its expression was detectable under most of the previously tested conditions, although at a lower level .
dual-function rnas are transcripts which assume a regulatory function as srna and additionally act as short protein-coding mrna.
exploring this possibility for norf <dig>  we checked the accumulation of norf <dig> transcripts during the removal of combined nitrogen.
northern blot analysis showed the existence of a prominent transcript of ~200 nt which declined initially .
due to the localization of the rna probe used in the detection of norf <dig> transcripts, this prominent transcript corresponds to the coding part of tu <dig>  however, with increasing duration of the nitrogen stress, we noticed the overaccumulation of a longer transcript, of about 600–800 nt that appeared more diffuse .
quantitative analysis of transcript accumulation showed that this longer norf <dig> transcript was only transiently accumulated, with a peak at the 24 h time points .fig.
<dig> the norf <dig> peptide.
a datasets from the previously performed primary transcriptome analysis showed that norf <dig> expression responded positively to nitrogen depletion in both synechocystis  <dig>  <cit>  and synechocystis  <dig>  <cit> , when  <dig> different growth conditions were tested.
the mrnas of norf <dig> and gap <dig> are co-regulated and overlap by several hundred nt.
the previously mapped transcriptional start sites are labelled by black arrows.
b northern blot analysis of norf <dig> mrna accumulation in a time course experiment up to 72 h after the removal of nitrogen.
the same rna samples were used as in fig.
<dig>  c the signals obtained from the northern blots  were evaluated densitometrically after normalization by the level of 5s rrna.
the relative norf <dig> expression is shown with respect to the maximum expression after transfer to nitrogen-free conditions .
the bands at 200 nt  and at 800 nt  were analyzed separately from each other.
d multiple sequence alignment of  <dig> homologs from  <dig> different cyanobacterial genome sequences
with very few amino acid substitutions, norf <dig> is extremely conserved, including a predicted transmembrane region .
homologs can be detected in  <dig> cyanobacterial genome sequences from all  <dig> morphological subsections, comprising free-living unicellular as well as multicellular strains, marine and freshwater isolates, thermophiles and symbionts.
the presence of norf <dig> in the two available genome sequences of candidatus atelocyanobacterium thalassa suggests their positive selection in these highly streamlined genomes  <cit> .
however, homologs are lacking in α-cyanobacteria, which are mainly marine synechococcus and prochlorococcus.
the homologs from the two used synechocystis strains are identical, except for a possible n-terminal extension by  <dig> amino acids in synechocystis  <dig> .
however, such extensions appear questionable also in other strains, because the start codon corresponding to the synechocystis  <dig> orf is 100% conserved.
moreover, the homologs in  <dig> microcystis genomes are identical to each other, as are the homologs in five crocosphaera watsonii and in two fischerella genome sequences.
our data suggest that norf <dig> is a previously unknown membrane-bound μ-protein and that the norf <dig> transcript may play a dual role, with a mainly coding function during nitrogen-sufficient conditions and a possibly rna-mediated regulatory function on the gap <dig> mrna during nitrogen stress.
hlir <dig> and ssr1169
hlir <dig> was chosen because of its very high induction under high light  and the gene location upstream of sodb encoding superoxide dismutase.
whereas the homologs from the two synechocystis strains are conserved in length, sequence  and the likely presence of a transmembrane region , no possible homologs were detected beyond the genus synechocystis.
the location upstream of sodb and the shape of the read coverage in transcriptome analysis  suggested a possible link between the two genes.
indeed, northern analysis confirmed the inducibility by high light  and in addition showed the presence of two major transcripts, ~ <dig> and 1400 nt in length.
the longer form should encompass also the complete sodb gene.
thus, transcription from the upstream located hlir <dig> promoter will lead by read-though to an enhanced sodb gene expression under high light.
hence, it is tempting to speculate, that hlir <dig> is a membrane-bound peptide with a regulatory function on the superoxide dismutase.fig.
<dig> the hlir <dig> peptide in synechocystis  <dig>  a pairwise sequence alignment of the hlir <dig> peptides from synechocystis  <dig> and synechocystis  <dig>  a predicted transmembrane region is boxed.
b data replotted from the primary transcriptome analysis of synechocystis  <dig> suggest that hlir <dig> expression is induced by high light and that transcripts may extend into the subsequent tu <dig> covering the sodb gene  <cit> .
c northern analysis of hlir <dig> mrna accumulation upon transfer to high light  or normal light .
d quantification of the hlir <dig> mrna accumulation shown in panel c after normalization to the 5s rrna level.
relative values refer to the maximum level at  <dig>  h after hl shift
the previously annotated short gene ssr <dig> was chosen because of its expression under several different conditions  and its physicochemical characterization as a hydrophobic protein.
features of all  <dig> investigated μ-proteins are summarized in table  <dig>
homologs of ssr <dig> are frequently encoded by a small gene family and exist in plants , in e. coli  and in many other bacteria and other eukaryotic organisms, including yeast and c. elegans.
expression of the homologs rci2a and rci2b in a. thaliana became induced upon exposure to low temperature, dehydration, salt stress, or abscisic acid  <cit> .
ssr <dig> homologs possess two transmembrane helices  that form a pmp <dig> domain and might be a stress induced proteolipid membrane modulator.fig.
<dig> sequence alignment of ssr <dig> homologs from cyanobacteria with those from arabidopsis thaliana, desulfococcus oleovorans hxd <dig>  and identical proteins in  <dig> strains of rhodospirillum rubrum.
putative transmembrane domains were predicted using tmhmm v.  <dig>   <cit>  and are boxed
all five μ-proteins can be expressed from their native promoters in a regulated fashion
in the previous sections we verified the transcription of the five selected μ-protein encoding genes  as well as their translation from an mrna harboring the regulatory sequence elements  of the pete gene .
however, despite verifying a stable accumulation of the translated protein the latter approach renders the possibility of translating all rnas as long as they contain an open reading frame.
to exclude this possibility, we repeated the experiment from fig.
<dig> but placed all five flag-tagged μ-orfs under control of their own, native promoter and 5′utrs.
after introduction of these constructs into synechocystis  <dig> we subjected the resulting cultures to an inducing condition according to the transcriptome analysis.
samples from cultures grown at standard conditions or the inducing conditions were taken and analyzed by western blot experiments .
the results showed unambiguously the expression of all five μ-proteins when placed under control of their own promoters and 5′ utrs, i.e., their expression was not artificially induced by the ectopic fusion of their orfs to the pete promoter and 5′ utr.
we noticed a strong upregulation of nsir <dig> accumulation 24 h after transfer to nitrogen starvation and of hlir <dig> accumulation 6 h after exposure to high light as well as a mild upregulation of norf <dig> accumulation 24 h after transfer to nitrogen starvation .
the accumulation of norf <dig> increased somewhat 6 h after the shift to darkness.
these data show that the observed regulation of gene expression at rna level has a strong effect on the amounts of three of the respective proteins and a milder on one of the other two.fig.
<dig> detection of μ-proteins upon expression from their native promoters and 5′ utrs.
recombinant synechocystis  <dig> cells carrying the respective genes under control of their own promoters and 5′utrs on vector pvz <dig> were collected from cultures grown at standard conditions  and after transfer to the respective inducing condition at indicated time points  or in case of ssr <dig> after 24 h at standard condition.
the western blot was probed with specific anti-flag® m2-peroxidase  antibody.
all samples were separated on the same gel and transferred to the same membrane but the part probed for norf <dig> had to be exposed longer because of its lower expression.
prestained protein ladder  was used as molecular weight marker
discussion
for synechocystis  <dig> alone, more than  <dig> independent proteomic studies identified a total of  <dig> proteins at least once , representing  <dig> % of the entire predicted proteome.
however, the percentage of identified proteins was only  <dig> % for small proteins  of high hydrophobicity  <cit> .
in addition, as we show in this study, very short protein-coding genes might not even be modelled and annotated at all.
thus, due to the challenges in their identification and biochemical detection, μ-proteins were in the past either not detected or were ignored.
however, systematic genome-wide approaches have recently reported an increasing number of μ-proteins in pro- and eukaryotes  <cit> .
besides the short orfs within 5′ leader and 3′ trailer sequences of mrnas, known for a long time , μ-peptides were recently also described to originate from long ncrnas, i.e.
transcripts, which were previously assumed to be non-coding  <cit> .
in e. coli approximately  <dig> genes encoding μ-proteins have previously been reported  <cit> .
expression profiling showed that many μ-proteins accumulate under specific growth conditions or are induced by stress  <cit> .
a particular group of small proteins are toxic due to their integration into the cell membrane as peptide component of a type i toxin-antitoxin system .
in the cyanobacterium synechococcus elongatus, four small secreted proteins have been suggested to be involved in biofilm development  <cit> .
small proteins of the type ii toxin-antitoxin category in synechocystis  <dig> have been catalogued separately  <cit>  but the majority of them are somewhat larger than the here considered μ-proteins.
here, we found  <dig> candidate genes for small proteins ≤ <dig> amino acids in the model cyanobacterium synechocystis  <dig> and demonstrate the synthesis of five examples by c-terminal flag-tagging and immune detection.
three of these five small proteins are predicted to contain one or two transmembrane helices , placing them in the category of proteins that are particularly challenging to verify by proteomic approaches  <cit> .
hence, our list of predicted proteins provides a solid basis for functional studies.
regulated expression suggests involvement in stress adaptation for some of the here investigated small proteins.
this applies especially to hlir <dig>  nsir <dig> and norf <dig>  whose expression is activated in response to high light, nitrogen stress or transfer into darkness .
the fact that some of the here described proteins are part of tus much longer than needed points to the possibility that some of them could constitute dual function rnas.
such dual-function rnas that in addition to their role as a regulatory rna molecule also encode a functional peptide, have been identified in bacteria.
a prominent example for a dual function rna is the  <dig> amino acid peptide sgrt encoded in the 5′ region of the e. coli sgrs transcript, which regulates the glucose transporter ptsg at protein level, whilst the sgrs 3′ region contains a regulatory domain that targets the ptsg mrna by base-pairing  <cit> .
in bacillus subtilis, sr <dig> is a highly conserved dual-function srna that acts as a base-pairing regulatory rna on the ahrc mrna  and in addition encodes the  <dig> amino acid peptide sr1p.
interestingly, this peptide binds gapa , thereby stabilizing the gapa operon mrna  <cit> .
in analogy, it is interesting to note that the here described cyanobacterial norf <dig> μ-protein overlaps the gap <dig> mrna and appears to be co-regulated with it.
the high total numbers of predicted μ-orfs, together with the distribution, conservation, regulation of gene expression and the physicochemical properties of the five examples studied here in more detail, underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies.
CONCLUSIONS
synechocystis  <dig> is a widely used model cyanobacterium that possess with  <dig> genes encoding small proteins ≤ <dig> amino acids and potentially  <dig> proteins ≤ <dig> amino acids a high number of such μ-orfs.
these numbers are certainly no overestimation: due to the previous extensive work to elucidate all subunits of the photosynthetic apparatus, 52% of the small proteins ≤ <dig> amino acids have a known function.
this sets the small proteome of cyanobacteria apart from that of other bacteria: in addition to the  <dig> photosynthesis-related small proteins only five other in the size category ≤ <dig> are functionally annotated .
hence, about half of the predicted small proteins are uncharacterized.
when analysing small proteins up to  <dig> aa, we found  <dig> of the  <dig> predicted small proteins  without annotation.
the experimental results and expression data for the five here selected proteins  underline that it is worthwhile to study small protein functions directly in cyanobacteria.
the here provided data and strains will be useful for such studies in a systematic way.
abbreviations
hlir1high light inducible rna 1
norf <dig> and norf4novel orf  <dig> and 4
nsir6nitrogen stress-induced rna 6
tutranscriptional unit
uefunique expression factor

 proteins with less than amino acids in prokaryotes or amino acids in eukaryotes are defined as short proteins. during standard genome annotation these short protein-coding genes are frequently neglected and proteomics-based analyses fail to detect this class of peptides routinely. as a result, μ-protein-coding genes are a systematically underestimated class of gene products. in strong contrast is the finding that small orfs constitute the most frequent essential genomic component in bacteria, even more than conventional orfs. indeed, the functional characterization of selected examples of μ-proteins has revealed their critical involvement in processes such as quorum sensing or interspecies communication, regulatory functions and in the formation of multi-subunit protein complexes. an increasing number of μ-proteins is being discovered also in eukaryotes, and archaea, indicating their ubiquity in all three domains of life. nevertheless, the likely diverse functions of short proteins are largely unknown, even for simple unicellular bacteria. photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 μ-proteins of less than amino acids, that play a role in photosystem ii, psbi, psbl, psbj, psby, psbx, psb, psbn, psbf, psbk ), in photosystem i, photosynthetic electron transport, or have accessory functions ). the shortest annotated protein conserved in cyanobacteria is with amino acids the cytochrome b 6 f complex subunit viii, encoded by petn. several cyanobacterial model species have been studied by transcriptomics and proteomics approaches but there are no reports specifically targeting μ-proteins. based on extensive comparative transcriptome and genome information we used the model cyanobacterium synechocystis sp. pcc and the closely related strain synechocystis sp. pcc for the prediction of possible μ-orfs. we found transcriptional units containing candidate small orfs ≤ codons in synechocystis including all known mrnas encoding small proteins of the photosynthetic apparatus. we chose examples from synechocystis for experimental analysis. these were norf and norf, nsir and hlir, the latter two transcripts originally considered non-coding as well as the short gene ssr which was predicted as protein-coding in the current version of the genome sequence. all five proteins could be detected after flag tagging in vivo. their modes of regulation, conservation and physico-chemical properties make these five μ-proteins interesting candidates for functional studies. methods strains and growth conditions synechocystis substrain “pcc-m”, served as wt and was grown in cu2+-free, tes-buffered liquid bg medium with gentle agitation or on agar-solidified bg supplemented with % sodium thiosulfate at 30 °c under continuous illumination with white light of ~40 μmol photons m− s− to induce expression of flag - tagged μ-proteins from the cu2+-responsive pete promoter 2 μm cuso was added to exponentially growing cells. different environmental conditions were applied for induction of gene expression under control of native promoters: high light, 300 μmol photons m− s−1; dark, flasks wrapped with aluminium foil; nitrogen deficiency, cells were pelleted by centrifugation, washed once and resuspended in no3 −-free bg samples for protein extraction were taken just before and 6 h or 24 h after induction of gene expression. ssr was expected to be most expressed in exponential growth phase, hence samples were taken from exponentially growing cells at two consecutive days. synechocystis strain pur-ppetj-3xflag-sfgfp was used as positive control for the detection of flag-tagged proteins by western blots. e. coli strains top10f’ and j53/rp were used for generating synechocystis mutant strains by conjugation. in liquid bg medium 5 μg ml− gentamicin or 50 μg ml− kanamycin and 5 μg ml− gentamicin were used to maintain recombinant strains. for examination of gene expression by northern blot analysis, exponentially growing wt cells were transferred to the different environmental conditions described above. cultivation under high light was followed by a shift back to standard light conditions ). cultures grown in the dark as well as nitrogen deprived cultures were additionally aerated with ambient air through a glass tube and a sterile filter for constant and fast growth. computational methods small orfs and their orthologs were identified and annotated in synechocystis and in three steps.blastn searches returning hits with e values ≤1e− were performed against the ncbi nt database for all intergenic regions covered by tus. from the blast results, multiple alignments were created with clustalw and analyzed for their coding potential with rnacode. the significant small orf candidates were manually curated. to annotate candidate small orfs, blastp queries with e values ≤1e− were done against the ncbi nr database. orthologs of existing and newly detected small orfs were identified in synechocystis and via a reciprocal best hit approach using blastp with a minimum e value ≤1e− and allowing a difference in length of ≤20% and a maximum length of amino acids in both strains. genes of small orfs that were covered by a predicted tu were considered to be expressed. transmembrane helices were predicted with tmhmm server v.. generation of mutant strains gene constructs for ectopic expression of flag - tagged norf under control of the pete promoter or the native promoter were generated via gene synthesis. the constructs consisted of the upstream sequence of pete or the upstream sequence of norf, the norf coding sequence omitting the stop codon, a 3xflag coding tag followed by a stop codon, the norf 3′utr and the bacteriophage lambda oop terminator. the obtained ppete::norf1::3xflag::toop and pnorf1::norf1::3xflag::toop constructs were digested with xhoi and hindiii and introduced into self-replicating vector pvz. the resulting plasmids were transferred into synechocystis wt via triparental mating with e. coli j53/rp and top10f’. these two recombinant strains were selected on bg agar containing 10 μg ml− gentamicin. to establish ectopic expression of flag - tagged nsir hlir ssr and norf the respective genomic sequences were amplified using the primer pairs nsir6_fw/nsir6_rev, ppete::hlir1_fw/3xflag_hlir1_rev, ppete::ssr1169_fw/3xflag_ssr1169_rev and ppete::norf4_fw/3xflag_norf4_rev. all oligonucleotides used in this study are listed in table  the pete promoter was amplified separately for each construct to generate overlaps with the particular μ-orfs using the primer puc19-xbai_ppete_fw in different combinations with nsir6::ppete_rev, hlir1::ppete_rev, ssr1169::ppete_rev or norf4::ppete_rev. the 3′ segments consisting of the sequence encoding the 3xflag tag, the 3′utr of the norf mrna and the oop terminator were amplified from the plasmid obtained via gene synthesis described above using the primer 3xflag_psti-puc19_rev in combination with nsir6_3xflag_fw, hlir1_3xflag_fw, ssr1169_3xflag_fw or norf4_3xflag_fw, respectively. fragments belonging together were combined by gibson assembly® master mix according to the manufacturer’s instructions utilizing xbai and psti digested puc as vector backbone. for expression of the small proteins under control of their native promoters the obtained plasmids served as templates for amplifying corresponding coding sequences associated with the 3′ segment described above using the primer 3xflag_psti-puc19_rev in combination with cdsnsir6::pnsir6_fw, cdshlir1::phlir1_fw, cdsnorf4::pnorf or cdsssr1169::pssr1169_fw. upstream sequences of nsir hlir norf and ssr considered as promoter sequences were amplified from synechocystis genomic dna with the primer pairs puc19::pnsir6_fw/pnsir6::cdsnsir6_rev, puc19::phlir1_fw/phlir1::cdshlir1_rev, puc19::pnorf4_fw/pnorf4::cdsnorf4_rev or puc19::pssr1169_fw/pssr1169::cdsssr1169_rev. related fragments were combined by gibson assembly® master mix as described above. all resulting cassettes were released by restriction, introduced into pvz and transferred into synechocystis wt via triparental mating. additionally, the empty vector pvz was introduced into the wild type to create a control strain. the recombinant strains were selected on bg agar containing 10 μg ml− gentamicin and 50 μg ml− kanamycin.table list of oligonucleotides orf generating overlaps with ppete and 3xflag::3′utr n orf1::toop ssr or n orf4 rna extraction and analysis synechocystis cells were harvested by vacuum filtration on hydrophilic polyethersulfone filters, immediately immersed in 1 ml pgtx and frozen in liquid nitrogen. rna extraction was performed by 15 min incubation at 65 °c followed by chloroform washing and isopropanol precipitation as previously described. northern hybridization with 32p-labelled, single-stranded transcript probes was carried out as described. oligonucleotide sequences for pcr amplification of probe templates used for in vitro transcription are listed in table  protein purification and immunodetection cells for protein extraction were collected by centrifugation, resuspended in pbs buffer in the presence of protease inhibitor cocktail and immediately frozen in liquid nitrogen. cells were mechanically disrupted by using glass beads and a precellys® homogenizer at 6000 rpm and 4 °c applying six cycles of 3 × 10 s homogenization. glass beads were removed by centrifugation. to solubilize membrane proteins, samples were heated for 30 min at 50 °c with 2% sds followed by determination of the protein concentration using direct detect spectrometer. proteins were separated by sds-page on 15% polyacrylamide gels and stained with gelcode® blue stain reagent. pageruler™ prestained protein ladder was used as molecular weight marker. for immunoblot analysis, separated proteins were transferred to nitrocellulose membranes. membranes were blocked over night at 4 °c with 5% low fat milk powder in tbs-t and subsequently probed with monoclonal anti-flag® m2-peroxidase antibody raised in mouse in tbs-t for 1 h at room temperature in the dark. all washing steps were performed with gentle agitation in tbs-t tween-20) at room temperature. signals were detected with ecl™ start western blotting detection reagent on a chemiluminescence imager system and subsequently visualized using fusion-cap and quantity one software. reporter gene assays to measure promoter activity as a function of bioluminescence the putative norf promoter sequence and its 5′utr was fused to promoterless luxab reporter genes by pcr, followed by cloning into the promoter probe vector pila as described. the resulting pila derivative was used for transformation of a synechocystis strain expressing the luxcde genes encoding enzymes for the synthesis of decanal, the luciferase substrate, under control of the strong promoter of the ncrna yfr2a. cells were grown in the presence of 10 mm glucose to provide energy for the luciferase reaction also in darkness. bioluminescence was measured in vivo at different time points after inducing dark conditions as described.  comparative transcriptomics for the identification of μ-proteins in synechocystis the extensive comparative transcriptome and genome information for the model cyanobacterium synechocystis and the closely related strain synechocystis was utilized for the prediction of possible μ-orfs. in our previous studies transcriptional units had been defined, combining information on the transcriptional start sites, the lengths of transcribed utrs, operons, coding and non-coding regions. here we judged all possible non-coding transcripts by the program rnacode for their protein-coding potential. rnacode detects protein-coding regions in any given sequence on the basis of multiple sequence alignments and the evolutionary signatures that are associated with a coding sequence. after combination with the pre-existing annotation, this analysis led to the prediction of potential small proteins with a maximum of amino acids in synechocystis and possibly in synechocystis.fig. scheme of computational prediction of small orfs in cyanobacteria. small orfs were detected based on the transcriptome information for expressed intergenic regions and their coding potential evaluated with rnacode. this information was merged with the pre-existing annotation. orthologs between the two small orf populations were detected when they were identified as reciprocal best hits by blastp with e ≤ 1e−2 the resulting sets of candidate μ-proteins were compared against the predicted proteome of the respective other synechocystis strain, against e. coli and the higher plant arabidopsis thaliana as reference organisms for proteins possibly conserved among bacteria or among photosynthetic organisms. this procedure led to the identification of 146 μ-proteins shared between the two synechocystis strains, as well as and 29 μ-proteins which are shared between synechocystis and a. thaliana or e. coli, respectively. interestingly, we found the proteins shared with higher plants to be identical in both synechocystis strains. in contrast to observations in other bacteria, a relatively high number of the predicted proteins in the smallest fraction had assigned functions and a matching protein in the higher plant arabidopsis thaliana or in e. coli.table predicted and previously annotated proteins ≤ amino acids in synechocystis 6803 petn 6 psbt, ycf8 psam 1148230 1148325 - 31 norf4 norf4 norf4 y n n petl 6 psbm petm 6 1606868 1606978 + 37 hlir1 hlir1 hlir1 y n n rpl36 petg 6 psbi psbl psbj psby psbx ycf12 psaj psai ndhp psbn psbf psbk rpl34 ndhq hlic, scpb 298826 298972 - 48 norf1 norf1 norf1 n n the start and end positions according to the chromosomal or plasmid sequences in genbank files, the location on the forward or reverse strand and respective length are given, followed by the locus tag id, gene name and product if assigned. location on chromosome or one of the plasmids is prefixed by “chr” or the name of the plasmid. the existence of homologs in synechocystis a. thaliana and e. coli is indicated by “y” for yes or “n” for no. homologs tagged and detected in this study are highlighted in boldface letters. names of genes tested in this work are in boldface in vivo tagging and detection of cyanobacterial μ-proteins we chose examples for closer analysis: norf nsir hlir ssr and norf norf and norf were previously defined based on transcriptomic evidence. the protein ssr was previously modelled as part of the existing annotation, but there is no information on possible functions nor that their very existence was shown thus far. nsir and hlir are not annotated in the genome but were found by transcriptomics. although these rnas harbor potential open reading frames they were initially indicated as non-coding. after flag - tagging and inducing their expression in synechocystis all five proteins were detected by western blotting. hlir and ssr showed a tendency for aggregation, even under the used denaturing conditions, possibly related to their hydrophobicity and the predicted presence of transmembrane regions.fig. western blot detection of small proteins. recombinant synechocystis cells carrying the genes of interest under control of the pete promoter on pvz vector were collected before or 24 h after induction of gene expression for the extraction of total proteins. flag-tagged superfolder gfp under the control of the petj promoter served as positive control, a wt strain carrying an empty pvz vector was used as negative control. theoretical protein masses are listed in table  two gels were run in parallel. a proteins were separated on a 15% sds polyacrylamide gel and subjected to colloidal coomassie g- staining as a loading control. b immunoblot with the same loading order probed with specific anti-flag® m2-peroxidase antibody the nsir transcript is highly induced upon nitrogen deprivation nsir was not previously known as a protein-coding gene. its mrna originates from a tss at position 729645f in the chromosome of synechocystis. previously, we introduced the uef to identify genes whose expression was enhanced at a single from ten tested environmental conditions. this factor gives the ratio of the transcriptome read counts for the condition with the highest and the one with the second highest expression for a single tu. thus, tus with a high uef respond strongly to a particular stimulus. for nsir the uef was, ranking on position of the most-strongly induced genes, both in synechocystis as well as in strain, when the cells were deprived of sources of combined nitrogen. this induction was confirmed by independently performed northern blots, indicating a rapid induction of expression, reaching a peak at 6 h with an about 10-fold higher transcript accumulation, followed by a declining abundance which remained higher than at the beginning of the experiment. the nitrogen-stress-dependent induction is likely mediated via a conserved ntca binding site 5′-gtaacatttgtgac-3′, centered 42 nt upstream the transcription initiation site in both strains. ntca-binding sites frequently overlap the − promoter region and are centered close to position − with respect to the tss when they mediate activation. homologs of nsir are widely conserved throughout the cyanobacterial phylum and in the paulinella chromatophora chromatophore genome, consistent with its occurrence in the genomes of α-cyanobacteria, but not in any other bacteria or plants. the alignment of these homologs shows two pairs of conserved cysteine residues which might be involved in redox control, protein-protein interactions or structure formation. two pairs of cysteine residues occur also in another short protein, the amino acid cp protein, which mediates the formation of a complex between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase in response to changes in light intensity, characterizing it as a thioredoxin-mediated metabolic switch. in cp the cysteine pairs confer the redox input via post-translational thiol-disulfide bridge conversion. the arrangement ‘cpvc’ of the first cysteine pair matches the c-2-c motif, which frequently is involved in metal-binding. hence, the putative cysteine pairs in nsir may confer redox control or metal binding.fig. heatmap indicating the expression of the genes encoding the five investigated small proteins in synechocystis strains pcc and pcc under different growth conditions: exponential and stationary growth phase; cold and heat stress for 30 min each; depletion of inorganic carbon, cells were washed times with carbon-free bg and cultivated further for 20 h; dark, no light for 12 h; fe2+ limitation, addition of iron-specific chelator desferrioxamine b and further cultivation for 24 h; high light, 470 μmol photons m−2s− for 30 min; nitrogen depletion, cells were washed times with nitrogen-free bg and cultivated further for 12 h; phosphate depletion, cells were washed times with phosphate-free bg and further incubated for 12 h. data derived from previous genome-wide expression analysis by differential rna-seq. values indicate sequencing read counts for the primary 5′ end of the corresponding transcriptional unit. the tss positions are given for the synechocystis genomes available under accession numbers ba and cp the colour varies from red to yellow to green fig. the nsir peptide. a transcriptomic datasets indicated high read coverage in a region without annotation in synechocystis, which contains the here defined nsir gene. the homolog in synechocystis is d082_. shown is the read coverage resulting from previous transcriptome analysis, including the respective transcriptional units defined in that work and a putative ntca binding site, centered 42 nt upstream the transcription initiation site in both strains. relevant transcription initiation sites appear as steep increase in read coverage and are labelled by a black arrow. the length of the 5’-utrs is 26 nt in both strains. other non-coding tus are colored orange. there is transcription in antisense orientation in both strains but with much lower coverage. b northern blot showing the nitrogen stress-induced transcript accumulation of the nsir mrna in synechocystis over 72 h. time point refers to the nitrogen-replete condition. c time course of nsir mrna accumulation after normalization to 5s rrna. the data are presented as relative to the signal at 6 h after diminishing n. d sequence comparison of nsir homologs from the two synechocystis strains, cyanothece atcc crocosphaera watsonii wh and the two marine synechococcus strains wh and wh which harbor an identical protein. four conserved cysteine residues are highlighted by arrows. these are conserved in all homologs detected throughout the cyanobacterial phylum norf is highly induced upon dark incubation norf is specific for cyanobacteria but widely conserved throughout this phylum. it is present in of cyanobacterial genomes available in the jgi database. homologs are lacking in early-branching cyanobacteria such as gloeobacteria and thermophilic synechococcus ja-2-3b’a and ja-3-3ab and also in marine picocyanobacteria. an alignment of representative homologs is shown in fig. 5a.fig. sequence comparison and regulated expression of the norf gene. a sequence comparison of norf homologs from the two synechocystis strains, cyanothece atcc different strains of crocosphaera encoding an identical protein and anabaena sp. pcc b expression of norf in synechocystis is strongly upregulated after transfer to darkness. c bioluminescence of a synechocystis reporter strain harboring a transcriptional fusion of pnorf and luxab genes in response to transfer to darkness. representative bioluminescence dataset indicating means ± sd of measurements for two biological replicates. a strain carrying a promoterless luxab was used as a negative control strong accumulation of the norf mrna was observed in response to darkness. the uef for this condition was in synechocystis but the gene was expressed also under the other tested conditions. to examine whether the dark-related expression of norf is under transcriptional control, we conducted reporter gene assays. the upstream sequence of synechocystis norf was fused to luxab reporter genes encoding luciferase, and expression was monitored as bioluminescence in vivo. indeed, the promoter activity showed a positive response after transfer into darkness as seen for the mrna accumulation. we conclude that the observed induction of norf in response to shifts from light exposure to darkness is under transcriptional control. the high expression of the norf gene in darkness sets it apart from the vast majority of genes. among the previously tested different growth conditions, in synechocystis only out of tus and in synechocystis only out of tus defined in total had their maximum expression after dark incubation. the norf μ-protein is highly conserved and its mrna overlaps the gap gene norf is encoded within a tu much longer than is needed to encode the amino acids: tu in synechocystis is 704 nt and tu in synechocystis is 534 nt. these tus partially overlap the gap gene encoding glyceraldehyde 3-phosphate dehydrogenase on the complementary dna strand. as a result, these tus overlap the gap mrna by and 373 nt, respectively. transcriptomic evidence suggested that both the gap and the norf mrnas were co-regulated with each other, with a mild up-regulation upon the removal of nitrogen. thus, the norf transcript does not function as an antisense rna with a co-degradation function, which was observed previously for other pairs of overlapping transcripts in synechocystis. however, co-regulation between an asrna and its cognate mrna was previously observed for the psba asrna protecting its 5′ leader from rnase e-mediated degradation. the expression of norf was stimulated upon removal of nitrogen, but its expression was detectable under most of the previously tested conditions, although at a lower level. dual-function rnas are transcripts which assume a regulatory function as srna and additionally act as short protein-coding mrna. exploring this possibility for norf we checked the accumulation of norf transcripts during the removal of combined nitrogen. northern blot analysis showed the existence of a prominent transcript of ~200 nt which declined initially. due to the localization of the rna probe used in the detection of norf transcripts, this prominent transcript corresponds to the coding part of tu however, with increasing duration of the nitrogen stress, we noticed the overaccumulation of a longer transcript, of about 600–800 nt that appeared more diffuse. quantitative analysis of transcript accumulation showed that this longer norf transcript was only transiently accumulated, with a peak at the 24 h time points.fig. the norf peptide. a datasets from the previously performed primary transcriptome analysis showed that norf expression responded positively to nitrogen depletion in both synechocystis and synechocystis, when different growth conditions were tested. the mrnas of norf and gap are co-regulated and overlap by several hundred nt. the previously mapped transcriptional start sites are labelled by black arrows. b northern blot analysis of norf mrna accumulation in a time course experiment up to 72 h after the removal of nitrogen. the same rna samples were used as in fig.  c the signals obtained from the northern blots were evaluated densitometrically after normalization by the level of 5s rrna. the relative norf expression is shown with respect to the maximum expression after transfer to nitrogen-free conditions. the bands at 200 nt and at 800 nt were analyzed separately from each other. d multiple sequence alignment of homologs from different cyanobacterial genome sequences with very few amino acid substitutions, norf is extremely conserved, including a predicted transmembrane region. homologs can be detected in cyanobacterial genome sequences from all morphological subsections, comprising free-living unicellular as well as multicellular strains, marine and freshwater isolates, thermophiles and symbionts. the presence of norf in the two available genome sequences of candidatus atelocyanobacterium thalassa suggests their positive selection in these highly streamlined genomes. however, homologs are lacking in α-cyanobacteria, which are mainly marine synechococcus and prochlorococcus. the homologs from the two used synechocystis strains are identical, except for a possible n-terminal extension by amino acids in synechocystis. however, such extensions appear questionable also in other strains, because the start codon corresponding to the synechocystis orf is 100% conserved. moreover, the homologs in microcystis genomes are identical to each other, as are the homologs in five crocosphaera watsonii and in two fischerella genome sequences. our data suggest that norf is a previously unknown membrane-bound μ-protein and that the norf transcript may play a dual role, with a mainly coding function during nitrogen-sufficient conditions and a possibly rna-mediated regulatory function on the gap mrna during nitrogen stress. hlir and ssr1169 hlir was chosen because of its very high induction under high light and the gene location upstream of sodb encoding superoxide dismutase. whereas the homologs from the two synechocystis strains are conserved in length, sequence and the likely presence of a transmembrane region, no possible homologs were detected beyond the genus synechocystis. the location upstream of sodb and the shape of the read coverage in transcriptome analysis suggested a possible link between the two genes. indeed, northern analysis confirmed the inducibility by high light and in addition showed the presence of two major transcripts, ~ and 1400 nt in length. the longer form should encompass also the complete sodb gene. thus, transcription from the upstream located hlir promoter will lead by read-though to an enhanced sodb gene expression under high light. hence, it is tempting to speculate, that hlir is a membrane-bound peptide with a regulatory function on the superoxide dismutase.fig. the hlir peptide in synechocystis a pairwise sequence alignment of the hlir peptides from synechocystis and synechocystis a predicted transmembrane region is boxed. b data replotted from the primary transcriptome analysis of synechocystis suggest that hlir expression is induced by high light and that transcripts may extend into the subsequent tu covering the sodb gene. c northern analysis of hlir mrna accumulation upon transfer to high light or normal light. d quantification of the hlir mrna accumulation shown in panel c after normalization to the 5s rrna level. relative values refer to the maximum level at  h after hl shift the previously annotated short gene ssr was chosen because of its expression under several different conditions and its physicochemical characterization as a hydrophobic protein. features of all investigated μ-proteins are summarized in table  homologs of ssr are frequently encoded by a small gene family and exist in plants, in e. coli and in many other bacteria and other eukaryotic organisms, including yeast and c. elegans. expression of the homologs rci2a and rci2b in a. thaliana became induced upon exposure to low temperature, dehydration, salt stress, or abscisic acid. ssr homologs possess two transmembrane helices that form a pmp domain and might be a stress induced proteolipid membrane modulator.fig. sequence alignment of ssr homologs from cyanobacteria with those from arabidopsis thaliana, desulfococcus oleovorans hxd and identical proteins in strains of rhodospirillum rubrum. putative transmembrane domains were predicted using tmhmm v. and are boxed all five μ-proteins can be expressed from their native promoters in a regulated fashion in the previous sections we verified the transcription of the five selected μ-protein encoding genes as well as their translation from an mrna harboring the regulatory sequence elements of the pete gene. however, despite verifying a stable accumulation of the translated protein the latter approach renders the possibility of translating all rnas as long as they contain an open reading frame. to exclude this possibility, we repeated the experiment from fig.  but placed all five flag-tagged μ-orfs under control of their own, native promoter and 5′utrs. after introduction of these constructs into synechocystis we subjected the resulting cultures to an inducing condition according to the transcriptome analysis. samples from cultures grown at standard conditions or the inducing conditions were taken and analyzed by western blot experiments. the results showed unambiguously the expression of all five μ-proteins when placed under control of their own promoters and 5′ utrs, i.e., their expression was not artificially induced by the ectopic fusion of their orfs to the pete promoter and 5′ utr. we noticed a strong upregulation of nsir accumulation 24 h after transfer to nitrogen starvation and of hlir accumulation 6 h after exposure to high light as well as a mild upregulation of norf accumulation 24 h after transfer to nitrogen starvation. the accumulation of norf increased somewhat 6 h after the shift to darkness. these data show that the observed regulation of gene expression at rna level has a strong effect on the amounts of three of the respective proteins and a milder on one of the other two.fig. detection of μ-proteins upon expression from their native promoters and 5′ utrs. recombinant synechocystis cells carrying the respective genes under control of their own promoters and 5′utrs on vector pvz were collected from cultures grown at standard conditions and after transfer to the respective inducing condition at indicated time points or in case of ssr after 24 h at standard condition. the western blot was probed with specific anti-flag® m2-peroxidase antibody. all samples were separated on the same gel and transferred to the same membrane but the part probed for norf had to be exposed longer because of its lower expression. prestained protein ladder was used as molecular weight marker discussion for synechocystis alone, more than independent proteomic studies identified a total of proteins at least once, representing % of the entire predicted proteome. however, the percentage of identified proteins was only % for small proteins of high hydrophobicity. in addition, as we show in this study, very short protein-coding genes might not even be modelled and annotated at all. thus, due to the challenges in their identification and biochemical detection, μ-proteins were in the past either not detected or were ignored. however, systematic genome-wide approaches have recently reported an increasing number of μ-proteins in pro- and eukaryotes. besides the short orfs within 5′ leader and 3′ trailer sequences of mrnas, known for a long time, μ-peptides were recently also described to originate from long ncrnas, i.e. transcripts, which were previously assumed to be non-coding. in e. coli approximately genes encoding μ-proteins have previously been reported. expression profiling showed that many μ-proteins accumulate under specific growth conditions or are induced by stress. a particular group of small proteins are toxic due to their integration into the cell membrane as peptide component of a type i toxin-antitoxin system. in the cyanobacterium synechococcus elongatus, four small secreted proteins have been suggested to be involved in biofilm development. small proteins of the type ii toxin-antitoxin category in synechocystis have been catalogued separately but the majority of them are somewhat larger than the here considered μ-proteins. here, we found candidate genes for small proteins ≤ amino acids in the model cyanobacterium synechocystis and demonstrate the synthesis of five examples by c-terminal flag-tagging and immune detection. three of these five small proteins are predicted to contain one or two transmembrane helices, placing them in the category of proteins that are particularly challenging to verify by proteomic approaches. hence, our list of predicted proteins provides a solid basis for functional studies. regulated expression suggests involvement in stress adaptation for some of the here investigated small proteins. this applies especially to hlir nsir and norf whose expression is activated in response to high light, nitrogen stress or transfer into darkness. the fact that some of the here described proteins are part of tus much longer than needed points to the possibility that some of them could constitute dual function rnas. such dual-function rnas that in addition to their role as a regulatory rna molecule also encode a functional peptide, have been identified in bacteria. a prominent example for a dual function rna is the amino acid peptide sgrt encoded in the 5′ region of the e. coli sgrs transcript, which regulates the glucose transporter ptsg at protein level, whilst the sgrs 3′ region contains a regulatory domain that targets the ptsg mrna by base-pairing. in bacillus subtilis, sr is a highly conserved dual-function srna that acts as a base-pairing regulatory rna on the ahrc mrna and in addition encodes the amino acid peptide sr1p. interestingly, this peptide binds gapa, thereby stabilizing the gapa operon mrna. in analogy, it is interesting to note that the here described cyanobacterial norf μ-protein overlaps the gap mrna and appears to be co-regulated with it. the high total numbers of predicted μ-orfs, together with the distribution, conservation, regulation of gene expression and the physicochemical properties of the five examples studied here in more detail, underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies.  synechocystis is a widely used model cyanobacterium that possess with genes encoding small proteins ≤ amino acids and potentially proteins ≤ amino acids a high number of such μ-orfs. these numbers are certainly no overestimation: due to the previous extensive work to elucidate all subunits of the photosynthetic apparatus, 52% of the small proteins ≤ amino acids have a known function. this sets the small proteome of cyanobacteria apart from that of other bacteria: in addition to the photosynthesis-related small proteins only five other in the size category ≤ are functionally annotated. hence, about half of the predicted small proteins are uncharacterized. when analysing small proteins up to aa, we found of the predicted small proteins without annotation. the experimental results and expression data for the five here selected proteins underline that it is worthwhile to study small protein functions directly in cyanobacteria. the here provided data and strains will be useful for such studies in a systematic way. abbreviations hlir1high light inducible rna 1 norf and norf4novel orf and 4 nsir6nitrogen stress-induced rna 6 tutranscriptional unit uefunique expression factor 
the distribution and conservation of these five genes as well as their regulation of expression and the physico-chemical properties of the encoded proteins underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies. photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of small proteins of less than amino acids. from these transcriptional units, are shared between the two strains, are shared with the higher plant arabidopsis thaliana and with e. coli. upon activation of expression via the cu2+.responsive pete promoter or from the native promoters, all five proteins were detected in western blot experiments. despite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than amino acids are a systematically underestimated class of gene products in bacteria. in analogy, previously unstudied small orfs with similar degrees of conservation might encode small proteins of high relevance also in other functional contexts. pcc also including the known mrnas encoding small proteins of the photosynthetic apparatus. cyanobacterianitrogen deprivationphotosynthesissynechocystissmall proteinsissue-copyright-statement© the author 2016 pcc for the prediction of small orfs. we found transcriptional units containing candidate small orfs ≤ codons in synechocystis sp.
 despite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than amino acids are a systematically underestimated class of gene products in bacteria. photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of small proteins of less than amino acids. in analogy, previously unstudied small orfs with similar degrees of conservation might encode small proteins of high relevance also in other functional contexts.  here we used comparative transcriptomic information available for two model cyanobacteria, synechocystis sp. pcc and synechocystis sp. pcc for the prediction of small orfs. we found transcriptional units containing candidate small orfs ≤ codons in synechocystis sp. pcc also including the known mrnas encoding small proteins of the photosynthetic apparatus. from these transcriptional units, are shared between the two strains, are shared with the higher plant arabidopsis thaliana and with e. coli. to verify the existence of the respective μ-proteins in vivo, we selected five genes as examples to which a flag tag sequence was added and re-introduced them into synechocystis sp. pcc these were the previously annotated gene ssr two newly defined genes norf and norf as well as nsir6 and hlir, which originally were considered non-coding. upon activation of expression via the cu2+.responsive pete promoter or from the native promoters, all five proteins were detected in western blot experiments.  the distribution and conservation of these five genes as well as their regulation of expression and the physico-chemical properties of the encoded proteins underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies. keywords cyanobacterianitrogen deprivationphotosynthesissynechocystissmall proteinsissue-copyright-statement© the author 2016 